© 2016 Gene Codes Corporation, Inc.

All rights reserved

Sequencher® is a trademark of Gene Codes Corporation
Microsoft® is a registered trademark of Microsoft Corporation.
Macintosh® is a trademark of Apple Computer, Inc.
All other trademarks are the properties of their respective owners.
1. Preface 1

Gene Codes 1

2. Using this manual 2

Conventions used in this manual 2

Installing Sequencher 2

Sequencher Help 2

Navigating Sequencher Help 2

Context-Sensitive Screens 3

The button bar 3

Sequencher’s menus 4

Sequencher (Mac only) 4

File menu 4

Edit menu 4

Select menu 4

Assemble menu 4

Contig menu 4

Sequence menu 5

View menu 5

Window menu 5

Help menu 5

Context- Sensitive menus and buttons 5

Contextual menus 6

Keyboard shortcuts 6

3. The Project Window 7

Sequencher concepts 7

About the Project Window 7

i
 Creating a new project 7

Opening an existing project 8

Working with items in the Project Window 8

Icon types 8

Project views 9

View as Large Icons 9

View as Small Icons 10

Moving icons 10

Cleaning up 11

View as a list 11

Project Window Columns 12

Viewing constituent sequences 13

Viewing the information for a sequence or contig 13

Editing the information for a sequence or contig 14

The Project Window button bar 14

Templates 15

Save Project As Template 15

New Project From Template 15

Import from Template 15

4. Importing data 16

Quick and easy importing of data 16

Dragging and dropping files 16

Copy and paste 16

How to import using menu commands 16

Importing sequences 16

Importing GenBank features 17

Importing lists of sequences 17

ii
 Folder of sequences 18

Projects in specific formats 19

Sequencher projects 19

Files from VecBase 20

Creating a new sequence 20

Double-clicking a project icon 21

Importing Confidence Scores 21

Importing PHRED called data 21

Importing PHRAP Files 21

Removing sequences and contigs from a project 22

Closing an editor window 22

Closing a project 23

5. Organizing project views 24

Working with icons and lists 24

Changing between project views 24

Collecting sequences in refrigerators 24

Selecting sequences 24

Selecting sequences and contigs 24

Selecting with the mouse 25

Selecting by typing 25

Selecting multiple items with a marquee 25

Selecting using menu commands 26

Selecting all icons 26

Selecting all items containing sequence 26

Finding assembled sequences 27

Renaming a sequence or contig 27

Renaming using the mouse 27

iii
 Renaming using the menu 27

Labeling items in the Project Window 28

Saving your work 28

Saving your Project 28

Auto-Save 29

Saving a duplicate of a current project 29

Reverting to the saved version of the project 29

Closing the Project Window 29

Exporting data 29

Exiting the program 30

6. Preparing Your Data for Assembly 31

Removing undesirable data 31

Trimming poor quality data 31

Selecting and trimming sequence data 31

Performing a trim with default settings 31

Automatically selecting sequences for ends trimming 32

How to set the Ends Trimming criteria 32

Using Confidence to Trim Ends 33

Reviewing the Trim 33

Sorting items 34

Executing the trim 34

Trim Ends without Preview 35

Trimming vector contamination 36

Selecting sequences for Vector Trimming 36

Specifying a vector using VecBase 36

Specifying the insertion site manually 38

Saving and loading vector sites 38

iv
 Setting Vector Trimming Criteria 38

How Sequencher screens sequences 39

Executing the trim command 40

Trim Log 40

7. The Reference Sequence 41

What is a Reference Sequence? 41

Why use a Reference Sequence? 41

Reference Sequence properties 41

Basic Reference Sequence properties 41

Working with a Reference Sequence 43

How to mark a sequence as a Reference 43

Assemble to Reference 43

To Reference by Name 43

Trim to Reference 43

Contig Editing with a Reference Sequence 44

Reference Sequence Translation 45

8. The Sequence Editor 46

Opening an existing sequence for editing 46

Locked Editors 46

Basic sequence editing 46

Setting the base numbering 48

Set Circular Genome Size 48

Splitting a sequence 48

Duplicating a sequence 49

Reverse and Complement 49

About experimental data 49

Creating a baseline 49

v
 Resetting a baseline 50

Viewing experimental data 50

Reverting to experimental data 50

Viewing experimental data for Chromatograms 51

Viewing summary information in the Sequence Editor 51

Selecting Bases 52

Finding Ambiguous Bases 52

Finding Open Reading Frames (ORF) 53

Sequence features 53

Looking at Codon Maps 53

Looking at Restriction Maps 54

Annotating Sequences 55

Customizing your sequence view 56

Formatting Ruler 56

Margins 56

Translation 57

Base grouping 57

Line spacing 57

Case and Font 57

Voice verification 58

Selecting a voice file 58

Identifying a speaker 58

Audible keystrokes 58

Read sequence selection 58

9. Sequence assembly 60

The Assembly Strategy 60

Setting the Assembly Conditions 60

vi
 Setting assembly parameters 60

Assembly Algorithms 61

Assembling with Dirty Data 62

Assembling with Clean Data 62

Assembling with Large Gap 63

Refining the Assembly conditions 63

Minimum Match Percentage 63

Minimum Overlap 63

Maximum Loop Out Size 63

Optimization of gap placement 64

Performing the assembly 64

Automatic assembly 64

Adding selected items to others – incremental building of contigs 64

Assemble to Reference 65

Interactive assembly 65

Performing an interactive assembly 65

Changing parameters for an interactive assembly 68

Mindlessly Join 68

MUSCLE 69

Using MUSCLE to align sequences 69

10. Assemble by Name 70

The Assemble by Name Strategy 70

Setting the Assemble by Name Conditions 71

Configuring Assembly Handles using a Single Delimiter 71

Configuring Assembly Handles using Advanced Expressions 72

What is a regular expression? 72

Setting your Assembly Handle Names 73

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 Choosing a New Assembly Handle 74

Enabling Assemble by Name 74

Setting Assembly Parameters 75

Performing an Assembly with Assemble by Name 75

Aligning sequences with Clustal 76

Using Clustal to align sequences 77

Using Clustal with Assemble by Name 77

11. The Contig Editor 79

Contig editing 79

Overview of the contig 79

The contig overview window 79

Selection marquee 80

Checking coverage in the Overview 80

Overview features 81

Bases 81

Summary 81

Sort 81

Overview options 81

Scale Diagram To Window Size 82

Diagram Key 82

Labels Of Fragments 82

Names Of Fragments 82

Names Of Fragments, At Left 82

Positions Of Fragments 83

Start & Stop Codons 83

Base Numbers At Transitions 83

Base Numbers At Every x Bases 84

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 Condensed Base Numbers 84

Contig Coverage Graph 84

Graphic Feature Maps 84

Getting more information 85

Find 85

Get Info 86

The Bases View 86

Working in the Bases View 86

Viewing sequence names 88

Before you start to edit 88

Setting the base numbering 88

Confidence Histograms 88

Consensus calculation 89

Consensus Inclusively 89

Consensus By Plurality 90

Consensus to Forensic Standards 91

Consensus by Confidence 91

Assigning a Reference Sequence 92

12. Editing Contigs 93

Finding bases which need attention 93

Viewing an individual sequence from the contig 93

Finding disagreeing ambiguities 93

Finding Disagreeing Bases 94

Finding Low Confidence Bases 94

Finding Edited Bases 94

Performing edits 94

The Edit Command 94

ix
 Editing modes 95

Viewing Base Edits 95

Collect Gaps 96

Moving Bases and Gaps 96

Using ReAligner to clean up contigs 97

Moving sequences 97

Deleting bases 98

Deleting bases from the 5’ or 3’ ends 99

Inserting gaps or bases into a contig 99

Making edits from the consensus line 100

Split After Selection… 100

Auto Match 101

Create new sequence from a consensus 101

Removing sequences from a contig 102

Dissolving a contig 102

13. The Variance Table 103

The Variance Table Strategy 103

What is a Variance Table 103

The Variance Table and consensus sequences 103

The Translated Variance Table 103

The Structure of the Variance Table 104

Structure of the Translated Variance Table 105

Creating a Variance Table 106

Variance Table for sequences in the same contig 106

The consensus Variance Table 107

Translated Variance Table for sequences in the same contig 108

The Translated consensus Variance Table 109

x
Setting the Variance Table Conditions 110

Restricting the Comparison Range 110

Restricting the Translation Range 111

Setting the Variance Table Display Options 115

Setting the Consensus Calculation for a Variance Table 116

Variance Table Options 117

Working with the Variance Table 117

Navigating a Variance Table 117

Sorting the Variance Table 117

Removing columns from the Variance Table 118

Review Mode 118

The Review Mode and Compare Consensus to Reference 119

The Review Mode and the Translated Variance Table 120

The Review Mode and Sister Tables 121

Variance Table Reports 121

Variance Table Report 122

Individual Variance Reports 123

Variance Detail Report 124

Working with Variance Table Reports 126

Printing a Variance Table Report 126

Printing selected data from a Variance Table 127

Exporting a Variance Table Report 128

Exporting selected sample sequences 129

Exporting data for selected variants 130

Translated Variance Table Report 131

Translated Variance Table Report 131

Working with a Translated Variance Table Report 131

xi
 Exporting a Translated Variance Table Report 131

Exporting selected sample sequences from a Translated Variance Table 132

Exporting data for selected variants 133

Removing data from a table before export 134

14. The Summary Report 135

The Summary Report view 135

View by Summary 135

Summary report display options 136

Compare To Consensus or Reference Sequence 136

Icons 136

Bullets, Pluses and Dashes 137

Consensus Sequence 137

Fragment Sequences 137

Matching Bases as Dashes 137

Protein translations and the Summary Report 138

Enabling the protein display 138

Protein translations for the Consensus or Reference Sequence 138

Protein translations for Fragments 139

Matching Proteins As Dashes 139

Using the formatting ruler 139

Reverse and Complement a contig 139

15. Chromatograms 140

Working with automated sequencer data 140

Viewing chromatograms from a Sequence Editor 140

Viewing chromatograms from a contig editor 140

Understanding the Trace Display 141

Display secondary peak 142

xii
 Select Next Commands 144

Editing bases from traces 144

Reverting to experimental data 145

Reverting assembled sequences back to experimental data 146

Adjusting trace positions 147

16. NGS for DNA and RNA-Seq 148

Sequence File Formats 148

FastA and FastQ formats 148

Paired-end data 149

GSNAP list of known SNPs file 149

GSNAP FastA format file 150

SAM format 150

AFG Format 150

Location of Results Files – The External Data Home 150

Strategies 152

External Data Browser 152

Using the button bar 153

Runs pane 153

Log File/Notes pane 154

Performing Reference-Guided alignments 154

FastQ Quality Control Reports 154

Creating a reference sequence database or index 156

Reference-guided alignment using the MAQ algorithm 159

Reference-guided alignment using GSNAP or BWA 161

Looking at Reference-guided alignment results 163

Advanced Parameters for GSNAP 164

GSNAP and Indels 166

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 Advanced Parameters for BWA-MEM 166

Further Analyses with Reference-guided Alignments 168

SNP hunting with Maq 168

SNP tolerant alignment with GSNAP 170

Methylation studies with GSNAP 171

RNA A-to-I tolerant alignment with GSNAP 173

Variant Calling with SAMtools 174

Viewing VCF files in Tablet 175

Reference-Guided Aligners and Multiplex IDs 176

Format of barcode file 176

Enabling Multiplex ID mode 177

Running GSNAP with Multiplex data 178

Running BWA-MEM with Multiplex data 179

Performing de novo assemblies 181

Assembling Single-end data with Velvet 182

Assembling Paired-end data with Velvet 182

Looking at Velvet results in Tablet 183

Advanced Parameters for Velvet 183

Using Sequencher to improve a Velvet alignment 184

Velvet and Multiplex IDs 185

Format of barcode file 185

Enabling Multiplex ID mode 185

Running Velvet with Multiplex data 186

RNA-Seq and Differential Expression 187

RNA-Seq Workflow 188

What is a GTF file 189

Obtaining GTF files from UCSC 190

xiv
 Obtaining a FastA file of transcripts 191

Managing your RNA-Seq analyses with the External Data Browser 191

Cufflinks 194

Advanced options for Cufflinks 196

Viewing Cufflinks results files 197

Cuffmerge 198

Cuffdiff 199

Advanced options for Cuffdiff 202

Viewing RNA-Seq Differential Expression results 203

The Volcano and Scatter Plot 205

The Bar Chart 207

Searching for specific genes in a results table 208

Sorting and plotting selected results 208

The alternate route to analysing your RNA-Seq data 209

Cuffquant 209

Advanced options for Cuffquant 210

Cuffnorm 210

Advanced Options for Cuffnorm 212

17. Restriction Maps 213

Displaying a Restriction Map 213

Restriction Map display options 213

How to select enzymes 213

How to show cut positions or fragment sizes 214

How to set the map style 214

Setting the map width 215

Setting the map caption 216

Getting more information about an enzyme 216

xv
 Changing the selected enzyme 217

Changing the default enzymes 218

Adding and editing enzymes 218

Changing the recognition sequence 218

Salt concentration effects 219

Saving new or modified enzymes 219

Setting the sequence selection in a Sequence Editor 219

Setting the sequence selection in a contig editor 219

Copying the map 220

18. Simple BLAST 221

How to use BLAST with a sequence 221

How to use BLAST with a range of bases 221

How to use BLAST with a specific segment of sequence 222

How to use BLAST with a contig consensus 223

19. Motifs and Features 224

Motifs 224

Entering motifs 224

Quick motif entry 225

Displaying motifs 225

Highlighting 226

Features 226

What is a feature 226

Creating and editing features 227

Editing an existing feature 228

Quick feature creation 228

Showing and hiding features 229

Listing a sequence’s features 229

xvi
 Listing several sequence’s features 229

How Features are displayed 230

20. Finding items 231

Finding the Project Window 231

Finding open windows with menu commands 231

Finding open windows in the Project View 231

Finding the current selection 231

Searching in Open Windows 232

Finding a subsequence 232

Find Amino Acids 232

Refining a subsequence search 233

Exact matching 233

Matching ambiguous bases 233

Any ambiguous match 233

Include Reference Sequence in Find 233

Finding bases by number 234

Extend Selection 234

Other search options 234

Project Window selection 234

Finding an item by name 234

Selecting all items that… 235

Contain a subsequence 236

Contain Items Named 236

Contain Items With Names Containing 236

Have Chromatograms 237

Have Comments Containing… 237

Have Labels Containing… 237

xvii
 Have Names Containing… 238

Were Edited On Or Since… 238

21. Exporting Data 239

Exporting data from Sequencher 239

Exporting Sequences 239

Exporting a Consensus 240

Exporting Contigs 240

Exporting Selection as subproject 241

Export Selected Bases 241

Export Options 241

Export Formats 242

Exporting protein translations 242

Export Overviews 244

Exporting contig summaries 244

22. Output 245

Copy picture 245

Page setup 245

Print Trace in One Page 245

Printing in detail 245

Setting header, footer and margin options 245

Report formats 246

Page breaks 246

23. Customizing Sequencher & User Preferences 248

View Preferences 248

Ambiguous bases 248

Edited Bases 248

Display Color Bases 248

xviii
 Colors as Backgrounds 249

Display Base Confidences 249

Display Features 249

Display Motifs 249

Setting up custom codes 249

Standard coding system 249

Configuring Ambiguity Codes 249

Replacing an existing character keystroke code 250

Saving and loading custom codes 250

Show and hide ambiguity helper 251

Choosing a genetic code 251

Editing a genetic code 251

Abbreviations 252

Remember Window Layout 252

User Preferences 253

General 254

Settings 254

Auto-Save 254

Confidence 255

External Data 255

Label & Name 256

Menu 257

New Project 257

Sound 258

Display 259

Chromatogram 259

Contig Chromatogram 260

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 Contig 261

Feature, Motif 261

Format Ruler 262

Start-Stop 263

Variance Table 264

Input/Output 264

File Import 264

Report 265

24. Forensic Features 266

Working with mtDNA profiles 266

Validating mitochondrial DNA profiles 266

Create Reports 267

Export CMF 267

Further Commands of Interest 268

New Project From Template 268

Consensus to Forensic Standards 268

Display secondary peak 269

Set Circular Genome Size 270

How to mark a sequence as a Reference Sequence 270

25. Sequencher Connections 272

Sessions 272

Channels 273

Invoking a Sequencher Connections session for the first time 273

Using GenBank Accession numbers 274

Running Sequencher Connections 275

Viewing results with Sequencher Connections 276

Adding or Removing Channels 277

xx
 Viewing saved sessions 279

Deleting saved Sessions 279

BLAST 280

Primer-BLAST 280

Primer-BLAST options 281

Saving Primers to Sequencher Project 282

Local-BLAST - Mac 286

Local-BLAST - Windows 287

Working with Local-BLAST 289

Connections Schematic 289

Sequencher Connections, Group sequences and MUSCLE 291

MUSCLE options 293

Sequencher ConnEctions MUSCLE Tree View 294

26. Appendix – Keyboard shortcuts 297

Windows 297

Mac 300

27. Appendix – Advanced Expressions 305

28. Appendix - IUPAC-IUB Standard Codes 307

29. Appendix – Default Feature Styles 308

30. Appendix - Feature Keys & Qualifiers 313

GenBank Feature tables 313

Feature table qualifiers 316

31. Appendix - Default Feature Qualifiers 318

32. Glossary 323

xxi
 1. PREFACE
Sequencher is the premier software choice for DNA and RNA sequence assembly and analysis.
Its capabilities include:
• Multiple, configurable DNA assembly algorithms
• Comprehensive DNA sequence editing tools
• Complete RNA-Seq workflow for differential expression
• Full support of sequence data confidence values
• Powerful Reference Sequence and Variance Table to find SNPs quickly and easily
• Restriction Mapping
• Extensive data import & export capabilities, including customizable GenBank Feature
handling
• Specialized tools for Forensic mtDNA profiling
Over 25 years of daily use by biologists in labs around the world have refined Sequencher's
tools and interface. You get the power and the speed to get the best, most accurate results
from your DNA analysis, and get back into the lab more quickly.

GENE CODES

Gene Codes is a biotechnology-oriented software developer whose headquarters are in Ann

Arbor, Michigan. Our goal is to write powerful software tools that are easy to use. If you have
any suggestions on how to improve this product, please contact us. You can telephone us at
(734) 769-7249, fax us at (734) 769-7074, or write to us at:
Gene Codes Corporation
775 Technology Drive, Suite 100A
Ann Arbor Michigan 48108 http://www.genecodes.com
We welcome your comments or support questions by electronic mail at:
[email protected] and general inquiries at: [email protected]

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 1
 2. USING THIS MANUAL

This manual is for use on both Windows and Macintosh operating systems. We’ll document the
differences in conventions or behaviours for each platform where applicable. We’ll rotate
Windows and Mac images in every other chapter unless the images are different enough to
warrant including both the Windows and Mac images.
This chapter will introduce you to the conventions we will be using in this manual and how to
get help. We will then introduce you to the button bar and Sequencher’s menus, which offer
you a rich range of options while maintaining a scientist-friendly interface.

CONVENTIONS USED IN THIS MANUAL

This manual follows certain conventions for displaying text and pictures and for alerting you to
special information. Where keyboard shortcuts are mentioned in the text, we will show
Windows shortcuts like Ctrl+key and Mac shortcuts like Cmd+key.

INSTALLING SEQUENCHER

Sequencher is easy to install using the installer which you can download from our Free
Download website page. Follow the instructions in the installation guide to install your
software. You must have Administrator privileges in order to install Sequencher.
If your Sequencher license is based on a license tied to a Sequencher key (dongle), the key,
which you will be receiving, will have to be plugged into either the computer serving licenses
to other computers, a KeyServer Network key, or to a computer locked to the key, a
Standalone key.

SEQUENCHER HELP

Sequencher offers help through a Help viewer. To display Help, go to the Help menu and
choose Sequencher Help or press F1 (Windows) or Command+Shift+? (Mac).
Sequencher Help is organized into two main areas: application screens and the Sequencher
User Manual. The Sequencher application screens contain context-sensitive links to relevant
sections of Sequencher Help.
Note: You can leave the Help window open and return to Sequencher by clicking on an open
window.

NAVIGATING SEQUENCHER HELP

The Sequencher Help window consists of three main parts. There is a button bar at the top of
the window. There is also a navigation pane on the left side of the window and a content pane
on the right side of the window.

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 2
The button bar has Back and Forward icons which function like a web browser’s Back and
Forward buttons. If you click on the Home button, you will return to the Welcome screen. You
can also type keywords into the Search input field.
The navigation pane displays a Table of Contents. Click on the relevant icon to display any
subtopics. Click on a page link icon in the navigation pane to display the Sequencher Help
article in the content pane. Some of these pages contain underlined hyperlinks to related
topics in the manual and a hyperlink to the following topic in the Table of Contents.
You can also get information on specific topics such as Assembly Parameters or the Sequence
Editor from context-sensitive screens.
To search for a specific topic, type the topic name into the search field at the top right corner
of the Help Viewer. When your search is complete, double-click on an item to display it.

CONTEXT-SENSITIVE SCREENS

Context-sensitive screens provide easy links to information about the region of Sequencher
you are currently using. If you have the Project Window displayed when accessing Help, then
the Project Window context page will appear. This will be the case for most of Sequencher’s
major windows.
If you see a flagged icon, this means you can click on a link to get further information. If you
place the cursor over this icon, a Tool Tip with the title of the Help article will be displayed.

THE BUTTON BAR

The button bar contains buttons for frequently used commands and a drop-down menu for
setting assembly modes. It appears just below the top of many windows. (See Figure 2-1)

Figure 2-1 The Project Window button bar

Clicking on Assembly Parameters displays the Assembly Parameters dialog where you can
change your assembly settings. Assemble Automatically, Assemble Interactively, and
Assemble to Reference are one-click buttons which initiate your chosen assembly on the
selected items in the Project Window. These buttons are context sensitive based on your
assembly mode setting.
Clicking on the Trash Can icon will remove the selected items in the Project Window if you
confirm that is what you want to do.
Clicking either of the font sizing icons will increase or decrease the size of the fonts on the
items in the Project Window.

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 3
SEQUENCHER’S MENUS

SEQUENCHER (MAC ONLY)

The Sequencher menu gives you information on the version of Sequencher you have, contact
information for Gene Codes Corporation, Sequencher technical support, access to the User
Preferences, and the Quit Sequencher command.

FILE MENU

The commands in the File menu let you open, close, and save projects as well as import and
export data. You can also open and close windows, specify page setups, and print from the File
menu.

EDIT MENU

The commands in the Edit menu let you cut, copy, and paste selected data. You can also
duplicate sequences, mark motifs, reposition sequences, and choose your editing mode from
the Edit menu.

SELECT MENU

The commands in the Select menu cover a variety of items you might want to find and/or
highlight to help you in your analysis. These commands use a wide variety of criteria to help
you locate items from sequence fragments, subsequences, and individual bases.

ASSEMBLE MENU

The commands in the Assemble menu contain the essential items for assembling your Sanger
and NGS sequences. These include the assembly parameters, assembly algorithms, and the
alignment commands for tools such as Clustal and the next-generation sequencing-alignment
algorithms.

CONTIG MENU

The commands in the Contig menu facilitate most of your work in contig editing. You can set
the consensus mode, dissolve and rename your contigs, remove selected sequences, as well as
Trim to Reference Sequence, Compare Consensus to Reference, or Compare Translation to
Reference. The menu also contains commands for creating a new sequence from the
consensus and displaying NGS data.

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 4
SEQUENCE MENU

The commands in the Sequence menu facilitate most of your work in sequence editing. You
can set up data entry preferences such as trimming poor quality data or vector contamination,
edit features, create and rename sequences, set up a Reference Sequence, set up base
numbering, and Revert To Experimental Data. You can also Compare Bases to Top
Sequence, Consensus, or Reference Sequence, these commands will trigger the appearance
of the Variance Tables. In the Analyses submenu, you will be able to view the results of your
Next-Generation sequencing analyses or view the Frequency Histogram or Report. NCBI Blast
Search is designed for short, rapid queries of NCBI’s nr database. The search is limited to the
first 7800bp (Mac OS X) or 1600bp (Windows) of the selected sequence or consensus
sequence.

VIEW MENU

The commands in the View menu let you control how data is displayed as you work on it. You
can add extra information to the display, specify the format and marking of the data, and
organize project files from the View menu. Some of the functions in the View menu have
options which you can change by going to User Preferences (see Chapter 23 “Customizing
Sequencher and User Preferences” for more information).

WINDOW MENU

The commands in the Window menu offer access to different kinds of user help, to reference
information on various aspects of your data, to different windows you have opened, and to
User Preferences.

HELP MENU

The command in this menu on Mac offers access to user help. The commands in this menu on
Windows offer access to user help, gives you information on the version of Sequencher you
have, and contact information for Gene Codes Corporation and Sequencher technical
support.

CONTEXT- SENSITIVE MENUS AND BUTTONS

Sequencher has a number of menus and buttons that may change depending on the context.
Some items will become enabled. The labels of other items will change. This feature is
designed so that Sequencher can offer you extensive options within the same easy-to-use
interface.

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 5
CONTEXTUAL MENUS

Most windows in Sequencher offer contextual menus. You can invoke these by clicking within
the window you are reading while holding down the right hand mouse button. If you select
one or more items and then click the right hand mouse button, you will see other menus.
Note: If you have a mouse with only one button, you may be able to mimic the effect of a right
hand button with the use of a modifier key.

KEYBOARD SHORTCUTS

Sequencher has a number of keyboard shortcuts for major menu items and other important
features and allows you to define some of your own. See Appendix 26 “Keyboard Shortcuts”
for a complete listing. See Chapter 23 on “Customizing Sequencher and User Preferences” for
more information about how to define your keyboard shortcuts.

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 6
 3. THE PROJECT WINDOW

In this chapter, we will introduce you to Sequencher’s Project Window. We will also show you
how to get started with Sequencher by creating a new project or opening an existing one. This
chapter also describes how you view the constituent sequences or contigs in your project and
how to annotate information in your project for later editing and record keeping.

SEQUENCHER CONCEPTS

ABOUT THE PROJECT WINDOW

The concept of the project is central to Sequencher. Users work within the framework of a
project. A Sequencher project is comprised of a collection of DNA sequences and contigs
(contiguous alignments of overlapping sequences) that are built from those sequences. A
project can be as large or as small as you want.
Sequencher stores the sequences you enter, the information on how these sequences fit
together to create any contigs you have formed from them, and information on user-specified
parameters that control the alignment operations, all combined into a single data file. The
Project Window displays all of your sequences and contigs.

CREATING A NEW PROJECT

When you launch Sequencher by double-clicking the program icon, a new, empty project is
created (see Figure 3-1).

Figure 3-1 An empty Sequencher Project Window

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SEQUENCHER 5.4.6 User Manual © 1991 – 2016 Gene Codes Corporation, Inc. All rights reserved 7
Until you create or import sequence fragments, the new project will remain empty. As you add
sequences to your project and build contigs from them, the Project Window begins to look
more like Figure 3-2. All the sequences and contigs added to this particular sequencing project
are displayed in this window.

Figure 3-2 A new project

Note: If you have so many open windows on the screen that it’s difficult to find the Project
Window, choose Window > Project Window. This brings the current project to the front.
As you work, if you close an existing project and then want to start a new project, go to the File
menu and choose New Project. Sequencher will open a new untitled project. Sequencher will
remember the last projects you opened. You can see these as a list by going to the File menu
and selecting the Open Recent command.

OPENING AN EXISTING PROJECT

To open an existing project, go to the File menu and choose Open Project and select the
project you want to work with. Click on the Open button in the lower right area of the dialog,
or click the Enter/Return key.

WORKING WITH ITEMS IN THE PROJECT WINDOW

To work with items in the Project Window, first select the item(s) you need by clicking on
them. Then choose the appropriate menu command.

ICON TYPES

In the Project Window, a sequence is represented by an autorad icon. However, there are
other data types you may have in your project. To get familiar with some of the icons
Sequencher uses for these data types, see Figure 3-3.

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 Figure 3-3 Examples of Sequencher icons

A sequence fragment

A sequence fragment with edited comments

A contig

A refrigerator

A Reference Sequence

A sequence in the inverse-complement orientation

Other information may be displayed in addition to the basic icon. For example, the downward-
pointing arrow on a sequencing image icon shows that this sequence is stored in its original
orientation (the sequence travels “down” the gel). The arrow points up when the data is
inverse-complemented as shown in Figure 3-3 above.

PROJECT VIEWS

VIEW AS LARGE ICONS

To display large icons in your Project Window, go to the View menu and then choose Project
Icons As. Then select Large Icons from the submenu. The Project Window then displays the
sequences and contigs as shown in Figure 3-4.

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 Figure 3-4 Project Window with Large Icons

Note: A new project defaults to the icon view unless you previously specified a different view.

VIEW AS SMALL ICONS

To fit more information onto the screen, you can view project items as smaller icons. Go to the
View menu and choose Project Icons As and then choose Small Icons from the submenu. The
Project Window then displays the sequences and contigs as shown in Figure 3-5.

Figure 3-5 Project Window with Small Icons

MOVING ICONS

To reposition a sequence or contig icon, click the icon and drag it to the new location.
Note: Icons can be positioned on top of each other, thereby obscuring one or more of them
from view.

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CLEANING UP

Sequencher has clean-up commands to help you organize the icons in your Project Window.
Go to the View menu and choose the Sort/Cleanup command to see a submenu that will let
you rearrange the icons according to date edited, name, kind, size, etc. (See Figure 3-6.)

Figure 3-6 Small icons sorted by name

VIEW AS A LIST

You can show your project items as a list rather than as icons. Go to the View menu and
choose Project Icons As… and then select A List from the submenu. The Project Window will
look like the one shown in Figure 3-7.

Figure 3-7 Project Window with list view

You can navigate the list by using the up and down arrow keys. To select a continuous list of
sequences, choose the first sequence in the list and then, holding down the Shift key, select
the last sequence in the list. To select a discontinuous list of sequences, hold down the Ctrl
(Windows) or Command ( M a c ) key while clicking on the sequences.

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PROJECT WINDOW COLUMNS

When you are in the list view, Sequencher displays the attributes, such as the item name or
kind, of each sequence as a series of columns. You can control which columns are displayed.
Go to the View menu and choose the Project Window Columns command. Then choose your
desired option from the submenu.
If you are working with ABI sequences, you can view the sample name by selecting Project
Window Columns and then choosing Sample.
The column labeled Quality displays a value for each sequence that has confidence scores. The
Quality value is the percentage of bases in a sequence that is above the Low Confidence Range
threshold. The default setting is 20. You can alter the Low threshold value in the Confidence
pane of the General settings in User Preferencews. See Chapter 23 “Customizing Sequencher
and User Preferences” for more details. If you have used Consensus by Confidence for the
consensus calculation, then you will see a % quality value in the Quality column.
You can sort your list by item name, size, quality, kind, label, or modification date. Click on the
title of the column you want to use for your sort. For example, to sort by size, click the word
Size at the top of that column. Sequencher will reorder the list by size in ascending order. If
you shift+click the top of the column again, the column will re-sort in descending order. You can
also sort the list by going to the View menu, choosing Sort/Cleanup, and then selecting from
the options on the submenu.
If you sort by Kind, the order of precedence is, first any Contigs which contain Reference
Sequence fragments, then any unassembled Reference Sequences, then contigs without
Reference Sequences, and finally Sequence Fragments.

Figure 3-8 Project Window Columns submenu

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Note: You can toggle between types of views if you hold down the Alt (Windows) or option
(Mac) key and click in the header area, just above the icons and below the parameter
information.

VIEWING CONSTITUENT SEQUENCES

When you have contigs in list format, click the triangle to the left of the contig icon (Figure 3-9)
to see a sub-list of the sequences incorporated into that contig. That triangle will turn to point
downward and the sequence list will expand (see Figure 3-9). To hide a sub-list of sequences,
click the triangle again; it turns to point horizontally and the sequence list will collapse.
Go to the View menu and click on the Expand All and Collapse All commands to either
display or hide all of the contents of the project’s contigs.

Figure 3-9 Viewing a sub-list of a contig's constituents

VIEWING THE INFORMATION FOR A SEQUENCE OR CONTIG

The Get Info command displays additional information about a selected or open sequence or
contig.
To view additional information about an open sequence or a Contig Editor window, go to the
File menu and choose Get Info. If you do not have an open sequence or contig editing
window, you can select the icons of your sequences or contigs from the Project Window and
then go to the File menu and choose Get Info.
Comments from contigs created with Next-Generation sequence alignment algorithms, for
example, will contain information about the algorithm used, the data used, and the path to the
data together with the data and the time generated.

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 Figure 3-10 The Get Info window

If you have sequences from an ABI sequencing system, Sequencher can display information
from the ABI data sheet. In the Get Info window shown in Figure 3-10, note the Show ABI Info
button.

EDITING THE INFORMATION FOR A SEQUENCE OR CONTIG

You can annotate the sequence by clicking anywhere in the Comments: box and typing the
information. To store more information than the Comments: box allows, go to the Edit menu
and choose Edit Comments…. Sequencher opens a small text editing window that stores
about 250 characters of text for each sequence. After you have added text with Edits
Comments…, a small “I” (for “Info”) appears in the upper left corner of the icon.
If you want to remove the information in the Comments: box, select the text and then go to
the Edit menu and choose Contents from the Clear menu.

THE PROJECT WINDOW BUTTON BAR

The button bar contains buttons for frequently used commands. It appears just below the top
of the Project Window. You can use the smaller left-hand button, which has an up arrow on it,
to hide the button bar. Click on the button and the button bar will “hide” behind the title bar.
When the button bar is hidden, you can still see the bottom of the left-hand button under the
title bar. It will show a down-pointing arrowhead. Click on the arrowhead and the button bar
will reappear.
If you click on the Assembly Parameters button, the Assembly Parameters window will
appear. Clicking on the Assembly Mode drop-down menu will enable the Standard, Assemble
by Name, or Multiplex ID function. Assemble Automatically, Assemble Interactively, and
Assemble to Reference are used to assemble sequences you have selected. If Assemble by

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Name is enabled, then the Assemble Automatically and Assemble to Reference buttons will
have different labels. If you select a sequence and then click on the Trash Can button, that
sequence will be removed from the project.

TEMPLATES

A template is a special type of project that can be set with your choice of parameters and
preferences. Templates can contain ordinary sequences or a Reference Sequence. Preferences
can include Feature settings, enzyme sets, and display settings. With templates, you can also
set Assembly Parameters, Assemble by Name handles, and Trim parameters. These will be
discussed later in this manual.
A template can be used as a new project or imported into an existing project. You can use
templates to set up standard operating procedures and methods to reuse or distribute
throughout your lab.

SAVE PROJECT AS TEMPLATE

You can save any project as a template by using the Save Project As Template command. Set
the User Preferences and parameters that you want to use. You can include a Reference
Sequence or ordinary sequences.
Once you are satisfied with your choices, go to the File menu and click on the Save Project As
Template… command. A new dialog called Template Name: will appear. Type a name for
your template in the input field and then click on the OK button. Your project will now be
saved in a Templates folder. The name of the template will appear in the New Project From
Template submenu.

NEW PROJECT FROM TEMPLATE

The New Project From Template command allows you to open a new project containing all
the sequences, settings, and preferences associated with your chosen template. Go to the File
menu and click on New Project From Template. Select a template from the submenu. A new
blank Project Window will open.

IMPORT FROM TEMPLATE

You can add preferences, settings, and sequences to an existing project by importing a
template. If you have an open project, go to the File menu and click on the Import command
and then choose From Template. Select a template from the submenu. The settings and/or
sequences from the template will be applied to your open project automatically.

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 4. IMPORTING DATA

In this chapter, we will discuss how to import data from automated sequencing equipment and
other programs into Sequencher projects. The different techniques we will discuss include
dragging and dropping, copy and paste, and using import commands. We also explain how to
deal with projects in specific formats, how to import confidence scores, and how to remove
unwanted sequences and contigs from a project.

QUICK AND EASY IMPORTING OF DATA

DRAGGING AND DROPPING FILES

You can drag files from the desktop right into the Project Window and Sequencher will
import and load those files. Sequencher will alert you if it cannot read and load the files.
If you drag sequences onto the Sequencher icon on your desktop, you will open a new Project
Window containing your sequences. If Sequencher is already open, the sequences will be
imported into the open project.
Note: Sequencher always shows a newly imported file as selected, so if you do not want to use
it right away, de-select it by clicking elsewhere in the project.

COPY AND PASTE

One of the easiest ways to move data between Sequencher and other programs is to use the
clipboard’s Copy and Paste commands. First, launch both Sequencher and the other program.
Then, select and copy the bases of interest into the other program.
Go to the Sequence menu and choose Create New Sequence. Type a name for your sequence
into the New sequence’s name: input field and then click on the OK button. Now go to the
Edit menu and choose Paste. When you close the window, you will see a dialog with two
buttons. If you have finished entering your data, click on the Record As Experimental Data
button. Otherwise, click on the Not Yet Finished button. Sequencher then removes the
window.

HOW TO IMPORT USING MENU COMMANDS

IMPORTING SEQUENCES

Sequencher imports sequences in a variety of text and chromatogram formats. Some of the
most popular are ABI, MegaBase, CEQ, SCF, FastA, aligned FastA, FastQ, GenBank, EMBL, DDBJ
and plain text files.

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To import a sequence, first bring the Project Window to the front by clicking on it. Under File
menu, choose Import. Choose Sequences… and then browse to and select the file you want to
import. Click on the Open button in the lower right area of the dialog or press the Return key.
You may need to select a file format from the drop-down menu called Enable: in order to see
the files of interest in the dialog. To see all of the files in a particular location, choose All
Documents.
You can also select from a Folder of Sequences…, ACE Project…, CAF Project…, CEP
Project…, FASTA – aligned…, GCG Contig…, Sequencher P roject…, or Sequence from
VecBase…. (Working with specific formats is discussed below.)
If Sequencher cannot import the file, you will get a warning message to that effect.

IMPORTING GENBANK FEATURES

When you import a sequence in GenBank format, you will automatically import the features,
including the location, the feature keys, and feature qualifiers. The feature key used in
sequences published in GenBank, EMBL, and DDBJ describes the biological nature of the
annotated feature or indicates information about changes to the sequence. (For more
information on features and feature keys, see Chapter 19 “Motifs and Features” and Appendix
30 “Feature Keys and Qualifiers”.)
Where feature locations described in the GenBank file are ambiguous, Sequencher will warn
you that it cannot import these features and will list the features in a dialog you can print out
for your records.

IMPORTING LISTS OF SEQUENCES

Sequencher’s functionality has been modified to take advantage of the file system. If you use
the Import menu item with the Sequences… option, you can select multiple files for a single
import command. To select a continuous list of sequences, choose the first sequence in the list
and then, holding down the Shift key, select the last sequence in the list. The import window
selects the two sequences and all of the sequences in between. Click on the Open button to
import all of the selected sequences into your project.
To select a discontinuous list of sequences, hold down the Ctrl (Windows) or Command (Mac)
key while clicking on the file. Click on the Open button to import all of the selected sequences
into your project.
The Files of type (Windows) or Enable (Mac) drop-down menu allows you to filter the files
that are displayed in the file browser. On Mac, the filters are All Importable Documents, All
Documents, With Chromatogram Sequences, and Without Chromatogram Sequences. On
Windows, the filters are Supported File Formats, All Files (*.*), With Chromatogram
Sequences, and Without Chromatogram Sequences. On Mac, you may see some files
becoming grayed out with different file filters because these files do not meet the import
criterion of the current filter (see Figure 4-1 where With Chromatogram Sequences is set but
some of the files are text only files).

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 Figure 4-1 File import Enable option set to With Chromatogram Sequences

Note: To import traces, you must import the actual trace file. If you import a text file
containing ASCII characters, you will not import traces. Text files will be only a couple of
kilobytes in size, whereas trace files will be well over 50 kilobytes.
Sequencher uses a very efficient algorithm to compress the imported traces. This conserves
disk space so a project file containing several sequences from an automated sequencer may be
smaller than any one of the original trace files.

FOLDER OF SEQUENCES

When you have many sequences to import, you can import all those files from within a single
folder. Select the Project Window and go to the File menu and choose Import and Folder Of
Sequences… from the submenu. The standard Choose a Folder dialog appears on Mac and
the Browse for Folder dialog appears on Windows; select the folder that holds the data you
want to import and click on the Choose button on Mac and the OK button on Windows.
Note: On Mac OS X, the contents of a folder selected for import will be grayed out.
Use the Include .TXT and .SEQ files checkbox on Windows or the Include All Text Files
checkbox on Mac to include or exclude text files from your import.
Sequencher tells you how many files are in the folder and asks whether you want to import
them all. If you do, click on Import All Files in Folder. Sequencher imports all the files,
assigning a separate icon to each.
You can specify that you want files imported into the Project Window or directly into the trim
window (see Chapter 6 “Preparing your Data for Assembly”).

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 Figure 4-2 Browse for a Folder dialog

PROJECTS IN SPECIFIC FORMATS

Sequencher supports importing projects as ACE projects, Contig Express projects, GCG
contigs, and as CAF projects. The contents of these files include confidence values, feature
annotation, and assembly information. Select the Project Window, go to the File menu and
choose Import, and then choose the specific project format from the submenu. The standard
Open dialog appears; select the folder that holds the data you want to import and click on the
Open button.
Read more about importing confidence values in the section “Importing Confidence Scores”
later in this chapter.

SEQUENCHER PROJECTS

At times you may want to merge two projects or combine your own work with that of a
colleague. To combine Sequencher projects, first open one of the projects. Go to the File
menu and choose Import and then Sequencher Project… from the submenu. Select your
other project and click on the Open button. Sequencher displays all the imported projects in
the initial Project Window, retaining all comments, edits, and even the relative positions of the
icons. To keep track of which sequences came from which project, you may want to establish a
naming convention for sequences before combining them. Since the imported sequences and
contigs are highlighted after import, you could also use sequence Labels to differentiate them.
(See Chapter 23 “Customizing Sequencher and User Preferences”.)

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FILES FROM VECBASE

VecBase is a file of vector sequences in your Sequencher application folder. You can import a
vector from VecBase just as if it were any other DNA sequence. Go to the File menu and
choose Import then the sub-command Sequence From VecBase. The Open dialog appears.
Choose the desired file and click on the Open button. If you wish to import more than one file,
for continuous selection of files, you can use Shift+click (Windows or Mac). For discontinuous
selection of files, use Ctrl+click (Windows) or Command+click (Mac).

CREATING A NEW SEQUENCE

Another way to bring new data into Sequencher is to create a new sequence fragment. To do
this, go to the Sequence menu and choose Create New Sequence…
Sequencher displays a dialog asking you to name the new sequence (Figure 4-3). The default
name, in this case “Frag[0001]”, can be set as a user preference. A sequence name can be a
maximum of 255 characters, including any punctuation and spaces, but 31 characters is
recommended for export functions.
When you have typed the name you want (or if you accept the default name), click on the OK
button or press the Enter key. A new Sequence Editor dialog appears showing the name you
selected in the title bar. The new Sequence Editor lets you enter and change sequence data.
Just type as you would with a standard word processor.
When you are finished entering and/or editing a sequence, click on the close button in the top
left corner of the window at the left end of the title bar on Mac or in the top right corner of the
window at the right end of the title bar on Windows, or go to the File menu and choose Close
Window. Sequencher asks if you have finished editing. At this point, you can record your data
as experimental data or you can tell Sequencher you have not yet finished editing by selecting
the appropriate button. Sequencher then removes the Sequence Editor window from the
screen. The sequence is now represented by a sequencing icon in the Project Window.

Figure 4-3 Create New Sequence dialog

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DOUBLE-CLICKING A PROJECT ICON

When you double-click on a sequence project icon, this launches Sequencher and opens the
project. If a Project Window is already open, when you double-click on a project icon
Sequencher will ask whether you want to save changes to your current project before closing
it. Click on the Cancel button if you do not want to proceed. If you want to close the project
and save any changes you have made, click on the Yes button. If you want close the project
without saving your changes, click on the No button.

IMPORTING CONFIDENCE SCORES

Many base callers generate confidence values associated with each base call. Sequencher
supports confidence values from PHRED, ACE, and Trace Tuner files, as well as SCF, FastQ, ABI
3730, and ESD files.

IMPORTING PHRED CALLED DATA

Sequencher can read in the SCF, FASTA, and Qual files generated by PHRED and supports
PHRED base calls and confidence values. To view the confidence levels, you must first set the
user preferences for displaying confidence values (See Chapter 23 “Customizing Sequencher
and User Preferences” for more information). Then go to the View menu and choose Display
Base Confidences.
To import the data into Sequencher, it must first be organized in a folder containing three
subfolders. These subfolders are chromat_dir, which contains the trace files, edit_dir, which
contains the text files, and phd_dir, which contains the quality scores. Select File and then go
to Import and then Folder of Sequences to import the phd_dir folder. To import the
sequences you would like to view, you may also go to the File menu and choose the Import
command with the Sequences... submenu.

IMPORTING PHRAP FILES

Sequencher can import the ACE projects created by PHRAP. To import an ACE project select
File then go to Import and then the ACE Project… submenu. Browse to a file that ends with
".ace" or ".ace.x" (such as .ace.1,.ace.2, etc.). This file will typically be located in the subfolder
"edit_dir". Select the highest numbered ACE file to account for all editing and reassembling.
Sequencher automatically imports and incorporates as much auxiliary data as it can find. The
auxiliary files must be in the same folder as the ACE file or arranged in the traditional PHRAP
assembly hierarchy (chromatogram files in a chromat_dir folder and PHD files in a phd_dir
folder). If the .singlets file, the .qual file, the PHD files, and the chromatogram files all exist and
are available, Sequencher will import them.
Alternatively, you can import parts of the PHRAP project by going to the File menu and
choosing Import and then Sequences or by going to the File menu and clicking on Import and
then Folder of Sequences.

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Note: When using ftp to transfer your data to your local computer, it is ALWAYS important to
transfer these files in binary mode. If you are not familiar with ftp, speak with your network
administrator.

REMOVING SEQUENCES AND CONTIGS FROM A PROJECT

To remove items from the project, select the items you want to remove. Go to the Edit menu
and choose Remove From Project… or click on the Trash Can button in the button bar.
Removing an item from a project is permanent so Sequencher asks whether you really want to.
Note: Remember, you cannot back out of this action with a simple Undo command, but if you
close the project without saving, all changes including removed items will be forgotten. The
Revert to Saved Project command has the same effect.
If you import the chromatogram files from the "chromat_dir" folder, these fragments will
have PHRED base calls and chromatogram data but no base qualities.
If the "_.phd" files are loaded and "_. fasta.qual" files are available, you will have imported all
of the fragments from the PHRAP project. They will have base quality and chromatogram data.
The.qual files alone will not load your sequences.
If you import PHD files from the "phd_dir" and the corresponding chromatogram files are
available, these files will import with PHRED base calls, chromatogram data, and base qualities.

CLOSING AN EDITOR WINDOW

When you have finished entering and/or editing a sequence, click on the close button in the
top left corner of the window at the left end of the title bar on Mac or in the top right corner of
the window at the right end of the title bar on Windows (see Figure 4-4) or choose Close
Window from the File menu. Sequencher then removes the Sequence Editor window from
the screen. Once you have closed the window, a sequencing icon will represent your
sequence. (Figure 4-4)

Figure 4-4 Clicking the Close button

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CLOSING A PROJECT

If you have finished working with the project but not with Sequencher, choose Close Project
from the File menu. Sequencher will ask if you want to save the changes to your project. Click
Yes to save your changes and close the project. Click No to close the project without saving
your changes. Click Cancel if you wish to continue working with the project. You can then start
working with another project by going to the File menu and choosing New Project or Open
Project.
If you have finished working with Sequencher, go to the File menu and choose Exit (Windows)
or to the Sequencher menu and choose Quit Sequencher (Mac).

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 5. ORGANIZING PROJECT VIEWS

In this chapter on organizing project views, you will learn more about the structure of a
Sequencher project as you begin to work with your data. We will discuss how to select
individual and multiple sequences using various techniques, renaming items, and saving your
work.

WORKING WITH ICONS AND LISTS

CHANGING BETWEEN PROJECT VIEWS

Sequencher provides you with a variety of ways to view and organize the contigs and
unincorporated data in your Project Window. You can view your data as large or small icons or
in list form (for more information, see Chapter 3 “The Project Window”). You can toggle
quickly between types of views by holding down the Alt (Window) or Option (Mac) key and
clicking in the header title area, just above the icons and below the parameter information.
You can display a list of sequences in a single contig in the list view by clicking on the triangle
to left of the contig icon. You can see a list of sequences in all your contigs by going to the
View menu and choosing Expand All.

COLLECTING SEQUENCES IN REFRIGERATORS

Sequencher lets you create “refrigerators” in the Project Window to hold certain subsets of
your project. Refrigerators hold only raw sequences, not contigs or other refrigerators. To “put
away” a few sequences in “cold storage,” select the sequences and then go to the Edit menu
and choose Refrigerate. Sequencher collects the sequences and puts them in the refrigerator.
To remove sequences from the refrigerator, open the refrigerator by double-clicking on it. You
will see a list of items. Select the items you want to remove and then click the button called
Move Selected Items To Project Window to execute the command.

SELECTING SEQUENCES

SELECTING SEQUENCES AND CONTIGS

To select sequences you can go to the Select menu and use the Select All, Select None, and
Invert Selection commands. Invert Selection will give you the opposite of your previous
selection.
There are also selection functions that are increasingly specific. You can choose data based on
All Items That… and then further specify the data by choosing narrower classes such as

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Contain Subsequence…, Contain Items Named…, or Have Chromatograms from the Select
menu. You can indicate if you want these names to be case sensitive or not.
Note: The subcommand Contain Items with Names Containing… only works on contigs.
For more detailed information on these commands, see Chapter 20 “Finding Items”.

SELECTING WITH THE MOUSE

First make sure the Project Window is at the front. When you click on an icon, or hold down
the Shift key while clicking on several icons, Sequencher highlights the icon(s). When you have
finished selecting a group of icons, release the Shift key before you try to do anything with the
group.
You can deselect an icon by clicking on it while holding down the Ctrl (Windows) or Cmd ( Mac)
key. If you do not hold down the Ctrl (Windows) or Cmd ( Mac) key, Sequencher deselects the
last highlighted icon(s) as soon as you click on another. If you click in the white space between
icons and names, Sequencher deselects all the highlighted icons.
When you display the list view of a Project Window or a Contig, you can select a range of
sequences by using Shift+click. You can make a discontinuous selection by using the Ctrl
(Windows) or Cmd ( Mac) key and clicking on the individual sequences.

SELECTING BY TYPING

Type the name of a sequence fragment or contig. As you type, Sequencher searches the
Project Window for an icon with that name. If it finds the name, it scrolls to the point at which
the icon is visible and highlights the icon.
Note: If you pause for more than a second or two while typing a name, Sequencher assumes
you have started typing a different name and starts looking for the new one instead.

SELECTING MULTIPLE ITEMS WITH A MARQUEE

You can select multiple sequences by drawing a box around the icons or list items you want.
Position the mouse so it is not touching any icons or names. The cursor should be
approximately where one corner of the box should be. Hold the mouse button down and drag
diagonally across to where you would expect the opposite corner of the box. As you drag, the
marquee appears (as shown in Figure 5-1). As you move the mouse, any icons or names
touched by the marquee will be selected.

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 Figure 5-1 Selecting with the marquee

SELECTING USING MENU COMMANDS

SELECTING ALL ICONS

You can change the icons selected in the Project Window by going to the Select menu and
clicking on the Select All, Select None, and the Invert Selection commands.

SELECTING ALL ITEMS CONTAINING SEQUENCE

To select all items in the Project Window that contain a particular subsequence, go to the
Select menu and choose the All Items That command. Then select Contain Subsequence…
from the submenu. Sequencher displays a dialog that lets you type two sequences; the box has
buttons to specify how you want the sequences matched to items in the Project Window. You
can use the drop-down menus to specify restriction enzyme recognition sequences.

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FINDING ASSEMBLED SEQUENCES

To locate data assembled in a contig, go to the Select menu and click on either the All Items
That>Contain Subsequence… or Contain Items With Names Containing…. Sequencher will
select the contig(s) that match your selection criteria.
You can also use the Select and then Item Named… menu option to locate which contig
contains a particular fragment. With this command, you must type the full name into the Find
What: box and then press the Find button. The contig containing the fragment will be
highlighted.

RENAMING A SEQUENCE OR CONTIG

RENAMING USING THE MOUSE

To rename a sequence sequence or contig with the mouse, click twice on the name of the
item. This selects the icon and puts a textbox around the name. Just type in the new name and
when you are done, click elsewhere in the Project Window.
If you only want to change part of the name, drag the cursor over what you want to replace
and then type the replacement text in the highlighted area.
You can also use the Copy and Paste functions to rename sequences.

RENAMING USING THE MENU

If you wish to rename a sequence, you can go to the Sequence menu and choose the Rename
Sequence… command. To rename a contig, go to the Contig menu and click on the Rename
Contig… command. Sequencher displays a dialog (Figure 5-2) with a field for you to enter the
new name.
Click the OK button when you have finished.

Figure 5-2 Renaming a sequence

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LABELING ITEMS IN THE PROJECT WINDOW

You can organize items visually in the Project Window by applying Labels to the icons. Before
using Labels for the first time, you should customize the list of labels under the General
section of User Preferences. You will see a list of Label & Name options. You can also select
from the default labels available under the Edit menu and Label submenu. (See Chapter 23
“Customizing Sequencher and User Preferences” for more information.)
When viewing your project as a list, you will see the name of the label in the list. Labelling your
icons allows you to sort or find your information easily. For instance, you might define red
labels as cDNA data and blue labels as genomic data. Clicking on the label heading in the
Project Window will sort your project items by label (see Figure 5-3).
You can also find sequences bearing a particular label by going to the Select menu and
choosing the All Items That command with the Have Labels Containing… suboption.

Figure 5-3 Viewing labels as a list

SAVING YOUR WORK

SAVING YOUR PROJECT

To save a project, go to the File menu and choose Save Project, which saves all your changes
to the current project file. When you use the Save Project command for a new project,
Sequencher will prompt you to name your project with the Save As (Windows) or Save:
Sequencher (Mac) dialog. Enter a name for the new project and then click Save.

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AUTO-SAVE

If you are working on complex projects, you may find it helpful to have Sequencher
automatically save your work at pre-set time intervals. You will need to enable the Auto- Save
user preference from User Preferences… from the Window menu. (See the Chapter 23
“Customizing Sequencher and User Preferences” for more details.)

SAVING A DUPLICATE OF A CURRENT PROJECT

If you wish to create a second copy of a current project, you can use the Save Project As…
command to create the new copy of your project with a different name. When you save the
new project, a new project icon appears on your disk.

REVERTING TO THE SAVED VERSION OF THE PROJECT

If you are working in a project and want to cancel all the changes you have made since you last
saved it, go to the File menu and choose Revert To Saved Project… Sequencher displays a
dialog that tells you when your project was last saved. If you click on the Yes, Revert to Saved
Version button, you will lose the most recent changes. Click on the Cancel button to cancel
the command.
Note: Remember, if you click the Yes, Revert to Saved Version button, you cannot undo this
action.

CLOSING THE PROJECT WINDOW

When you have finished working on an open project and want to work on another, you must
first close the open project. You can either close the project by going to the File menu and
clicking on Close Project or you can close the Project Window by clicking on the close box. In
either case, Sequencher will prompt you to save your work if you have made any changes since
your last save.
To continue working in Sequencher, either go to the File menu to create a new project by
choosing New Project or click on Open Project to work on a previously saved file.
Note: If you want to save the data you have entered, you must save the project. Saving a
project is different from recording a sequence as experimental data. Only saving the project
actually stores data on the disk!

EXPORTING DATA

You can save your work in a file format other than Sequencher. You can export the entire file
or a subset of the project. Click on the File menu and choose an Export submenu option. A
dialog will allow you to specify the file format in which you want to export your selection
(Figure 5-4). For example, if you choose Selection as Subproject, you can specify CAF format
(for which you can set a number of options), Fasta concatenated format, or older versions of

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Sequencher. This is especially helpful if you want to share data with colleagues working with
older versions of Sequencher.
Note: It is important to remember that any version-specific features you may have used will be
lost if you save to an earlier version of Sequencher. (See Chapter 21 “Exporting Data ” for
more information.)

Figure 5-4 Export sequences format menu

EXITING THE PROGRAM

When you have finished working with Sequencher, make sure you have saved your project.
Then go to the File menu and choose Exit (Windows) or to the Sequencher menu and choose
Quit Sequencher (Mac).

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 6. PREPARING YOUR DATA FOR ASSEMBLY

In this chapter, we introduce you to Sequencher’s powerful tools for trimming poor quality
data or vector contamination from your sequences. You will learn how to use these functions
when you add sequence data to a project. We discuss the various criteria you can use such as
trimming off ambiguities, trimming off low confidence data, how to screen for specific vector
contamination, and how to change all the trim tools’ criteria to your own specifications.

REMOVING UNDESIRABLE DATA

Sequencher’s trimming tools allow you to trim one sequence at a time or thousands of
sequences at a time. The two trimming commands under the Sequence menu are Trim Ends…
and Trim Vector.... Both of the Trim dialogs share a similar interface. All the sequences to be
examined are collected in a single table. The criteria that you set determine how much data
you trim off.
The trimmed data are fully recoverable because Sequencher always stores two copies of every
imported file, the original sequence and the edited version. In this way, you can always use the
Revert to Experimental Data command from the Sequence menu to recover the original
untrimmed sequence.

TRIMMING POOR QUALITY DATA

SELECTING AND TRIMMING SEQUENCE DATA

Select one or more sequences(s) in the Project Window by clicking on them. Then go to the
Sequence menu and choose the Trim Ends… command. Note that if you select contigs, the
Trim Ends… function only affects unassembled sequences. All of the sequences are collected
into a single table. At the top of the table is a button bar (Figure 6-1) that you use to manage
Trim criteria, review which bases will be trimmed, and perform the trim command.

PERFORMING A TRIM WITH DEFAULT SETTINGS

You can specify how much dirty data should be trimmed off as a function of the number of
ambiguous base calls per run of bases by altering the Ends Trimming criteria. Each pane in the
Ends Trimming window shows how much sequence should be trimmed off. There will be a
checkbox at an end if it requires trimming. Bases to be trimmed will be marked in red.
You can proceed with the trim by clicking on the Trim Checked Items button. Once you have
trimmed your data, dismiss the window with the command from the File menu called Close
Window.

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 Figure 6-1 The Ends Trimming button bar

AUTOMATICALLY SELECTING SEQUENCES FOR ENDS TRIMMING

You can set the User Preferences so that sequence files are automatically added into the
trimming window as they are imported. During Import, Sequencher loads the sequences that
require trimming directly into the trim window. To determine which sequences are directly
imported into the trim window, go to the Window menu and, under User Preferences, click
on Input/Output and then select the submenu File Import. Figure 6-2 shows the criteria you
use to specify which sequences are candidates for direct import to the Ends Trimming
window. (For more information, see Chapter 23 “Customizing Sequencher and User
Preferences”.)

Figure 6-2 User Preferences for setting trim criteria when files are imported

HOW TO SET THE ENDS TRIMMING CRITERIA

The amount to be trimmed is based on the criteria you set for both the 5’ and 3’ ends of the
sequence. These criteria can be set independently of each other. To review or change those
criteria, go to the Ends Trimming window and click on the Change Trim Criteria button.
You will see a series of buttons across the top of the Ends Trimming Criteria window. These
buttons allow you to a) create a library of different trim criteria thereby saving time when you
are managing a series of projects or b) allow controlled trimming using specific criteria in a
certain order.
As you can see in Figure 6-3, you can trim off dye-primer peaks, set windows of acceptable
reliability, percentage of the highest peak height (3’ end), and even set an absolute number of

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bases. For example, you could specify that sequences must have fewer than 3 N’s in the first
20 and last 30 bases. To activate specific trim criteria, you must first select the checkbox that
governs the criteria fields.

Figure 6-3 The Ends Trimming Criteria window

USING CONFIDENCE TO TRIM ENDS

In addition to trimming data based on the number of ambiguities at the 5' and 3' ends, you can
trim sequence data based on the confidence scores assigned by your base caller. Confidence
scores (also called quality values) are numbers associated with each base call and which define
the likelihood that a base call is incorrect. The most common scale is from 1-60, where “60”
represents a 1/106 chance of a wrong call and 20 represents a 1/102 chance. Depending on the
program used, the confidence score may be based on peak height, the presence of more than
one peak, and/or the spacing between the peaks. (See Figure 6-3, the Ends Trimming Criteria
window for trimming by confidence criteria.)

REVIEWING THE TRIM

Each pane in the trimming windows shows schematically how much sequence should be
trimmed off. Blue lines represent the acceptable sequence data. The blue line is then flanked
by red scissors, which mark both the 5’ and 3’ suggested trims based on the current Trim
criteria. The remaining red regions of the sequence define the portion of the sequence to be
trimmed.

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Click on the Show Bases button to see more information on the bases. After you have clicked
Show Bases, the button toggles to Show Overview. Sequencher also provides additional
control within the Trim Ends window. If you want Sequencher to ignore the trim command for
one or the other ends of the fragment(s) without modifying the overall specifications, just click
off the checkbox associated with that trim position. Figure 6-4 shows a portion of the base
detail for these sequences. You can double-click on the sequence icon and invoke a Sequence
Editor to examine the data in more detail.
Figure 6-4 Bases view in Ends Trim window

SORTING ITEMS

In order to assist you with the review of your data prior to trimming, you can sort the
sequences. Click on the Sort Items button at the top of the Ends Trimming window to order
the sequences being trimmed. Figure 6-5 shows the sort criteria.

Figure 6-5 Sort Fragments dialog

EXECUTING THE TRIM

Once you have reviewed your data and are happy with the criteria you have set, you proceed
with the trim by clicking on the Trim Checked Items button. Sequencher warns you that this
command cannot be reversed with the Undo command. To proceed, click on the Trim button.

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Any red lines or bases will disappear from the Ends Trimming window. Now you can dismiss
the window with the command from the File menu called Close Window.
Remember to save your work at this point by going to the File menu and using the Save
Project command, since none of your trim work will have been recorded onto disk.
Alternatively, use the Auto-Save function to save your work at user-defined intervals (see
Chapter 23 “Customizing Sequencher and User Preferences”).

TRIM ENDS WITHOUT PREVIEW

Once you have established a set of trim criteria for your data, you do not need to perform the
same process each time. You can use the Trim Ends Without Preview command. This
command executes the trim without leaving the Project Window. If you are working with
Confidence scores, then you will notice that the Quality improves after the trim. This will be
the only visible indication that the trim has been successful.

BATCH REVERT TRIM ENDS

If you find that the Trim Criteria you used are too stringent, you can use the Batch Revert Trim
Ends… command to selectively restore bases on either or both of the 5’ or 3’ ends. Select the
sequences from the Project Window whose bases you want to revert. Choose the
Sequence>Batch Revert Trim Ends… command and enter the number of bases you wish to
revert into the textboxes. Finally click on the Revert button.

Figure 6-6 Batch Revert Trim Ends dialog

You should see that the values in the Project Window Quality and Length columns change.
Batch Revert Trim Ends does not act on Reference Sequences, Refrigerators or Contigs. If a
sequence cannot be reverted using your Batch Revert criteria, Sequencher will warn you and
ask if you want to use Revert to Experimental on those sequences.

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TRIMMING VECTOR CONTAMINATION

SELECTING SEQUENCES FOR VECTOR TRIMMING

Select one or more sequence(s) in the Project Window and go to the Sequence menu and
choose the Trim Vector… command. Note that even if you select contigs, the Trim Vector
function only affects unassembled sequences.

SPECIFYING A VECTOR USING VECBASE

After you have selected the Trim Vector… command and if you have not previously specified
vector insertion sites, a window will appear. Click on the Choose Insertion Site Now button if
you are ready to work with the vector trim function. Otherwise, click on the OK button to
dismiss the window.
You can also go to Window>Specify Vector Insertion Sites… to see this dialog.
Once you have pressed the Choose Insertion Site Now button, the Vector Insertion Sites
dialog appears. In the bottom part of the window you will see that you can screen for up to
five vectors at once.
Load vector information from the VecBase database by clicking the Use VecBase File button in
the Vector Insertion Sites window. Sequencher displays a list of the vectors. Select the vector
you want and click on the Select button.

Figure 6-7 Selecting a vector

The polylinker region of the vector you select is displayed in the window shown in Figure 6-7.

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 Figure 6-8 The polylinker window

Note: Not all vectors in VecBase have polylinker information. Sequencher cannot work with
VecBase vectors that do not include polylinker data. Add these manually (see below).
Click the enzyme restriction site (s) where your sequence was inserted into the vector. The
name of a selected site(s) changes to green and is boxed. If you change your mind and decide
on a different site, you must click the selected site again to deselect it. It will then return to its
original color. The bases from the selected site(s) are automatically entered into the Vector
Insertion Sites dialog, shown in Figure 6-8.
If the cloning technique you are using places your insert between two sites, replace the vector
sequence between the two sites with a single bullet (•), just as your insert replaces this
sequence.
Figure 6-9 Vector Dialog displaying vector and insertion site

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SPECIFYING THE INSERTION SITE MANUALLY

If the vector you select in VecBase does not have polylinker information, or if you are
screening for contamination using a sequence not already in VecBase, you must enter the
information manually. In the Specify Vector Insertion Sites window, paste the sequence
(maximum 250 bases) into the Polylinker window. Insert a “bullet” (“•”) by holding down the
Shift (Windows) or Option ( Mac) key and the 8 key (not the one from the number keypad) to
represent the sequence you wish to retain.

SAVING AND LOADING VECTOR SITES

You can save manually entered vector information to a file by clicking on the Save Sites
button. You can reload the file in the future by using the Load Sites button. This allows you to
save your preferences and parameters for a later session.

SETTING VECTOR TRIMMING CRITERIA

Sequencher has a set of default criteria to facilitate the recognition of any contaminating
sequence. These criteria are listed in the top region of the Vector Insertion Sites window. If
these parameters do not work for your specific circumstances, you can alter the vector
screening parameters. As an example, if there is ambiguity in the region of the vector junction,
you can increase the likelihood that Sequencher will recognize the vector sequence by
decreasing the approximate match percentage. If it is essential to remove even a single base of
vector contamination, you can decrease the minimum overlap to be considered as vector
contamination. You can change any of the five independent parameters in order to improve
your trim. Elevator buttons are used to increase or decrease the parameter value. The
following table describes the recognition criteria for contaminating sequence.

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 Table 6-1 Description of Trim parameters

Parameter Description

Minimum overlap to The smallest amount of overlap to consider as vector

consider as vector contamination. The default value is three bases.
contamination

Approx. match percentage The percentage match Sequencher needs to find

to consider as contamination before considering the matching sequence to be
vector contamination. Ambiguities do not count as full
matches or mismatches.

Minimum overlap allowed The smallest number of matching bases within which
without exact matches an inexact match is permitted. If fewer bases than this
match, they must match exactly.

Additional bases to remove Extra material, if any, to be trimmed off the 5' end of
from a contaminated 5' end the remaining DNA sequence along with the vector
contamination. The default is zero bases.

Additional bases to remove Extra material, if any, to be dropped off the 3' end of
from a contaminated 3' the remaining DNA sequence along with the vector
contamination. The default is zero bases.

Once you have finished choosing vectors and setting recognition criteria, close the Vector
Insertion Sites window.

HOW SEQUENCHER SCREENS SEQUENCES

When you execute the Trim Vector command, Sequencher screens the selected sequences for
vector contaminants using the vector sequences you specified. Sequencher starts at the
designated insertion site, the bullet, and then scans outwards. Any sequences that are found
to contain vector bases are collected in the Vector Contamination window. (See Figure 6-10.)
Please note that any contigs you have selected will be ignored, as will any sequences
containing fewer than 21 bases.

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 Figure 6-10 The Vector Contamination window

You can print out the Vector Contamination window as a record for your lab notebook by
going to the File menu and choosing Print. This is particularly useful if you use the Show Bases
button to show which sequences are to be trimmed. You can also sort the window using
various criteria by clicking on Sort Items.

EXECUTING THE TRIM COMMAND

To perform the vector screen and trim, go to the Sequence menu and choose Trim Vector…
Once you have reviewed your data and are happy with the criteria you have set, proceed with
the trim by clicking on the Trim Checked Items button. Sequencher warns you that this
command cannot be reversed with the Undo command. To proceed, click on the Trim button.
Any red lines or bases will disappear from the Ends Trimming window. Now you can dismiss
the window with the command from the File menu called Close Window.
If you wish to revert some or all of your sequences to their original state, you can go to the
Sequence menu and click on the Revert to Experimental Data command.
Remember to save your work at this point by going to the File menu and using the Save
Project command since none of your trim work will have been recorded onto disk.
Alternatively, use the Auto-Save function to save your work at user-defined intervals (see
Chapter 23 “Customizing Sequencher and User Preferences”).

TRIM LOG

The results of any trim will be recorded in a trim log file called SequencherTrimEndsHistory
which can be found in the Home directory. You can locate the log file from within Sequencher
using the Open Trim Log Folder command from the Window menu.

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 7. THE REFERENCE SEQUENCE

In this chapter, we explain Reference Sequences – sequences or fragments used as baselines or

benchmarks for subsequent comparison. We will describe the special features of Reference
Sequences. We will then discuss how to designate a sequence as a Reference for your project,
how to assemble sequences to the Reference, and how to edit contigs containing a Reference
Sequence.

WHAT IS A REFERENCE SEQUENCE?

A Reference Sequence is any sequence that you have selected to act as a model for your
subsequent comparisons. You may have obtained this sequence from one of the public DNA
databases or you may have characterized it yourself. Once you have marked your sequence as
a Reference, Sequencher attaches certain properties to it.

WHY USE A REFERENCE SEQUENCE?

There are many situations where you might want to designate one sequence in a project as a
Reference Sequence. In forensics, human identification depends on comparing differences with
a standard Reference Sequence. In medical genetics, a lab might want to compare the same
gene from family members to the specific variant that is known to be part of a disease process.
In a population study, a lab might compare the same gene from thousands or tens of
thousands of individuals to a wild type sequence. In all of these circumstances, using
Sequencher’s Assemble to Reference command with a Reference Sequence allows you to
pinpoint mutations and variants rapidly.

REFERENCE SEQUENCE PROPERTIES

Before you start work with the Reference Sequence, you should be aware of a number of
important properties.

BASIC REFERENCE SEQUENCE PROPERTIES

When you assemble sequences, which include a Reference Sequence, the base numbering of a
contig will be the same as the base numbering of the Reference Sequence.

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 Figure 7-1 Contig showing base numbering set by Reference Sequence

When a contig is built that includes a Reference Sequence, the contig will always remain in the
same orientation relative to that Reference. This way you can keep the contig from flipping as
more sequences are added. Because of this, there can only be one Reference Sequence in a
contig. You can easily spot the Reference Sequence in a Contig Editor because it is highlighted
along its entire length with a gray border.
When you assemble sequences that include a Reference Sequence, you will notice that gaps in
the Reference Sequence are given decimal numbers, thereby preserving the absolute
numbering of base positions.
The Reference Sequence does not contribute to the calculation of the consensus line of a
contig. For example, if a ‘T’ in the Reference Sequence overlaps a ‘G’ in another sequence, the
consensus line will show a ‘G’.

Figure 7-2 Base numbering showing decimal numbers

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WORKING WITH A REFERENCE SEQUENCE

HOW TO MARK A SEQUENCE AS A REFERENCE

To designate a sequence as a Reference Sequence, select the sequence icon in the Project
Window and, under the Sequence menu, select the Reference Sequence command. From
now on, that sequence icon will include a small letter "R" to remind you that it has been
marked as a Reference.

ASSEMBLE TO REFERENCE

Assemble to Reference is a powerful command which allows you to assemble all the samples
you select to a single Reference Sequence, regardless of any inconsistencies between the
individual sequences. Because it is a many-to-one comparison instead of the normal many-to-
many comparison, Assemble to Reference is much faster than the standard Assemble
Automatically mode.
To Assemble to Reference, select the sequences you want to assemble. Click on the Assemble
to Reference button or go to the Assemble menu and choose Assemble to Reference. You
will get a warning dialog if you have not included a properly designated Reference Sequence
among your selected sequences.
Note: You can use the Assembly Parameters to change the conditions under which the
assembly will take place. (See Chapter 9 “Sequence Assembly” for more information.)

TO REFERENCE BY NAME

To Reference by Name is a special case where the behavior of the Reference Sequence is
modified to work with the Assemble by Name command. The To Reference by Name
button will only be displayed if Assemble by Name has been enabled. (For more information
on Assemble by Name, see Chapter 9 “Sequence Assembly”.)
When the To Reference by Name command is used, each contig created will contain a
Reference Sequence and will also retain the numbering of the Reference Sequence.
To use To Reference by Name, select the sequences and Reference Sequence you want to
assemble. Click on the To Reference by Name button or go to the Assemble menu and
choose the Assemble Contigs command and then click on To Reference by Name from the
submenu.

TRIM TO REFERENCE

The Trim to Reference Sequence command is used to remove bases in a contig that flank the
Reference Sequence on both the 5’ and 3’ ends. To use this command, go to the Contig menu
and choose Trim to Reference Sequence. This command is only available when you are in a
contig that contains a Reference Sequence.

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Note: You cannot undo this action. If there are any sequences that do not overlap the
Reference Sequence, they will be removed from the contig. Sequencher will warn you which
sequences have been removed in a window, which you can print for your records.
Figure 7-3 Contig with Reference Sequence flanked by overhanging sequence (before and after)

CONTIG EDITING WITH A REFERENCE SEQUENCE

If you try to edit a Reference Sequence which has been assembled into a contig using either the
standard Assemble Automatically or Assemble to Reference, a warning dialog will be
displayed asking if you are sure this is what you want to do. You can proceed with the edit by
clicking on the Yes button. You can cancel the warning and continue to edit by clicking on the
No button.
The Reference Sequence is protected from any changes you make while you are editing in the
consensus line. Therefore, if you delete gaps from the consensus, this will not affect the
Reference Sequence unless it also has gaps at the position of the deleted bases. For example,
you can delete a range of bases from all sequences by selecting a range within the Consensus
sequence. When the deleted bases are in the middle of a sequence, they will be replaced with
gaps to maintain the alignment with the Reference Sequence. Since the action cannot be
reverted using the Undo command, you must use the Revert to Experimental Data command
from the Sequence menu to recover.

Figure 7-4 Filling gaps with the Reference Sequence

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REFERENCE SEQUENCE TRANSLATION

This command is only available when the Reference Sequence is assembled into a contig.
To see a translation of the Reference Sequence in the notation line below the consensus
sequence, go to the View menu and choose Reference Sequence Translation. The translation
has a pale gray border above and below the amino acids to distinguish it from the consensus
sequence translation.
Note: As you cycle through the Consensus translation button modes, you will notice one mode
which translates the consensus sequence in the same frame as the Reference Sequence. In this
mode, the button icon is marked with an “r”.

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 8. THE SEQUENCE EDITOR

In this chapter, we explain basic sequence editing, setting base numbers, and how to reverse
and complement your data. We then go on to discuss working with experimental data and how
Sequencher enables you to find ambiguities, work with codon and restriction maps, and
annotate features. You will also learn how to customize your sequence view and use voice
verification to help you check base calls.

OPENING AN EXISTING SEQUENCE FOR EDITING

To open an existing sequence, select a sequence icon and then go to the File menu and choose
Open Window. You can also just double-click the icon to open its editor window.

LOCKED EDITORS

If an editor displays experimental data or a sequence has been incorporated into a contig, it is
locked. A padlock icon (shown to the left of the word “Residue” in the information bar above
the sequence in Figure 8-1) shows that the file is locked.

Figure 8-1 A locked editor

A locked editor is read-only. You cannot directly edit data, you can only view it. However, if the
sequence has been incorporated into a contig, you can use the Contig Editor to make changes.
(See Chapter 12 “Editing Contigs”.)

BASIC SEQUENCE EDITING

The Sequence Editor, shown in Figure 8-2, is a text processor that is specially optimized to
work with DNA sequences.

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 Figure 8-2 The Sequence Editor

The default system for coding in Sequencher is the IUPAC-IUB coding system. You can define
your own coding system for bases and ambiguities. (See Chapter 23, “Customizing Sequencher
and User Preferences” for details.) The IUPAC-IUB codes are included in Appendix 28 of this
manual.
The Sequence Editor works like any other text editor. The insertion point can be moved using
either the mouse or the arrow keys.
Note: If you hold down the Alt key and use an arrow key, the insertion point moves three
bases. If you hold down both the Ctrl and Alt keys (Windows) or control and Alt keys ( Mac),
the insertion point moves nine bases. If you hold down the Ctrl (Windows) or Cmd ( Mac) key
and the left arrow, the insertion point moves to the beginning of the sequence. If you hold
down Ctrl (Windows) or Cmd ( Mac) key and the right arrow, the insertion point moves to the
end of the sequence.
The editor also performs typical operations including Cut, Copy, Paste, and Undo. Use the
mouse to highlight a selection; hold down the Shift key and click elsewhere in the sequence
with the mouse to extend the selection.
There are also two commands for extending a current selection to the beginning or end of a
sequence, To Left End and To Right End, both located under the Select menu in the Extend
Selection submenu.
Once you have made a selection, you can copy it by going to the Edit menu and using the Copy
Selection command. You can also cut the selection by using the Cut Selection command, also
located on the Edit menu.
The Paste function will remove non-sequence characters. You can define a sequence character
by going to the Window menu and then using the Ambiguity Editor from the Ambiguity/Key
Codes… command.
For instructions on trimming ambiguous data and vectors at the time you load your sequences,
see the chapters “Importing Data”, “Preparing Your Data for Assembly,, and “Customizing
Sequencher and User Preferences” elsewhere in this manual.

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SETTING THE BASE NUMBERING

Open the Sequence Editor and highlight a base. Go to the Sequence menu and choose Set
Base Number. From the submenu choose either As Base 1 or As Base Number. If you choose
As Base Number, you will see a dialog where you can enter a new number for the selected
base. Click OK to dismiss the dialog.
Note: If you set a base to be base 1, and this is not the first base in your sequence, every base
before it will have a negative number.

SET CIRCULAR GENOME SIZE

Sometimes you will be working with circular DNA such as plasmids or even small genomes. The
Set Circular Genome Size… command allows you to set the number of bases in your DNA
circle.
Select a sequence that has already been defined as a Reference Sequence. Then go to the
Sequence menu and choose the Set Circular Genome Size… command. The dialog in Figure
8-3 below will appear. Enable the circular number by clicking the checkbox. Then enter the
number of base pairs in your sequence. Dismiss the dialog by clicking on the OK button.

Figure 8-3 The Circular Genome Size dialog

Note: Circular numbering can only be used in conjunction with a Reference Sequence. You
must designate your sequence as a Reference Sequence before you can enable circular
numbering. See Chapter 7 “The Reference Sequence” for more information.

SPLITTING A SEQUENCE

You can split a sequence into two pieces, for instance, at a known exon boundary. Put the
cursor where you want to split the sequence. Go to the Sequence menu and choose Split
After Selection …. Sequencher will ask you if you want to split the sequence after the last
currently selected base. Click on the Split button to proceed. Sequencher will split the

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sequence into two pieces and label the new sequences for you. If your sequence was called
“MyPUC”, it will be divided into two sequences called “MyPUC 5’ end” and “MyPUC 3' end.”

DUPLICATING A SEQUENCE

You can duplicate a sequence. To do this, highlight the icon, then go to the Edit menu and
choose Duplicate Seq Fragment. Sequencher will use the name of the original sequence as a
basis for the new sequence’s name. If the sequence you want to duplicate has a chromatogram
associated with it, Sequencher will ask whether you also want to duplicate the chromatogram
or duplicate the sequence without chromatograms. Click on the appropriate button to
proceed.
If you wish to cancel the operation, simply press on the Cancel button. Chromatograms occupy
a lot of memory but Sequencher uses a very efficient algorithm to compress trace data.
However, we suggest that you only duplicate the text if you don’t need the chromatograms.
(See Chapter 15 “Chromatograms” for more information.)

REVERSE AND COMPLEMENT

The data you bring into Sequencher may not necessarily be in a particular biologically relevant
orientation. To change the orientation of the sequence, go to View menu and choose Reverse
& Comp or Sequenced Strand. To display the original orientation, go to the View menu and
choose Sequenced Strand.
Note: You will always have two copies of your data in Sequencher, the original data and the
edited version. This duplication allows you to revert to the original data, as discussed in more
detail below.

ABOUT EXPERIMENTAL DATA

CREATING A BASELINE

Sequencher notes the original base sequence when you import your data so that, after editing
the sequence, you can still discard all of your edits and revert to the baseline (experimental)
data. You can also do this for sequences that you enter from the keyboard.
When you close a Sequence Editor that has not had its data recorded as experimental data,
the program explicitly asks whether the contents of the Sequence Editor should be recorded
as experimental data (see Figure 8-4). Click on Record As Experimental Data if you are
finished editing the sequence. If you are not finished, click on the Not Yet Finished button.

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 Figure 8-4 The Record as Experimental Data dialog

Note: When a sequence becomes part of a contig, the data are automatically recorded as
experimental data if they have not previously been stored in that form.
Once a version of the sequence has been recorded as experimental data, all future changes are
considered to be “edits” to it. As a data quality tool, all such edits can be marked in color and
displayed in Bold Magenta or lower-case text. This feature is invoked from the View menu
under the Base Edits As command. The default is set to Bold Magenta but you can also
choose Not Highlighted or bOLD & cASE cHANGE.

RESETTING A BASELINE

If you decide that some of the original data was incorrect (perhaps a simple typing error that
has been corrected in the “edited” version of the sequence), go to the “Experimental Data”
version of the sequence by clicking on the Show Experimental button and then click on the
New Baseline button.
Note: Setting a new baseline for a sequence resets all of the bases to a not-yet-edited state.
None of the bases that were edited will appear in Bold Magenta letters.

VIEWING EXPERIMENTAL DATA

After you enter a new sequence, Sequencher archives a copy that is “locked” (not editable). In
the course of assembling your data, you may make a great number of edits to any particular
sequence. This locked version enables you always to refer back to the sequence as it was
originally entered. To see the Experimental Data version of the sequence, click on the Show
Experimental button in the Sequence Editor button bar.

REVERTING TO EXPERIMENTAL DATA

If you edit a sequence incorrectly, you can delete all the changes to that sequence and go back
to the archived baseline. To do so, open the Sequence Editor. Then choose Revert To
Experimental Data from the Sequence menu.
If you edit a contig incorrectly, you can delete all the changes to an individual sequence and go
back to the archived baseline. To do so, open the Contig Editor and select the sequence by
clicking on its icon which can be found on the left-hand side of the Contig Editor. You can now

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remove the sequence from the contig by using the Remove Selected Sequences… from the
Contig menu. Then choose Revert To Experimental Data from the Sequence menu. You can
then reselect the sequences and rebuild the contig.

VIEWING EXPERIMENTAL DATA FOR CHROMATOGRAMS

If your data came from an automated sequencer, click the button labeled Show
Chromatogram to view the experimental data of a sequence. This will display the original
trace data. Sequencher allows you to scroll either vertically (the default) or horizontally. To
change the scroll orientation, click the appropriate button in the left bottom corner of the
sequence Chromatogram window. (See Chapter 15 “Chromatograms” for more information.)

VIEWING SUMMARY INFORMATION IN THE SEQUENCE EDITOR

You can bring up summary information on your data from the Sequence Editor. Go to the File
menu and choose Get Info…. The Get Info… window (in Figure 8-5) provides summary
information on the sequence you are currently editing.

Figure 8-5 Sequence information window

If you want to annotate your data with more information than the comment box allows you to
see, go to the Edit menu and click on Edit Comments… Sequencher will open a small text
editing window that stores about 250 characters of text with each sequence. After you have
added text with Edit Comments, a small “I” (for “Info”) will appear at the upper left corner of
the icon.
Base counts are displayed at the bottom of the window. If your sequence also includes
confidence scores, you will also see a base count for each of the three confidence ranges. If

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you use an ABI sequencing system, click on the Show ABI Info button. Sequencher displays a
window with information from the ABI data sheet, shown in Figure 8-6.

Figure 8-6 ABI data sheet info

SELECTING BASES

Sequencher offers a number of ways to select bases. You can choose commands from the
Select menu or you can use a keyboard combination. To select bases in the opposite
orientation, press and hold the Shift key when using any Select Next… command (from the
menu or with a keyboard shortcut).

FINDING AMBIGUOUS BASES

One of the most frequently executed commands is Next Ambiguous Base under the Select
menu. You can use this command in the Sequence Editor or the Contig Editor. By using this
command in the consensus sequence of a contig, Sequencher will display the next position in
your contig that contains an ambiguity or disagreement. The shortcut for this command is
Ctrl+N (Windows) or Cmd+N (Mac). (For more on keyboard shortcuts, see Appendix 26
“Keyboard Shortcuts”.) If you use the Shift key with this key combination, you will go back to
the previous disagreement.
Once you have used any of the Select Next commands, Sequencher will activate the space bar
to execute that operation. If you use either the menu command or the shortcut key
combination two consecutive times, Sequencher will remind you that this easier alternative is
available.

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FINDING OPEN READING FRAMES (ORF)

Choose Next Met to Stop ( > 0b ) to highlight the next pair of start and stop codons in one of
the three forward reading frames. The number of bases shown in this command depends on
whether you specified a preference for a minimum length in User Preferences. (For
instructions on how to set a specified minimum length for open reading frames, see Chapter
23 “Customizing Sequencher and User Preferences”.) If you set preferences to highlight ORFs
only when they are of some minimum length, the menu command will display the number of
bases you specified.

Figure 8-7 ORF selected by Next Met to Stop command

SEQUENCE FEATURES

LOOKING AT CODON MAPS

Click on the Codon Map button located in the button bar at the top of the Sequence Editor or
go to the View menu and choose Display Codon Map. The three “outlined bars” at the top of
the editor window represent the three forward frames of translation. Each green “flag”
sticking partially into the top of a frame marks the position of a start codon, while each red line
cutting through the frame and extending below marks the position of a stop codon. Clicking
between any two markers highlights that section of the sequence.

Figure 8-8 Sequence Editor with Codon Map button selected

In the example shown in Figure 8-9, a click between a start and a stop codon of the first
reading frame has selected the corresponding bases in the editor. Note that User Preferences

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have been set to highlight ORFS of more than 100 bases. There is one such ORF in Frame 2,
marked with a hash pattern. To dismiss the map, click on the Codon Map button again.
Figure 8-9 Sequence Editor with Codon Map

LOOKING AT RESTRICTION MAPS

You can look at a restriction map for the sequence by clicking on the Cut Map button at the
top of the Sequence Editor. A restriction map replaces the sequence in the window. (See
Chapter 17, “Restriction Maps,” for a general discussion of restriction maps.)
Sequencher can add a pane showing the restriction map for a sequence into the same window
as the sequence itself. At the top of the Sequence Editor dialog is a button showing an icon for
a staggered enzyme cut (shown in Figure 8-10).

Figure 8-10 Restriction Map button selected with map and sequence shown

Click the button; a window with a multi-line restriction map appears. You can adjust the size of
the panes by dragging the splitter bar that separates the scroll bars for each pane (see Figure

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8-10). You can also invoke this command by going to the View menu and choosing Display Cut
Map Inset.
Like the full-page cut map, the window restriction map is linked to the Sequence Editor. If you
click between two cut sites, Sequencher highlights the resulting restriction sequence, as
shown in Figure 8-11. If you click on the name of an enzyme, Sequencher highlights the
recognition sites.

Figure 8-11 Marking restriction and recognition sites

ANNOTATING SEQUENCES

You can assign a standardized GenBank Feature Key in addition to personal feature
annotations to describe subsequences. Select the range of bases you wish to annotate, then
choose Mark Selection As Feature from the Sequence menu.
If you want to assign a personal annotation, select Sequencher from the drop-down menu
called Feature Key:. To give the feature a name, type your text into the Feature Name: box.
You can use the default display style or you can assign a Feature Color and Feature Style,
such as inverted case or underlined, from the dialog. You can also use the Display: radio
buttons to determine whether single stranded DNA is displayed or RNA, protein translation, or
the complement will be shown. (For more information, see Chapter 19 “Motifs and Features”.)
If you want to assign a GenBank feature key, make a selection from the drop-down menu called
Feature Key. To give the feature a name, type your text into the Feature Name: input field.
You can use the default display style or you can assign a Feature Color and Feature Style, such
as inverted case or underlined, from the dialog. You can also use the Display: radio buttons to
determine whether single stranded DNA is displayed or RNA, protein translation, or the
complement. (For more information, see Chapter 19 “Motifs and Features”.)

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CUSTOMIZING YOUR SEQUENCE VIEW

FORMATTING RULER

Formatting can be controlled with a ruler (shown in Figure 8-12) that lets you adjust several
display parameters. Click on the Ruler button in the Sequence Editor or Contig Summary
window. Alternately, you can go the View menu and choose Display Format Ruler.

Figure 8-12 The formatting ruler

MARGINS

You can drag the margin triangles on this ruler to set the width of your sequence. Figure 8-13
shows margins that are limited to 42 bases per line.

Figure 8-13 Sequence Editor with ruler and translations

The sequence margins you set with the formatting ruler are not the same as your absolute
print margins, which allow room at the left to show the first base number of a line. To change
the absolute limits, go to the File menu and choose Set Header & Footer… and enter new
values into the Margin boxes or, drag the triangular markers just under the Translation and
Base Grouping icons, as shown in Figure 8-13 above.

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TRANSLATION

On the left, just under the word “Residue” and above the ruler is a set of three small icons that
let you specify sequence representation: single-stranded, double-stranded, or single-stranded
with a protein translation.
If you select the third ‘translation’ icon, the numbered button at the right controls which frame
or whether all three frames will be displayed. You can also use the translation commands in
the Translation submenu of the View menu: Single Stranded, Double Stranded, Protein 1st
Frame, Protein 2nd Frame, Protein 3rd Frame, Protein All 3 Frames (see Figure 8-14).

Figure 8-14 Translation icons on the Formatting ruler

See Chapter 23, “Customizing Sequencher and User Preferences”, for information on editing
the genetic code or using one-letter abbreviations for amino acids.

BASE GROUPING

To the right of the translation display icons are the icons that let you choose blocking, the
number of bases to be displayed in a single group. The available settings are 3, 5, 10, 20 and
Fill-to-margin. In Figure 8-14 above, the icon for a grouping of 10 is selected.

LINE SPACING

You can choose single, double, or triple line spacing. The three small icons that let you control
the line spacing are located to the right of the base grouping icons. Figure 8-14 above shows
the icon for single spacing selected.

CASE AND FONT

To the right of the line spacing icons is a pair of icons that let you choose upper or lower case
for the display. In Figure 8-14 above, the icon for upper case is selected.
To the right of the upper and lower case icons is a pull down menu which lets you choose a
font and size. Only fixed-width fonts are displayed, ensuring that the rows of bases will line up
vertically.

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VOICE VERIFICATION

Sequencher provides two forms of audio feedback to help you maintain the quality of data
input when you are entering information manually from the keyboard. You can have
Sequencher announce each base as you type it in. You can have the computer “read” the
sequence aloud back to you while you look at the original source of the data. Sequencher
stores the sounds that it uses in a sound file stored in your Sequencher folder.

SELECTING A VOICE FILE

First, make sure your computer sound system is enabled and that the volume is not set on
mute. Then, go to the Sequence menu and choose Speech. Then go to the submenu Select
Voice File….You will not get any audio feedback until the Select Voice file is loaded.

IDENTIFYING A SPEAKER

The voices Gene Codes uses to provide audio for you may be familiar, such as Walter Gilbert. To
hear whose voice is being used, go to the Sequence menu and choose Speech and then the
Identify Speaker sub-command. You can only select this command if a sound file was selected
previously.

AUDIBLE KEYSTROKES

Any meaningful keystroke you type into a Sequence Editor dialog will be spoken aloud if you go
to the Speech command in the Sequence menu and select the Say Sequence Keystrokes
submenu. That is, if you press the “A” key, a voice will say “A,” but if you press the shift key,
you will not hear any voice.

READ SEQUENCE SELECTION

To check the sequence you have entered into the Sequence Editor, first select the bases you
want the computer to read to you. Then go to the Sequence menu, click on Speech and then
the Read Sequence Selection submenu.
Note: You must place the cursor somewhere in the small window that appears above the
sequence as in Figure 8-15. To pause the reading, just move the cursor out of the window.

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 Figure 8-15 Speak selection cursor and instruction menu

To speed up or slow down the rate at which Sequencher’s audio “reads” your data, go to the
Window menu and choose User Preferences... Under the General section, you will find an
item called Sound. Click on this to display a slider bar that controls the speed of read back in
bases per second. To end the read back, just click your cursor anywhere on the Sequence
Editor window.

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 9. SEQUENCE ASSEMBLY

In this chapter, you will learn how to set assembly conditions by using Sequencher’s rich
variety of assembly algorithms and parameters. You will learn about automatic assembly,
assembling interactively, assembling groups of sequences using sequence name information,
the large gap algorithm for assembling cDNA and genomic DNA, and assembling to Reference
Sequences. You will learn how to set assembly conditions for each of the different algorithms.

THE ASSEMBLY STRATEGY

Sequencher provides you with powerful options for performing your sequence assembly. In
many cases, the default options will perform well. However, experimental variation can affect
your data, so there will be times when you may need to change the assembly parameters. The
following is a basic strategy for assembling your data.
Start with strict parameters to minimize the possibility of incorrect matches. Select the
sequences you want to assemble and use the Assemble Automatically option. Automatic
assembly relies on an exhaustive search-and-compare algorithm.
• If some sequences do not assemble, decrease the stringency of the parameters and
try Assemble Automatically again.
• If the selected parameters become relaxed enough that the Assemble
Automatically process may result in scientifically insupportable alignments, you
should switch to interactive alignment so you can exert more control over the
process.
• The most frequently used options; Assemble Automatically, Assemble
Interactively, and Assemble to Reference are available as prominent buttons in
the Project Window. In addition, Auto Assemble by Name and To Reference by
Name replace Assemble Automatically and Assemble to Reference when
Assemble by Name is enabled. There are numerous other options under the
Assembly Parameters and the Assemble menu.

SETTING THE ASSEMBLY CONDITIONS

SETTING ASSEMBLY PARAMETERS

• To change the parameter settings, select the Project Window. Then either click on
the Assembly Parameters button in the Project Window or go to the Assemble
menu and choose the Assembly Parameters command. Sequencher displays the
dialog shown in Figure 9-1.

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 Figure 9-1 Assembly parameters dialog

Sequencher uses the values set in this window to control the way it assembles sequences.
Once you have set the options you require click the OK button to dismiss the Assembly
Parameters window.

ASSEMBLY ALGORITHMS

The algorithms in this section are devoted to working with traditional Sanger Sequencing. If
you are interested in working with Next-Generation sequencing data, refer to Chapter 16 “NGS
for DNA and RNA-Seq” for complete information.
To choose which assembly algorithm you want to use, click on the Assembly Parameters
button on the Project Window and then click on one of the three radio buttons at the top of
the window. Your choice will depend on your data.
For instance, if you click on the Clean Data radio button, contig assembly is faster but
Sequencher may miss some possible matches if the ends of your sequences contain a large
number of ambiguities. For best performance, make sure that you have trimmed poor quality
data from your sequences.

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The Dirty Data algorithm is somewhat slower because Sequencher is performing more
rigorous comparisons between the sequences. You should note that automated sequencers
create dirty data and Sequencher’s algorithm is optimized to deal with that.
The Large Gap algorithm allows you to assemble sequences that are expected to contain
insertions and deletions (gaps) of 10 or more bases long. Such gaps are typically found in
evolutionary studies and sequence comparisons from different organisms. The Large Gap
algorithm is similar to and slower than the dirty data algorithm but allows larger gaps to occur
when performing the assembly. Typical examples of assemblies that require the Large Gap
algorithm include the comparisons of a cDNA sequence to a genomic sequence and the
assembly of related genes with alternative splicing.

ASSEMBLING WITH DIRTY DATA

Go to the Assembly Parameters dialog and select the Dirty Data radio button. There are two
sliders for changing minimum match values. To set the proportion of bases that have to match,
use the Minimum Match Percentage slider. To set the minimum number of bases that must
overlap, use the Minimum Overlap slider.
Change the settings by positioning your cursor on the slider, holding the mouse button down,
and dragging the slider left or right. The value set will automatically update as you move the
slider.
For example, if you attempt to assemble a 17-base sequence that matches perfectly to a
selected 1000 base sequence with the minimum overlap set at 20 bases (Sequencher’s default
minimum overlap), the two sequences will not assemble. You must first reduce minimum
overlap to 17 bases or lower and then assemble the sequences.

ASSEMBLING WITH CLEAN DATA

When you select the Clean Data algorithm, an additional option called Maximum Loop Out
Size is available. This has an elevator button for adjusting size value (Figure 9-2 below). This
parameter is the largest number of consecutively mismatched bases that are acceptable in a
potential overlap. Change the setting by positioning your cursor on the appropriate
arrowhead, and holding the mouse button down. The maximum value, which can be set, is 6.
To set the proportion of bases that have to match, use the Minimum Match Percentage
slider. To set the minimum number of bases that must overlap, use the Minimum Overlap
slider.

Figure 9-2 The Clean Data algorithm with maximum Loop Out Size shown

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ASSEMBLING WITH LARGE GAP

The Large Gap algorithm is specifically for data that has or might have large insertions or gaps.
This might include data from evolutionary studies, different organisms or comparing cDNA to
genomic DNA. When you select the Large Gap algorithm, there are two sliders for changing
minimum match values. To set the proportion of bases that have to match use the Minimum
Match Percentage slider. To set the minimum number of bases that must overlap use the
Minimum Overlap slider.
Note: Assemble to Reference cannot be used with the Large Gap algorithm.

REFINING THE ASSEMBLY CONDITIONS

MINIMUM MATCH PERCENTAGE

The Minimum Match Percentage can be used with all three assembly algorithms. It is used to
set the proportion of bases which must match in candidate sequences before Sequencher
accepts the sequences as actually overlapping. The default value of 85% can be changed by
moving a slider.
If this value does not give you any overlaps, you may need to decrease the Minimum Match
Percentage by moving the slider to the left to lower the percentage.
If you have an incorrect or unexpected overlap, you may need to increase the Minimum
Match Percentage by moving the slider to the right (higher percentage).

MINIMUM OVERLAP

The Minimum Overlap can be used with all three assembly algorithms. It is used to set the
minimum number of bases that must overlap before Sequencher accepts the sequences as
actually overlapping. The default value of 20 can be changed by moving a slider. The default
maximum value is 100 but this can be changed to 500 by clicking the checkbox in the Contig
User Preference pane.
If this value does not give you any overlaps, you can decrease the Minimum Overlap by
moving the slider to the left to indicate a smaller number of bases that must overlap.
If you have an incorrect or unexpected overlap, you may need to increase the Minimum
Overlap by moving the slider to the right (larger number of bases that must overlap).

MAXIMUM LOOP OUT SIZE

The Maximum Loop Out Size is the largest number of consecutively mismatched bases (in this
instance a gap also counts as a mismatch) that are acceptable in a potential overlap. The
number of mismatching bases is set by moving an elevator button up or down. The maximum
value that can be set manually is 6. Remember that this option can be used only with the
Clean Data algorithm.

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OPTIMIZATION OF GAP PLACEMENT

ReAligner is an optional step which can be used with the Clean Data and Dirty Data
algorithms. It evaluates the disposition of gaps within a contig and optimizes their placement.
ReAligner facilitates editing in the consensus sequence and clearly displays the effect of
insertions and deletions.
To use ReAligner, select the checkbox in the Assembly Parameters dialog. In some areas of
DNA analysis, the standard is to gather the gaps to the right. If you require this, select Prefer 3’
Gap Placement.
Note: This option cannot be used with the Large Gap algorithm.

PERFORMING THE ASSEMBLY

AUTOMATIC ASSEMBLY

Bring the Project Window to the front and select the sequences you want to assemble. Click on
the Assemble Automatically button or go to the Assemble menu and choose the
Automatically command. Sequencher compares all the selected sequences, including reverse
complements, and assembles the best matches that fall within the chosen assembly
parameters.
When assembly is done, the advisory window appears. (See Figure 9-3.) Click Close to go back
to the Project Window.

Figure 9-3 Assembly done alert

ADDING SELECTED ITEMS TO OTHERS – INCREMENTAL BUILDING OF CONTIGS

The assembly option Add Selected Items To Others, which is a special case of automatic
assembly, is very useful when you have a growing data set. Sequencher compares all the
selected (new) items to the unselected (old) items. It will also compare the new items to each
other. It has been designed to increase the efficiency of handling large numbers of sequences

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in efforts such as DNA library clustering and genome assembly. Therefore, it will not compare
any unselected items to each other again.
Go to the Assemble menu and click on Add Selected Items To Others.

ASSEMBLE TO REFERENCE

The Assemble to Reference command allows you to assemble samples to a single Reference
Sequence regardless of inconsistencies between the individual sequencess. Because it is a
many-to-one comparison instead of the normal many-to-many comparison, Assemble to
Reference is much faster than the standard Assemble Automatically algorithm.
To use this command, you need to have a Reference Sequence already designated. Click on
your sequence and then go to the Sequence menu and select Reference Sequence. Now select
the sequences you want to assemble, including the Reference Sequence. Click on the Assemble
to Reference button or go to the Assemble menu and choose Assemble to Reference.

INTERACTIVE ASSEMBLY

The Assemble Interactively algorithm can be used whenever you want complete control over
the assembly of a batch of sequences. The Assemble Interactively window provides you with
detailed information regarding overlap, mismatch, and gapping for any given sequence and a
set of candidate sequences. The candidate assemblies are driven by your user-defined
assembly parameter settings.

PERFORMING AN INTERACTIVE ASSEMBLY

Bring the Project Window to the front and select the items you want to assemble. Click on the
Assemble Interactively button at the top of the Project Window or go to the Assemble menu
and choose Interactively….
Sequencher displays the Assemble Interactively dialog shown in Figure 9-4. The sequences
and contigs you selected are displayed in the Candidates list. The panel on the right, showing
a face in the upper left corner, is called the “agent.” It displays information about the
comparisons you have requested. In the center is the Matches panel that displays the possible
matches. For each match in the panel, Sequencher lists the % Match, the approximate
number of Overlap bases, the number of Mismatch bases, the number of Gaps, and the New
Contig Length.
The top line shows the Candidate sequence and the line below this shows the Match sequence.
Below these two lines is the consensus sequence. There is a small button to the right of the
consensus labeled with a black circle. If you click repeatedly on this button, you can display the
first, second, or third reading frame or all three reading frames at once. The label on the
button changes to reflect which of these choices is active.

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 Figure 9-4 Assemble Interactively dialog

To have Sequencher compare any single sequence or contig with the other items in the
Candidates list, click on the name of that sequence or the contig in the Candidates list. When
the comparison is complete, the possible matches appear in the Matches list to the right of
the Candidates list, as shown in Figure 9-5.
Figure 9-5 Interactive assembly showing a candidate and its matches

If Sequencher fails to find a match, as in Figure 9-6, the agent panel displays a message to that
effect.

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 Figure 9-6 Match not found

When you click a possible match in the Matches list, Sequencher recalculates the optimal
positioning for gaps. The agent then displays a message about the selected sequences and
their computed overlap.
The start of an overlap and the base consensus appear in the fields at the bottom of the
interactive assembly box (Figure 9-7). You can explore the alignment further by moving the
scroll bar to the right.
Figure 9-7 Interactive Assembly window showing overlap

If you choose to create this assembly, click on the Assemble button. Name the new contig in
the Set Name dialog and click OK. Sequencher takes only a short time to assemble (or
reassemble) a contig and the name of the new contig now appears at the top of the
Candidates list. Note that as a sequence is assembled, its name is removed from the
Candidates list.
The new contig will appear in the Project Window when you close the Assemble Interactively
dialog by clicking on the Done button.

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CHANGING PARAMETERS FOR AN INTERACTIVE ASSEMBLY

If you suspect that your sequences should form a contig but Sequencher cannot suggest
candidate matches, you can change the parameters for assembly. To do this while using
Assemble Interactively, click on the Assembly Parameters button. The Assembly
Parameters dialog will appear. When you have changed the settings to your satisfaction, click
OK to return to the Assemble Interactively window.

MINDLESSLY JOIN

Mindlessly Join allows you to put sequences together without using the conventional sets of
algorithms. Examples of situations where you might wish to do this include making a plasmid
or constructing an artificial cDNA sequence.
Select the sequences you want to join by dragging a box around them or Shift-clicking them.

Figure 9-8 The new contig displayed in the Assemble Interactively Window

Note: They will be listed in the new contig in the order in which you selected them.
Go to the Assemble menu and then select Mindlessly Join. If Assemble by Name is enabled,
the Mindlessly Join submenu item becomes Join by Name. Join by Name uses handles to
group the sequences appropriately.
Sequencher will then ask if you want all the sequences aligned at the left of the contig (All
Left), at the right of the contig (All Right), or joined End to End. Click the appropriate button.
The selected items will be assembled and replaced by a contig icon in the Project Window.

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 Figure 9-9 Join by Name dialog

MUSCLE

Unlike the algorithms Clean Data, Dirty Data, and Large Gap, MUSCLE is not built into
Sequencher but is an external tool. More information on adding MUSCLE to Sequencher can
be found in the DNA-Seq Tools Installation pdf on the Gene Codes website Support page.
MUSCLE is a multiple-sequence alignment algorithm and it will attempt to align the sequences
over their entire length. This can introduce a large number of gaps into your aligned
sequences, so you should be aware that it may not be the best algorithm to use for all
purposes.

USING MUSCLE TO ALIGN SEQUENCES

Select the sequences you wish to align from the Project Window. Then go to the Assemble
menu, select Align Using, and click on MUSCLE from the submenu. A new dialog appears which
tells you that MUSCLE is running. When the alignment is completed, this dialog is dismissed.
You will see a new contig in your Project Window which you can analyze with Sequencher’s
other tools such as the Variance Table.
Note: You can read more about MUSCLE in this paper. Edgar RC: MUSCLE: a multiple sequence
alignment method with reduced time and space complexity. BMC Bioinformatics 2004, 5:113.

Figure 9-10 Align Using MUSCLE

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 10. ASSEMBLE BY NAME

Many labs apply strict sample-naming conventions to their sequences, with the name
indicating something about the data. Assemble by Name harnesses the information from
your sequence names to automate the process of selecting and naming your contigs.
In this chapter, you will learn how to use Assemble by Name, a powerful tool that lets you
separately assemble and automatically name multiple contigs from a single selection and
assembly command. For example, in just a few clicks of the mouse, Assemble by Name will
create ninety-six appropriately named contigs from ninety-six forward and reverse sequences.

THE ASSEMBLE BY NAME STRATEGY

The names of your samples frequently contain information such as your primer, template,
clone name, or sample source. Sequencher provides you with tools to separate the descriptive
parts of your sequence name and use these to manage the assembly. These tools are the
Assembly Handles and the Name Delimiters.
An Assembly Handle is any set of characters from your sequence name that provides
information about that sequence, such as the primer or clone name. A Name Delimiter
separates each Assembly Handle from the following one. Sequence names frequently have
several Assembly Handles and Name Delimiters.
In its simplest form, a Name Delimiter may be a character such as a dash or an underscore.
When you use a Name Delimiter, it will look like this:
handle1-handle2-handle3

or

handle1_handle2_handle3

However, a Name Delimiter can also be a combination of letters, numbers, and characters.
To define your Name Delimiter, Sequencher provides a set of commonly used characters as a
drop-down menu in the Assemble by Name Settings dialog. You will see this menu if you go
to Assembly Parameters dialog and click on the Name Settings… button. When your naming
convention does not use simple characters as Name Delimiters, you can use formal
descriptors known as "regular expressions" instead, which we describe later in this chapter.
Once you have defined an Assembly Handle, you will be able to assemble all of the sequences
in your project. Only sequences that share the same Assembly Handle will be analyzed for
assembly into the same contig. In a clinical application, for example, several contigs might be
created with each contig representing sequence data from only a single patient, provided that
each sample (patient) has a unique Assembly Handle in its name.

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SETTING THE ASSEMBLE BY NAME CONDITIONS

CONFIGURING ASSEMBLY HANDLES USING A SINGLE DELIMITER

Starting in the Project Window, click on the Assembly Parameters button. At the bottom of
the Assembly Parameters dialog, click on the Name Settings… button. The Assemble by
Name Settings dialog appears in Figure 10-1 below.

Figure 10-1 Assemble by Names Settings window

If your Assembly Handles are separated by a single character included in the Name
Delimiters drop-down menu, choose that character from the list. Otherwise, follow the
directions for an Advanced Expression.

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 Figure 10-2 The Name Delimiters drop-down menu

CONFIGURING ASSEMBLY HANDLES USING ADVANCED EXPRESSIONS

You will need to create an Assembly Handle using a regular expression if your Assembly
Handle is not separated by one of the characters in the Name Delimiters drop-down menu.

WHAT IS A REGULAR EXPRESSION?

A regular expression is a way of describing a text pattern using letters, numbers, and special
characters. These expressions follow certain syntactical rules.
Starting in the Project Window, click on the Assembly Parameters button. At the bottom of
the Assembly Parameters dialog, click on the Name Settings… button. The Assemble by
Name Settings dialog appears.
Choose Advanced Expression… from the Name Delimiters drop-down menu. You will notice
that the Define… button is now enabled. Click on the Define… button.
The Advanced Expression for Name Parsing dialog appears. At the top of the dialog is an
input field where you can type in a regular expression. Assemble by Name uses regular
expressions in two ways. When the Expression is a delimiter box is checked, the regular
expression you type in lets you define a delimiter option other than one available from the
drop-down menu.
If the Expression is a delimiter box is not checked, your regular expression must completely
define each Assembly Handle and Name Delimiter in your sequence name. The regular
expression you write will not work unless it describes the entire name of your sequences.
When you type the regular expression into the text field, it should be in the form:
(Assembly Handle1)Name Delimiter(Assembly Handle2 )

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In the example in Figure 10-3 below, a regular expression is used to combine the first two parts
of the sequence name, Origin and Clone, to create a new Assembly Handle, Origin&Clone. The
Preview button displays the results of your regular expression. If you are satisfied, click on the
OK button.
Figure 10-3 Advanced Expression for Name Parsing dialog

For more details on regular expressions, see Appendix 27 “Advanced Expressions” and the
tutorials in the Tutorials folder in your Sequencher installation folder.

SETTING YOUR ASSEMBLY HANDLE NAMES

Starting in the Project Window, click on the Assembly Parameters button. At the bottom of
the Assembly Parameters dialog, click on the Name Settings… button. The Assemble by
Name Settings dialog appears as in Figure 10-4.
You can describe up to eight Assembly Handles of which only one is active at any one time.
For each Assembly Handle, you can use the default name Sequencher automatically gives
your contigs or you can type a descriptive title into the input field. Click on the radio button to
the left of the handle you wish to activate. Sequencher displays the number and the name of
the Active Handle at the bottom of this dialog. Click on the OK button to dismiss this dialog
and return to the Assembly Parameters dialog.

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 Figure 10-4 The Assemble by Name Setting window

CHOOSING A NEW ASSEMBLY HANDLE

There are two ways to choose an Assembly Handle. If you are in the Assembly Parameters
dialog, you can change the handle by choosing a new one from the drop-down menu.
Note: The probable number of contigs that can be formed using this handle is indicated in
square brackets to the right of the handle.
Figure 10-5 Assembly Handles drop-down menu

You can also click on the Name Settings… button and then click on the radio button next to
your preferred handle.

ENABLING ASSEMBLE BY NAME

You enable Assemble by Name from the Project Window by clicking on the Assembly
Modes drop-down menu in the button bar and choosing Assemble by Name. If you are in the
List View, you will notice a new column titled Handle, which lists the active handle for each
sequence. You will also see that the text of the assembly command buttons has changed to
Auto Assemble by Name and To Reference by Name. The new assembly status is also
reflected in the current parameters above the column headers.

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 Figure 10-6 The Project Window with Assemble by Name
enabled

SETTING ASSEMBLY PARAMETERS

Sequencher uses the values set in the Assembly Parameters dialog to control the way it
assembles sequences. Once you have set your required options, you can store these as your
default Assembly Parameters. Click on the Set as Defaults button at the bottom of the dialog
and Sequencher will use your preferred Assembly Parameters whenever it launches. If you
just click OK to dismiss the Assembly Parameters dialog, Sequencher will use your new
parameters only with this project or session of Sequencher.

PERFORMING AN ASSEMBLY WITH ASSEMBLE BY NAME

Once you have chosen your Name Delimiters and Assembly Handles, you are nearly ready to
perform your assembly. In addition to the standard Auto Assemble by Name command, you
can use the Assemble by Name function to assemble your sequences with a Reference
Sequence or to Mindlessly Join your sequences.
Under the Assemble menu, the following menu items will change if you have enabled
Assemble by Name. The Mindlessly Join command becomes Join by Name…. Similarly, the
Assemble to Reference button will now be labeled To Reference by Name.
Before you use the To Reference by Name command, you will need to define a Reference
Sequence. You will also need to choose and set your assembly algorithm and any associated
parameters. (For more details, see Chapter 9 “Sequence assembly”.)
Starting from the Project Window, go to the Select menu and click on the Select All command
to select all the sequences in your project. Click on the Auto Assemble by Name button, or
the To Reference by Name button if you have a Reference Sequence. The Assembly Preview
window will appear. This window contains the names of the contigs that may be generated
using your chosen handle and the probable number of sequences in each contig. Click on the
Assemble button to continue with the assembly, or click on the Cancel button. Sequencher
displays the Assembly Completed dialog when the assembly has finished assembling your
sequences.

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 Figure 10-7 The Assembly Completed dialog

This dialog gives a summary of the assembly process. If this information is sufficient, click the
Close button. This will dismiss the Assembly Completed window. If you require more detailed
information, click on the Details… button. You will see a report that contains further
information about the assembly. You can save a text copy of this report by clicking on the Save
As button. Dismiss the dialog by clicking on the Close button.

Note: The name of each contig formed using Assemble by Name will be based on the
Assembly Handle used.

ALIGNING SEQUENCES WITH CLUSTAL

Unlike the algorithms Clean Data, Dirty Data, and Large Gap, Clustal is not built into
Sequencher but is an external tool. More information on adding Clustal to Sequencher can be
found in the DNA-Seq Tools Installation pdf on the Gene Codes website Support page.
Figure 10-8 Clustal Options dialog

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USING CLUSTAL TO ALIGN SEQUENCES

Select the sequences you wish to align from the Project Window. Then go to the Assemble
menu, select Align Using, and click on Clustal from the submenu.
Click on either the Wilbur & Lipman radio button or the dynamic programming radio button.
Now set any other parameters by changing the values in the input fields or choosing from the
appropriate drop-down menu. At the bottom of the dialog, you will see a series of values and
parameter names, these are the settings that will be sent to Clustal. If you are not sure about
the meaning or effect of any parameter, then use the default value.
To start the alignment, click on the OK button. A new window appears which asks you to be
patient while the alignment takes place. The results will appear as a single contig in your
Project Window.

USING CLUSTAL WITH ASSEMBLE BY NAME

You can also use Clustal with Assemble by Name. Typically you would use Assemble by Name
when you have multiple sequences from different sources, taking this approach will save you
time but works best when you have a well standardised naming scheme for your sequences.
Click in the Assembly Mode drop-down menu on the button bar and choose Assemble by
Name. Then click on the Assembly Parameters button on the button bar at the top of the
Project Window and select the Name Settings… button.

Figure 10-9 Assemble by Name and Name Settings…

Next you will need to set your Assemble by Name parameters by choosing the Name Delimiter
from the drop-down menu and the Assembly Handle by clicking on the appropriate radio
button that represents the portion of the sequence name to be used for assembling the
contigs.

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 Figure 10-10 Assembly Handles with Name Delimiter chosen

Once these have been set and the Assemble by Name function has been enabled, you can
then proceed with the alignment.
Select the sequences you wish to align from the Project Window. Then go to the Assemble
menu, select Align by Name Using, and click on Clustal from the submenu. A new window
appears which asks you to be patient while the alignment takes place. When the alignment is
completed, you will see the Alignment Completed dialog. Click on the OK button to dismiss
this window.
You will see new contigs appear in your Project Window which you can analyse with
Sequencher’s other tools such as the Variance Table.

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 11. THE CONTIG EDITOR

In this chapter, you will learn about the overview and bases view of the Contig Editor. You will
learn how to use these options to explore your contig. We will discuss the steps to take before
you start editing: base numbering, setting a consensus calculation, and assigning a Reference
Sequence.

CONTIG EDITING

DNA sequencing is an error-prone process, and so the base calls from an automated DNA
sequencer may not correctly represent the sample being sequenced. This means that when
working with automated DNA sequencing data, it is usually necessary to check and edit your
contigs.
Sequencher provides a powerful interface between the user and the data so that you can
analyze and edit sequences and contigs according to your own specifications. You can move
directly from one ambiguity or disagreement to the next to analyze and override base calling
errors, eliminating discrepancies and ambiguities. Sequencher allows you to choose from a
number of consensus calculations and continually recalculates the consensus of the contig as
you edit the sequences.

OVERVIEW OF THE CONTIG

THE CONTIG OVERVIEW WINDOW

When you first open a contig by double-clicking on it, you will see an overview that shows how
the sequences have been assembled (Figure 11-1). There is also a button bar at the top of the
Overview window.
The schematic provides you with a wealth of useful information enabling you to assess and
manage your data. A horizontal line represents each sequence in the contig. The line will be
green and solid if the sequence is in the forward orientation and red and dotted if it is in the
reverse orientation. These orientations may change as more data is added to the contig, unless
you have used a Reference Sequence in your assembly. In this case, the Reference Sequence
determines the contig orientation. Below the sequences, you will see the coverage map and
below the coverage map you will see the start and stop map. Notice the diagram key at the
base of the window.
If your sequence(s) had a GenBank style feature table, then the features would automatically
be applied on import into Sequencher. You would see these features marked in the Overview.

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 Figure 11-1 Contig Overview

SELECTION MARQUEE

In the Overview, you will notice a dashed rectangle. This animated marquee (sometimes called
the marching ants) highlights the section of the contig displayed when you switch to the Bases
View. You can change the location you want to view by dragging the marquee to a new
location. As you move the cursor over the marquee, you will see it change to a hand icon. You
can now hold down the mouse button and drag the marquee to the desired location.

CHECKING COVERAGE IN THE OVERVIEW

The bar across the bottom of the Overview shows various levels of sequence coverage
indicated by different graphic patterns. For instance, a wide solid green line indicates that the
sequence coverage comprises both strands. A region with no color or pattern indicates a hole
in the coverage. The full array of patterns indicating coverage are shown at the bottom of the
Overview in the figure above.
In the example in the figure above, the Overview shows the contig starting out with a single,
unsupported sequence. Then a second sequence starts to overlap with the first. Next there is
an area with "full" coverage: both strands are sequenced with additional confirming
sequences. At the extreme right end of the contig, there are just two sequences in opposite
directions.

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OVERVIEW FEATURES

BASES

To get from the Overview to the view used for editing, click on the Bases button. You can also
go to the View menu and choose Bases from the Bases, Map, Overview, … submenu.
Sequencher displays the Contig Editing window.

SUMMARY

You can press the Summary button to view a compact report of the contig. To see this report,
go to the View menu, choose the Bases, Map, Overview, … submenu, and click on the
Summary Report option. The features of the Summary Report will be dealt with in more
detail later (see Chapter 12 “Editing Contigs” for more information).

SORT

You can sort the sequences in a contig vertically. Click on the Sort button to display a dialog of
options. Choose an option by clicking on its radio button and then pressing OK. You may also
use the sorting options in the View menu under the Sort/Cleanup submenu.

OVERVIEW OPTIONS

To choose the display options, click on the Options button or go to the View menu and click
on View Options. An Overview Options dialog will appear where you can set your choices.

Figure 11-2 Contig Overview options dialog

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SCALE DIAGRAM TO WINDOW SIZE

Selecting Scale Diagram To Window Size shrinks the whole overview to fit in the window,
even if the names of the individual sequences become unreadable. When you turn this option
off, you make the names of the sequences readable, but you may have to scroll the window to
see all the sequences in the diagram.

DIAGRAM KEY

Clicking this checkbox displays or hides a key that explains the Overview.

LABELS OF FRAGMENTS

You must have already defined and applied some labels to your sequences with User
Preferences (see Chapter 23, “Customizing Sequencher and User Preferences”) and the Label
command. Clicking Labels of Fragments will display or hide any fragment label names.

NAMES OF FRAGMENTS

Clicking this checkbox displays or hides the sequence names in the Overview. You may have
this and the previous item checked at the same time.

NAMES OF FRAGMENTS, AT LEFT

Clicking the At Left box in conjunction with Names of Fragments lists the fragment names
down the left side of the window instead of on top of each arrow (as shown in Figure 11-3). If,
for example, the sequences are also sorted by position, a faint blue line will connect the name
with its fragment arrow.

Figure 11-3 Names Of Fragments At Left

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POSITIONS OF FRAGMENTS

Clicking Positions of Fragments displays the numerical range positions of the sequences in
the Overview. If you do not wish to see this information, leave the box unchecked.

START & STOP CODONS

You can display a start and stop Codon Map showing all the start and stop codons in the three
reading frames of one strand. Start codons are indicated by green flags and stop codons are
denoted by vertical red lines. To display this map, check the Start & Stop Codons box. The
map will be positioned at the bottom in the Overview.
In Figure 11-4, the three-frame map of start and stop codons is located just above the diagram
key.
To see start and stop codons in the other strand, go the View menu and choose Reverse &
Comp. This will reverse and complement your sequences. Choosing Reverse & Comp again
will restore your contig’s original orientation.

Figure 11-4 Start and Stop Codon Map

BASE NUMBERS AT TRANSITIONS

Clicking Base Numbers at Transitions displays or hides the numbers showing approximate
positions of the coverage transitions.

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BASE NUMBERS AT EVERY X BASES

Selecting Base Numbers Every x Bases shows the base numbers at constant intervals.

CONDENSED BASE NUMBERS

Selecting the Condensed Base Numbers option changes the font of the base numbers to a
compressed version. This option can be used with either Base Numbers at Transitions or Base
Numbers at Every x Bases.

CONTIG COVERAGE GRAPH

As well as the standard Overview you can see a representation of your contig in graph form.
This is especially useful when you are dealing with large numbers of sequences. To see the
Coverage Graph, go to the View menu and choose Bases, Map, Overview, … and select the
Coverage Graph command.

GRAPHIC FEATURE MAPS

When you annotate your sequence with features or if you have imported a sequence
containing a GenBank Feature Table, Sequencher will present a graphical representation of the
features under the display of overviews and restriction maps.
The graphical feature map has two parts. Above the sequences (red/green lines) the features
are presented on one line. Below the coverage map the feature may be presented over several
lines. You can see this at the five prime end of the sequence in the image below, where there
are three features in blue, red and green which overlap. In the bottom map the features are
displayed on separate lines. If you allow the mouse cursor to stay over one of these features a
tool tip appears, containing information about the feature.

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 Figure 11-5 Graphic Feature Maps on Overview

GETTING MORE INFORMATION

FIND

You can search for a specific subsequence in your contig. Fragments, which contain the
subsequence, will appear with a heavier line in the Overview.
You can choose to find the first occurrence by clicking on the Find button. You can search for
subsequent occurrences using the Find Next button. Three radio buttons allow you to control
the specificity of the search by choosing Exact Matches, Specified Bases (which would allow
you to use ambiguity codes such as W to find T or A), or Any Ambiguous Base.
Use the Any Ambiguous Base command to find any degenerate DNA that may match. There is
also a drop-down menu from which you may choose restriction enzyme sequences.
If you have used a Reference Sequence and want to include it in the search, select the Include
Reference Sequence in Find checkbox. Close the dialog by pressing Done.

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GET INFO

Summary information about the open contig can be obtained by pressing the Get Info button
or go to the File menu and click on the Get Info command.

THE BASES VIEW

WORKING IN THE BASES VIEW

To get from the Overview to the view used for editing a contig, click on the Bases button.
Alternatively, you can go to the View menu and then go to the Bases, Map, Overview…
submenu and choose Bases. Sequencher displays the Contig Editing window shown in Figure
11-6.

Figure 11-6 The Contig Editing window

To return to the Overview, click on the Overview button or go to the Bases, Map, Overview…
submenu and choose Overview.
The labels in Figure 11-7 identify the different parts of the Contig Editor.

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 Figure 11-7 Identifying parts of the contig editor

Table 11-1 Parts of the Contig Editor

Label Name Function

A Button Bar Contains several buttons you click to perform frequently-

used functions.

B Sequence list Shows which sequences make up the contig you are
editing.

C Agent Describes current selection and the current status of the

spacebar shortcut.

D Splitter bar Controls the width of list/message area and the editing
area; dragging the splitter bar to the left or right changes
the size of these items.

E Sequence Displays the sequence bases aligned.

Bases

F Sequence Scrolls up and down through the sequence bases in the

Scroll contig.

G Base Numbers Shows which bases you are viewing, numbered from the
beginning of the contig.

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 Label Name Function

H Consensus line Shows the calculated consensus of the sequences that

were aligned to form this contig.

I Ambiguities Displays a plus (+) below any ambiguous base calls in the
contig line and a bullet (•) below any base calls with
disagreeing bases above them. This area also displays
protein translations with a bullet (•).

J Base Scroll Scroll left and right through the bases of the contig.

K Translation This button toggles between the display of ambiguities

Button and protein translations.

L Notation Line Displays translation of Reference Sequence and user text

notes

VIEWING SEQUENCE NAMES

If the names of your sequences are very long, you may want to move the splitter (item D in the
key) in the Contig Editor dialog.

BEFORE YOU START TO EDIT

SETTING THE BASE NUMBERING

You have control over the base numbering of the consensus. For instance, you can select any
base in the consensus and make it the “origin” base (base #1). All the bases before that
position will have negative numbers. To set the numbering, select a base, then go to the
Sequence menu and choose Set Base Number with an appropriate suboption.
Note: If you have already used a Reference Sequence in your assembly, this will set the base
numbering.

CONFIDENCE HISTOGRAMS

Many Sequencher users have data which contains confidence scores. Phred scores have a
range with a maximum of 60, other systems have a dynamic range which goes up to 100. To
see the histograms which are displayed above the bases in the Contig Editor, choose one of

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the following from the View menu. For Phred scores, choose Confidence Histogram (max 60).
For other systems, choose Confidence Histogram (max 100).

Figure 11-8 Confidence histograms in the Contig Editor

CONSENSUS CALCULATION

Under the Contig menu, there are four choices for calculating the consensus of your
sequences: Consensus Inclusively, Consensus by Plurality, Consensus to Forensic
Standards, and Consensus by Confidence. The consensus is displayed in the bottom line of the
Contig Editor and is continuously updated as you edit. Thus, the consensus will alter after any
change you make, whether that is altering bases or adding new sequences to your contig.
Bullets under the consensus highlight discrepancies and pluses indicate ambiguities.
If you choose a consensus calculation for a project and then save and close the project, the
consensus choice will be set as the default calculation for that project.

CONSENSUS INCLUSIVELY

With Consensus Inclusively, Sequencher determines the base in the consensus line by the
smallest category of ambiguity that covers all available data. In the example in Figure 11-9, a
column with one sequence containing a G and the other containing a C is shown in the
consensus line as an S (the IUPAC code for C or G). This consensus is particularly useful in any
work involving mutations.

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 Figure 11-9 Consensus inclusively example

CONSENSUS BY PLURALITY

Consensus By Plurality is Sequencher’s default setting. With this consensus calculation,

Sequencher determines the consensus line bases by majority rule. If there is no clear majority,
Sequencher chooses the smallest category that fits the available data.
In the example in Figure 11-10 below, a column with two sequences that contains a T and an A
is still shown in the consensus line as a W, because there is no clear majority. However, a
column with three sequences that contains a gap in one sequence is shown in the consensus
line as an ambiguity code in the consensus line only if the other two sequences do not agree.

Figure 11-10 Consensus by plurality example

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CONSENSUS TO FORENSIC STANDARDS

In certain applications, such as forensic mtDNA sequencing, there is an established standard

reference for human identification. This calculation is based on the coverage of sequences at a
given position and taken from work at the U.S. Armed Forces DNA Identification Laboratory
(AFDIL) in Rockville, Maryland.
All positions with a coverage of only one sequence are marked as N (ambiguous).

• All IUPAC codes apart from ACG and T are treated as N.

• If any positions disagree where the coverage is between two and four, this position
is marked as N. If the coverage is between five and seven, any position with a
disagreement will be marked as ambiguous.

• If there are two or more disagreeing bases at a position, this will be called an N
regardless of the coverage.

Figure 11-11 Consensus to Forensic Standards example

CONSENSUS BY CONFIDENCE

While Consensus by Plurality and Inclusively are good approximate calculations for creating a
snapshot of the consensus, they do not contain any information about the quality of the bases
chosen. With Consensus by Confidence, you can take advantage of the confidence scores
which your sequencer provides and which are imported with your data. With this method,
Sequencher looks at each column of data and calculates the consensus using the underlying
confidence data.
Additionally, if you have enabled Display Base Confidences from the View menu, you will see
light, medium, or dark blue backgrounds behind the reads and the consensus line.

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 Figure 11-12 Consensus by Confidence example

ASSIGNING A REFERENCE SEQUENCE

To designate a sequence as a Reference Sequence, select the sequence icon in the Project
Window and check the Reference Sequence item in the Sequence menu. From now on, that
sequence icon will include a small letter "R" to remind you that it has been marked as a
Reference Sequence (see Chapter 7 “The Reference Sequence” for more information).

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 12. EDITING CONTIGS

In this chapter, we explain how to find bases such as ambiguities and low-confidence bases.
We discuss how to perform your edits, move and delete bases and sequences, insert gaps or
bases, and create a new sequence from a consensus. You will learn how to compare sequences
to highlight differences and how to create customized reports.

FINDING BASES WHICH NEED ATTENTION

You are likely to see a number of issues in your data. Some will need to be examined and
perhaps edited. These issues might be indicated as an N, as a disagreement, as a gap, or as
having a low confidence rating. You can use one of the Next commands from the Select menu
in the consensus sequence of a contig to find any of these problem areas.
Once you have used any of the Next commands, Sequencher will activate the space bar to
execute that operation. If you use either the menu command or the shortcut key combination
two consecutive times, Sequencher will remind you that this easier alternative is available.

VIEWING AN INDIVIDUAL SEQUENCE FROM THE CONTIG

To look at the data for one of the sequences in your contig, double-click the sample name in
the sequence list. Sequencher opens a locked sequence viewer containing the data for that
sequence.
You can also select a sequence in the Contig Editor and then go to the File menu and choose
Open Window.

FINDING DISAGREEING AMBIGUITIES

You can find ambiguities quickly by typing Ctrl+N (Windows) or Cmd+N (Mac) or by clicking in
the consensus line and going to the Select menu and choosing Next Ambiguous Base. This
will find data that is indicated as an N or as a disagreement or gap. The Next Ambiguous Base
command will also find positions in the contig where the only contribution to the consensus is
from a base with a low confidence score. This type of base will be marked with a + (plus) or a •
(bullet) below the consensus line.
Each time you press Ctrl+N (Windows) or Cmd+N (Mac), Sequencher moves the cursor to the
next ambiguity in the consensus, starting after the currently selected character. After
establishing this function, you can move to the next ambiguous base by hitting the spacebar or
going to the Select menu and using the Repeat Select command. If you use the Shift key in
conjunction with a Next Ambiguous Base key combination, the cursor will move in the
reverse direction.

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FINDING DISAGREEING BASES

You can find disagreeing bases quickly by typing Ctrl+D (Windows) or Cmd+D (Mac) or clicking
in the contig line, going to the Select menu, and then choosing Next Contig Disagree. This
type of base will be marked with a • (bullet) below the consensus line.
Sequencher moves the cursor to the next disagreement in the consensus, starting after the
currently selected character and ignoring ambiguities.

FINDING LOW CONFIDENCE BASES

You can quickly locate low confidence bases by typing Ctrl+L (Windows) or Cmd+L (Mac) or by
going to the Select menu and choosing Select Next Low Confidence Base. You can set the
ranges for Low, Medium, and High confidence in the User Preferences (see Chapter 23
“Customizing Sequencher and User Preferences”).
Note: You can use the Next Low Confidence Base command to help direct your editing. Under
the Window menu, click on User Preferences…, select the Confidence option under General
settings , and set the Low Confidence range appropriately for the value range you wish to find.
Go to the Select menu and click on Next Low Confidence Base to move your cursor directly
to the bases that require attention.

FINDING EDITED BASES

You can quickly review edits already made to a contig by typing Ctrl+E (Windows) or Cmd+E
(Mac) or by going to the Select menu and choosing Next Edited Base.
Note: This function will not find deletions.

PERFORMING EDITS

THE EDIT COMMAND

To edit a base, first set the Edit mode you wish to use, then select the base by clicking on it
(see Figure 12-1). Now type the new character over the old. If you accidentally type the wrong
character, go to the Edit menu and choose Undo or just type over the mistake. Sequencher
will recalculate the consensus line automatically. If you resolve an ambiguity at a particular
position, the ambiguity marker below the consensus line disappears (see Figure 12-1).

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 Figure 12-1 A base is selected in the contig editor

EDITING MODES

The Edit command allows you to replace/overstrike, insert, or erase a base call. The specific
option you choose from the Edit menu will create that behavior. When you use Replace When
Editing, the base you have just edited will remain highlighted. When you use Overstrike
When Editing, the cursor will move one base to the right and this base will then be
highlighted. Insert When Editing allows you to insert bases before the currently selected base,
pushing all of the following bases forward. This also allows you to add bases beyond the end of
a sequence. Sequencher uses Replace When Editing as its default setting.
Note: If you insert or delete a base from any one sequence, the sequence may no longer align
with other sequences after the point where you made your change. The consensus line will
reflect this by showing lots of ambiguities in the form of bullets •. If you have made only one
change, go to the Edit menu and the Undo command will remove it.

VIEWING BASE EDITS

To assess the quality of your data, you need to be able to see how much modification has
already been done to your contig as you edit it.
When a base is edited, Sequencher changes its appearance by displaying it in magenta and
bold-faced text. If you want edits to stand out even more (for instance in a black and white
printout), you can also invert the case of the characters (e.g. change all edits from uppercase
to lowercase text).
To see the options for displaying edits, go to the View menu and click on the Base Edits As…
submenu to choose from Not Highlighted, Bold Magenta, or bOLD and cASE cHANGE.

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COLLECT GAPS

The Collect Gaps command allows you to select a range in a sequence or contig consensus and
move all of the gaps within this selection to either the 5’ or the 3’ end of the selection.
If you wish to collect the gaps within a single sequence, you must select the range within that
sequence. Go to the Sequence menu and choose the Collect Gaps command. To move the
gaps to the 5’ end, select Left from the submenu. To move gaps to the 3’ end of the selection,
choose Right from the submenu.
If you wish to collect the gaps within a region of a single contig you must select the range from
the consensus line. Go to the Sequence menu and choose the Collect Gaps command. To
move the gaps to the 5’ end, select Left from the submenu. To move gaps to the 3’ end of the
selection, choose Right from the submenu.
Note: The Collect Gaps command may have the effect of increasing the number of
ambiguities. Check the consensus line to see if the number of ambiguities has increased or
decreased. You can go to the Edit menu and use the Undo command to reverse the Collect
Gaps command.

MOVING BASES AND GAPS

You can move a selection within a single sequence using the lasso tool. The selection can be a
single base or gap or a number of bases or gaps.
Begin by making your selection by dragging the cursor across the bases or gaps you wish to
move. The region will be highlighted. Then hold down the Alt key and your cursor will turn into
a little “lasso” tool. Hold down the Alt key and click on the highlighted selection you want to
move. Then drag it to its new location. The selection will change to Bold Magenta text in its
new position.
Note If you have used the Large Gap algorithm to assemble sequences you can double click
anywhere within the gap region to select it.

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 Figure 12-2 The lasso tool around a base to be moved

USING REALIGNER TO CLEAN UP CONTIGS

The ReAligner button on the Contig Editor button bar lets you apply the ReAligner algorithm
to optimize gap placement in your sequences without first dissolving your contig. This is
especially useful when you are left with gaps and unaligned bases after removing one or more
sequences from a contig.
Click on the ReAligner button. Once the ReAlignment has finished, a new window called
ReAlignment Complete will appear. This window displays a summary of the disagreements,
gaps, and ambiguities before and after the ReAlignment.
Note: ReAlignment is not the same as reassembly. If you wish to reassemble your sequences,
you must first dissolve your contig. (See Chapter 9 “Sequence Assembly” for more
information.)

MOVING SEQUENCES

If you hold down the Ctrl (Windows) or Cmd (Mac) key, the “grabber” tool is invoked. It
resembles a small hand. You can use the grabber tool to drag an entire sequence to the left or
right. This method of moving entire sequences also works in the Overview. The grabber tool is
shown in Figure 12-3. You may notice that some or all of your sequence no longer aligns and
that a large number of bullets (•) appear in the consensus line. If you decide that the new
position is not correct, you can use the Undo command to restore it to its previous place.

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 Figure 12-3 Grabber tool

Note: Moving sequences in a low coverage region could leave a hole in the middle of the
contig. This is not recommended since the hole may cause the contig to align inappropriately
with other data. Sequencher will warn you if this about to happen.

DELETING BASES

You can delete bases in the middle of a sequence by simply highlighting them and then using
the Delete key. Sequencher asks if you want to fill the gap with bases moved from either the
left or the right of the void you have created. Click on either Yes, Fill Void From Left, or No,
Fill Void From Right button. (Figure 12-4) Sequencher then moves the rest of the bases
accordingly. Click on Cancel if you do not want to delete anything. You can also perform the
deletion by going to the Sequence menu, using the Delete Bases command, and then
choosing either Fill Void From Left or Fill Void From Right from the submenu.

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 Figure 12-4 Fill Void dialog

DELETING BASES FROM THE 5’ OR 3’ ENDS

Although you may have automatically trimmed your sequences for poor quality data when you
imported them (see Chapter 6 “Preparing your data for assembly”), you may still need to
delete a few 5’ bases to correct any mistakes made during the amplification process.
Select the bases you want to delete. If you are at the 5' (left end), then click the Forward
Delete key. Sequencher will delete those bases without moving the entire sequence to the
left. Similarly, if you choose bases on the 3‘ (right) end, use the Backspace key for Windows or
the Delete key on Mac and Sequencher will automatically delete those bases without
realigning your sequence.
Note: When editing from the consensus line, you do not have to worry about the bases shifting
to the left and right.

INSERTING GAPS OR BASES INTO A CONTIG

If you want to insert gaps, go to the Sequence menu and click on Insert Gaps & Move Bases,
then choose Right or Left from the submenu. You can also create gaps by selecting a number
of bases equivalent to the gap size you want to create. Press the Tab key if you want the
selected bases to move to the right. To move to the left, hold down the Alt (Windows) or
Option (Mac) key when you press Tab. The space created will be filled with gap marks (small
colon marks) that will appear without disrupting the rest of your contig.
Once your gaps are in place, you can either type the bases you want into the gap using
Overstrike When Editing mode or use the lasso and grabber tools to move existing bases into
the new position.

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MAKING EDITS FROM THE CONSENSUS LINE

You can simultaneously edit all the bases in the column above the position you choose in the
consensus line, instead of individually changing each one. To do this, select a base in the
consensus line and enter the character that represents the edit you want to make in that
column. If you currently have a base selected in a sequence, go to the Select menu and choose
Contig Column (Ctrl+K (Windows) or Cmd+K (Mac)).
Any bases in the sequences above the contig column you select that do not match the
character you type will be changed to match the character you type in the consensus line. Your
edits will appear according to the preferences you set in the View menu. Generally, this will be
magenta and bold-text.
You can also delete bases from the consensus line. You cannot use the Undo command to
reverse this action if your contig contains a Reference Sequence.
Note: Remember that changing bases from the consensus line edits only the sequences, after
which the consensus is recalculated and displayed. Your action does not change the consensus
directly. Changes in the consensus result from matching sequences with each other and
evaluating them according to the type of consensus calculation you have chosen (e.g. by
plurality, inclusively, forensic, confidence).
Further note that this type of editing does not change Reference Sequences.

Figure 12-5 The base is changed

SPLIT AFTER SELECTION…

You can split a sequence into two pieces. Put the cursor where you want to split the sequence.
Go to the Sequence menu and choose Split After Selection….
Sequencher will ask you if you want to split the sequence after the last currently selected
base. Click on the Split button to proceed. Sequencher will then split the sequence in two and
label the new sequences for you.

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You can also apply the Split After Selection… command to an entire contig by simply making
your selection in the consensus sequence, and then choosing Split After Selection… from the
Sequence menu.

AUTO MATCH

Sometimes you may wish to edit a number of sequences simultaneously, perhaps to match a
high quality segment of sequence. Sequencher allows you to perform this type of mass edit on
a range of bases using the Auto Match command.
First select the base or range of bases to which the other sequences should match, then go to
the Edit menu and choose Auto Match. This command will edit (and highlight) every
overlapping base so that it will agree with the selected sequence. To reverse this procedure, go
to the Edit menu and click on Undo.

CREATE NEW SEQUENCE FROM A CONSENSUS

You can create a new sequence from the consensus sequence by going to the Contig menu
and choosing Create New Seq From Consensus... In addition to creating a new sequence, you
can use this feature to track the changes made to your contig over time.
Note: If you have used the Consensus by Confidence calculation, then the newly created
consensus will also contain the confidence scores calculated by this consensus method.
Sequencher displays a dialog that tells you that you are about to create a snapshot consensus
of the selected contig. This dialog allows you to control some basic features of the new
sequence. Figure 12-6 shows the options dialog for this command.

Figure 12-6 Create New Sequence From Consensus options dialog

Check or uncheck the boxes to indicate how you want the new sequence to appear. If the
default setting Remove Gaps is checked, then Sequencher will remove all gaps (large and
small) when it creates the new sequence.

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If you want to keep large gaps independently from other gaps, then select the checkbox called
Retain Large Gaps. In Sequencher, a large gap consists of 10 or more consecutive bases. Such
a gap might appear if, for example, you have used the Large Gap algorithm to assemble
genomic and cDNA. The Retain Large Gaps option is not available if Remove Gaps is checked.
Note: To see the new sequence, go to the Project Window. Remember this is a snapshot of the
consensus at the time of its creation. Future edits will not be marked.

REMOVING SEQUENCES FROM A CONTIG

To remove sequences from a contig, click on the name of the sequence you wish to remove
from the list at the left side of the Contig Editor window or in the Overview window. To
remove more than one sequence at a time, hold down the Shift key while you click the names.
Next, go to the Contig menu and choose Remove Selected Sequences… Sequencher will ask
you if you are sure you wish to remove the sequence(s). Click on the Remove Fragments
button to delete the sequences from the contig. The sequences you just removed from the
contig are moved out of the contig and back into the Project Window. They will also stay
selected.
Note: This action may break a contig into multiple pieces. If this is about to happen,
Sequencher will display a dialog (Figure 12-7) to ask if this is really what you intend to do. Press
the Cancel button if you do not wish to proceed. Any new contigs generated in this way are
named by appending an alphabetic character to the name of the original contig.

Figure 12-7 Warning dialog when about to make a hole in a contig

DISSOLVING A CONTIG

To dissolve a contig, first select the contig’s icon from the project. Go to the Contig menu and
choose Dissolve Contig…. You can also perform this command while you are in the Contig
Editor.
Sequences from the dissolved contig are shown highlighted in the project. It is important to
remember that all edits made to a sequence while in the contig will be retained. If you want to
restore the original sequences, you must go to the Sequence menu and click on Revert to
Experimental Data.

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 13. THE VARIANCE TABLE

In this chapter, we explain how to use the Variance Table, an interactive display of the
differences among your sequences or contigs. We go on to discuss how you find differences
and work with your trace and confidence data. The Variance Table is useful for SNP analysis,
mutation detection, and clone checking.

THE VARIANCE TABLE STRATEGY

WHAT IS A VARIANCE TABLE

In its simplest form, a Variance Table compares and displays the differences between two
sequences. The data in the Variance Table are dynamically linked to the data in the underlying
contigs.
You can compare some or all of the sequences in a contig to the Consensus sequence, the
Reference Sequence, or the Top Sequence of that contig (see Figure 13-1 below). This
generates a Variance Table which will display the differences between the exemplar sequence
and your chosen sequence(s). The results may be from just one contig, but can represent an
assembly of hundreds or even thousands of sequences. If you are performing activities such as
de novo sequencing, clone checking, or resequencing, you may want to use this form of the
Variance Table.

THE VARIANCE TABLE AND CONSENSUS SEQUENCES

You can also create a Variance Table that summarizes all of the differences between selected
contig consensus sequences (see Figure 13-1 below) in your project and a common Reference
Sequence (consensus Variance Table). When you are working with multiple samples from
multiple sources and have used Assemble by Name with a Reference Sequence, you should
use this Variance Table. If you are performing activities such as comparative sequencing, clone
checking, or SNP analysis, you probably want to use this form of the Variance Table.

THE TRANSLATED VARIANCE TABLE

If you are interested in focusing on the differences between sequences or contigs at the amino
acid level, you should use the Translated Variance Table. This table differs from the Variance
Table by displaying both codons and their associated amino acid residues. Each row in the
Translated Variance Table summarizes the variations among all of the selected sequence
translations at a given amino acid position relative to the exemplar.
You can also create a Translated Variance Table that summarizes the differences between the
translations of selected contig consensus sequences in your project and the translation of a
common Reference Sequence (Translated consensus Variance Table). When you are working
with multiple samples from multiple sources, and have used Assemble by Name with a
Reference Sequence, you should use this form of the Translated Variance Table.

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 Figure 13-1 Variance Table menu commands

THE STRUCTURE OF THE VARIANCE TABLE

Each row in a Variance Table summarizes the variation among all of the selected sequences at
a given base position relative to the exemplar (see Figure 13-2). The exemplar can be a
Reference Sequence, a consensus sequence, or any other sequence you choose. In the
consensus Variance Table, the exemplar is limited to the Reference Sequence.
The range of bases included in a Variance Table is called the Comparison Range. The length of
the exemplar determines the default Comparison Range and sets the base numbering.
The extent to which the exemplar matches any sample sequence is called the Coverage Range.
The Coverage Range can be complete (full range) or incomplete. If the range is full, the entire
length of the sample sequence has been compared to the entire length of the exemplar
sequence.
If the range is listed as incomplete, the display of the sample sequence comparison runs along
only part of the exemplar sequence. If a sequence extends the full length of the Comparison
Range, its column header will be shaded in gray. If a sequence in the table does not extend the
full length of the Comparison Range, its column header will be shaded in pink.
The first column of the Variance Table displays the position and the second column displays
the base call of the exemplar sequence, but only at positions that differ. The additional
columns in the Variance Table are created by the sequences or contigs you select for
comparison.

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You can view or edit the data in any version of the Variance Table by double clicking on any of
the cells in that table. In Review mode, when you type a new base in the Variance Table, you
edit the underlying sequence or contig automatically.

Figure 13-2 Components of Variance Table

STRUCTURE OF THE TRANSLATED VARIANCE TABLE

The structure of the Translated Variance Table follows the format of the Variance Table but
its display consists of an amino acid and its codon in each cell (see Figure 13-3).
When you see a cell in the table with an amino acid and its codon written in black typeface,
this indicates a difference between your exemplar and your sample. You may also see some
cells in the table where the amino acid and codon are displayed in light gray. Amino acids in
these cells match the exemplar but use either an identical or synonymous codon.
The numbers that appear in the first column of the Translated Variance Table refer to the
position of the first base of the codon (in the upper location) and the position of the amino
acid (in the lower one).

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 Figure 13-3 The Translated Variance Table

Note: When Sequencher encounters a gap, it skips to the next available base to include it as a
component of the codon. If an N is in the first or second position of the codon, Sequencher
displays a question mark (?) instead of an amino acid. If the N is in the third position of the
codon, a residue may be displayed. This will depend on the genetic code redundancy.

CREATING A VARIANCE TABLE

VARIANCE TABLE FOR SEQUENCES IN THE SAME CONTIG

When you are ready to begin creating your Variance Table, either select individual sequences
from within a contig or go to the Project Window and select a contig by clicking on its icon.
This will select all of the contig’s sequences.
Then go to the Sequence menu and choose Compare Bases To and either Consensus,
Reference Sequence, or Top Sequence from the submenu. The Variance Table appears in a
new window (Figure 13-4).

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 Figure 13-4 Variance Table Created with Compare Bases To Reference Sequence

THE CONSENSUS VARIANCE TABLE

When you want to compare a consensus sequence to a Reference Sequence, you use the
Contig menu’s Compare Consensus to Reference command.
You can also use the Compare Consensus to Reference command to compare the consensus
sequences from multiple contigs that share the same Reference Sequence. These contigs are
the result of using the Assemble By Name command with a Reference Sequence.
For each of these operations, go the Project Window and select one or more contigs that
share a common Reference Sequence. Then, from the Contig menu, choose the Compare
Consensus to Reference command. The Variance Table appears in a new window (see Figure
13-5).
Each cell in the resulting Variance Table will display a consensus base when that base differs
from the Reference Sequence. Pink shading in the header and footer cells of the Variance
Table highlight contigs where the consensus sequence does not cover the span of the
Reference Sequence. The pink X’s mark specific cells where coverage is missing (see Figure
13-5). It is possible for a sequence to have partial coverage, a pink header and footer, but not
have any pink X’s in the column.
Note: When comparing multiple consensus sequences, the View menu’s Display Base
Confidences option is not relevant, because confidence values exist only in sequences and not
in consensus sequences.

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 Figure 13-5 Compare Consensus to Reference Variance Table

For more information about Assemble by Name and Reference Sequences, see Chapter 10
“Assemble by Name” and Chapter 7 “The Reference Sequence”.

TRANSLATED VARIANCE TABLE FOR SEQUENCES IN THE SAME CONTIG

When you are ready to begin creating your Translated Variance Table, either select individual
sequences from within a contig or go to the Project Window and select a contig by clicking on
its icon. This will select all of the contig’s sequences.
Then go to the Sequence menu and choose Compare Translation To and either Consensus,
Reference Sequence, or Top Sequence from the submenu. The Translated Variance Table
appears in a new window (see Figure 13-6).
The Translated Variance Table header summarizes the table’s contents including a
Description, the Base Positions, and the Amino Acid Positions. If a sequence in the table does
not extend the full length of the Comparison Range, its column header will be shaded in pink.
The pink X’s mark cells that are not covered by sequence data, indicating that the status of the
base at that position is unknown.

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 Figure 13-6 Translated Variance Table for sequences in the same contig

THE TRANSLATED CONSENSUS VARIANCE TABLE

Sequencher allows you to compare the translation of a consensus sequence to the translation
of a Reference Sequence. Go to the Contig menu and click on Compare Translation to
Reference.
You can also use the Compare Translation to Reference command to compare the
translations of consensus sequences from multiple contigs to the translation of their common
Reference Sequence. These contigs are formed when you use Assemble By Name with a
Reference Sequence.
For both of these operations, select one or more contigs in the Project Window that share a
common Reference Sequence. Then, from the Contig menu, choose Compare Translation to
Reference. The Translated consensus Variance Table appears in a new window (see Figure
13-7).

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 Figure 13-7 Translated variance Table for consensus sequences from different contigs

SETTING THE VARIANCE TABLE CONDITIONS

RESTRICTING THE COMPARISON RANGE

You can restrict the Comparison Range by Feature Key if you have any Features in your
exemplar. Construct a Variance Table then go to the button bar and click on the Comparison
Range button. The Comparison Range dialog appears (see Figure 13-8).
In the default setting the Unfiltered radio button is checked. Leave this radio button checked if
you decide to leave your table unfiltered.
If you want to restrict the Comparison Range, click the Filter Comparison by: radio button.
Once you have clicked on that button, the Feature Key: drop-down menu becomes active. You
can now choose which Filter Key to use by making a selection from this menu.
Once you have selected a Feature Key, you will see a list of features annotated with that key in
the pane located below the Feature Key: drop-down menu. You can select a single feature, a
continuous range or a discontinuous range of features. You can also select all of the features
within this pane by clicking on the Select All button.

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You can widen your Comparison Range by typing a number into the Flanking Bases box. For
example, by typing the number ten in this box, the Comparison Range will include 10 flanking
bases on either side of the chosen Feature Key.
Once you have made your selection, click on the OK button. The Variance Table will be rebuilt
automatically.

Figure 13-8 Comparison Range dialog

RESTRICTING THE TRANSLATION RANGE

Reference Sequences are often marked up with GenBank-style features. (See Chapter 19
“Motifs and Features” for further information.)
You can reduce the scope of your Translated Variance Table to a single feature. Some features,
such as mRNA or CDS, may be composed of several elements. These elements are
concatenated and treated as a single feature. Translation of the DNA sequence starts in the
first frame, with the first base in the feature range. Figure 13-9 illustrates an unfiltered
Comparison Range, a joined feature (blue), and a single feature (pink).

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 Figure 13-9 Restricting the Comparison Range using Features

View your Translated Variance Table and go to the button bar and click on the Translation
Range button. The Translation Range dialog appears. You will see that the Translate Entire
Sequence radio button is checked. This is your default. Leave this radio button checked if you
decide to leave your table unfiltered.
If you want to restrict the Comparison Range, click on the Filter Translation by: radio button.
Once you have clicked the Filter Translation by: radio button the Feature Key: drop-down
menu becomes active. You can now choose which Filter Key to use by making a selection from
this menu.
Once you have selected a Feature Key, you will see a list of features annotated with that key in
the pane located below the Feature Key: drop-down menu. You can select a single feature, a
continuous range or a discontinuous range of features. You can also select all the features
within this pane by clicking on the Select All button.
Once you have made your selection click the OK button. The Translated Variance Table will be
rebuilt automatically (see Figure 13-10).

Figure 13-10 Translated Variance Table restricted by Translation Range

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The table header indicates how the Comparison Range was restricted. For some Feature Keys,
the actual feature is composed of several components joined together (see Figure 13-9). The
Base Positions indicate the exact range of bases used for each component.
Features which share the same Feature Key, such as CDS, and which are joined (the Feature
has a Join qualifier), will appear in the Feature Listing as CDS [01], CDS [02]. The same Feature
will appear in the Translation Range dialog as a single item (see Figure 13-11).
See Chapter 19 “Motifs and Features” and Appendix 30 “Feature Keys and Qualifiers” for more
information.

Figure 13-11 Feature Keys with more than one component

If you prefer to have each component of a Joined Feature as a separate item, you must give
them different names. You can do this by editing the Feature using the Edit Features…
command (see figure below).

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 Figure 13-12 Appearance of features once they have been renamed

You can also create your own joined features. Use the Edit Features… command to create a
series of features which share the same name followed by a bracketed number, CDS [01], CDS
[02] (see Figure 13-13).

Figure 13-13 Creating a Joined Feature

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SETTING THE VARIANCE TABLE DISPLAY OPTIONS

Throughout Sequencher, you can customize the display of your sequences using the
commands from the View menu. When creating a Variance Table, you can also specify a
number of display options (see Table 13-1).
Table 13-1 View Options

Consensus
Translated Translated Consensus
Variance Variance
Variance Table Variance Table
Table Table
Base Ambiguities
No No No No

Base Edits Yes No No No

Display Color
Bases Yes Yes No No

Display Features
Yes Yes No No

Display Motifs No No No No

Colors As
Backgrounds Yes Yes No No

Display Base
Confidences Yes No No No

Labels Yes Yes Yes Yes

To highlight variants in your data, go to the View menu and click on Display Base
Confidences. This will add a background color to the cells in your Variance Table to indicate
that there is an associated confidence or quality score range for the variant. Variants that
require additional checking because they have a low confidence or quality score will be
highlighted with dark blue. Those with medium or high confidence scores will be highlighted
with a medium or pale blue background.
If you click on Display Color Bases and Display Colors as Backgrounds, it can help you to see
patterns in your data (see Figure 13-14). You can use Base Ambiguities As to distinguish
ambiguous or edited bases. You can also toggle the display of features from the View menu.
You can use the Label command to mark sequences in the table. Set your preferences using
the Label & Name preferences pane. Use the boxes grouped under Available Labels in the
preference pane to choose label colors and descriptions for marking individual sequences.
Select the sequence you want to label by clicking its column header. Go to the Edit menu and

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choose the Label submenu. The display name of the sequence will change to match the color
of the Label. (See Figure 13-4.) Notice how the tool tip provides the name of the sample, its
label, and other attributes.
(Refer to Chapter 23 “Customizing Sequencher and User Preferences” for more detailed
information.)

Figure 13-14 The Variance Table Display Options

SETTING THE CONSENSUS CALCULATION FOR A VARIANCE TABLE

You can generate a Variance Table where the exemplar is a consensus sequence rather than
the Reference Sequence by using the Compare Bases To command from the Sequence menu.
You generate a consensus Variance Table using the Compare Consensus to Reference
command from the Contig menu.
In either case, the method you choose to calculate your consensus can affect your results.
Generally, using the command Consensus Inclusively will report more variants than
Consensus by Plurality while Consensus by Confidence will use a method where the bases
are calculated to be the best based on confidence scores.
Sequencher recalculates the consensus on the fly, so if you change the methods you are using
to compute your consensus, you can reconstruct the Variance Table quickly. Just click on the
Refresh button in the button bar at the top of the Variance Table to display the results of the
new consensus calculation.
For more information on consensus calculations, see Chapter 11 “The Contig Editor”.

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VARIANCE TABLE OPTIONS

If you need to change User Preferences for your Variance Table, click on the Options button on
the Variance Table button bar. The User Preferences dialog opens in a new window. (See
Chapter 23 “Customizing Sequencher and User Preferences” for more detailed information.)

WORKING WITH THE VARIANCE TABLE

NAVIGATING A VARIANCE TABLE

To navigate through any form of Variance Table, use the arrow keys to move across the rows
or down the columns.
When you are working with any Variance Table and only want to focus on differences, use an
arrow key with the Alt key. This will skip any cells that match the comparison sequence. If you
try to move the cursor beyond the edge of the table, you will be alerted by a beep.
Note: If you are using the Compare Consensus to Reference command, you will see the
consensus base(s) for each contig you have selected in the same position. If you are using the
Compare Translation to Reference command, you will be viewing the consensus codon for
each contig you have selected in the same position.

SORTING THE VARIANCE TABLE

The Variance Table’s Sort functions are useful when, for instance, you need to segregate clone
sequences that perfectly match your Reference Sequence or cluster sequences of like alleles.
The Variance Table simultaneously displays differences and calculates summary information
about your selections. The Table lists the total number of differences for each sequence at the
bottom of that sequence’s column. The Variance Table also displays the total number of
differences for each position to the right of each row. There are two Total buttons, at the
bottom left and top right corners of the Variance Table. These act as Sort buttons.
The Total button, on the bottom left of the table, sorts the sample columns so that the
samples with the greatest number of differences move to the left. Click Total again, and the
columns sort in the opposite order.
There are two ways to define the sort order for the rows. You can sort by total number of
differences or you can sort by 5’-3’order. In the default, unsorted display of the Variance Table,
rows are sorted by base position in ascending (5'- 3') order. When you click on Total in the top
right corner of the Variance Table, the rows are rearranged so that those with the greatest
number of differences sort to the top. Click the Total button again to reverse the order.
To restore the order by base position, click on the button in the top left corner of the Variance
Table. Click here again to have the rows sort by position in descending (3’-5’) order.
Note: The text on the sort button is context sensitive and will change depending on the
Compare Bases To command used to generate the Variance Table.

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REMOVING COLUMNS FROM THE VARIANCE TABLE

As you start to work with your Variance Table, you may decide that some sequences are no
longer of interest. The Edit menu’s Remove From Table command allows you to delete
sequences selectively from your Variance Table.
To select a continuous range of sequences, choose the first sequence by clicking on its column
header and then, holding down the Shift key, select the last sequence in the same fashion. To
select a discontinuous list of sequences, hold down the Ctrl (Windows) or Cmd (Mac) key while
clicking on the sequences. Go to the Edit menu and choose the Remove From Table
command.
Note: The Remove From Table command does not remove sequences from the underlying
contig.

REVIEW MODE

In Review mode, the Variance Table lets you view the data supporting each cell (see Figure
13-15). You can access Review mode by clicking on the Review button in the Variance Table
button bar or by double-clicking on any cell in the table. Sequencher will open both the Contig
and Chromatogram editors with the associated base or column of bases selected.
You can specify the layout of these three Review windows, the Variance Table, the Contig
Editor, and the Contig Chromatogram Editor, and then set the new organization as your
default layout. After you have arranged your windows, go to the Window menu and choose
the Remember Window Layout command, then select Single Variance Table Review from
the submenu.
You can edit your data directly in the Variance Table in Review Mode. Select the cell to be
edited in the table, then type in your desired change. The change will be reflected in the
Variance Table and the Contig Editor immediately.
Note: You can create features in the Variance Table by going to the Sequence menu and
clicking on the Mark Selection as Feature command.

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 Figure 13-15 Comparing sequences in Review Mode

THE REVIEW MODE AND COMPARE CONSENSUS TO REFERENCE

The Contig menu’s Compare Consensus to Reference command creates a Variance Table
that reports differences from multiple contigs (see Figure 13-16). The Review mode lets you
navigate efficiently between a difference in one contig and the equivalent position in the next.
Each column in the Variance Table contains the differences from separate contigs. As you
change your selection across the Variance Table rows, Sequencher closes the Contig Editor and
the Contig Chromatogram Editor from your previous selection. It will then open the editors
corresponding to your current selection.
Just as you can edit contigs from the consensus line in a Contig Editor, you can also edit contigs
from a selection in the Variance Table Review mode. Your selection in the Variance Table
corresponds to a selection in the consensus of the underlying contig.
Note: Remember that when you type over a base in the Variance Table, the underlying data in
your contig will also change.

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 Figure 13-16 Comparing contigs in Review Mode

THE REVIEW MODE AND THE TRANSLATED VARIANCE TABLE

The Translated Variance Table also has a Review button in its button bar. When you click on
this button, you will see the associated Contig Editor and Chromatograms (see Figure 13-17).
Once you have arranged the individual windows to your satisfaction, you can save the layout
by going to the Window menu and choosing Remember Window Layout. Then click on
Single Table Variance Review from the submenu. When you click on an individual cell in the
table, its codon is highlighted in the Contig Editor and the first base is selected. You will notice
that the equivalent codon in the exemplar sequence is also highlighted.

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 Figure 13-17 The Translated Variance Table in Review Mode

Note: You cannot edit your sequences directly from the Translated Variance Table (although
you can do so in the ordinary Variance Table). Any edits to the Translated Variance Table must
be made in the Contig Editor. In order to see the results of your changes in the Translated
Variance Table, click on the Refresh button in the button bar.

THE REVIEW MODE AND SISTER TABLES

You can generate a “Sister Table” when you are working in an Individual Variance Table. If you
have generated a Variance Table, you can see the Translated Variance Table for the same data
set by going to the button bar and clicking on the Translation button (see Figure 13-18).
Similarly, if you have already generated a Translated Variance Table, you can see the Variance
Table for the DNA sequences of the same data set by going to the button bar and clicking on
the Bases button.
If you are in Review mode and have generated a Sister Table, you can arrange all four windows
and save the layout by going to the Window menu and choosing Remember Window Layout.
Then click on Double Table Variance Review from the submenu.

VARIANCE TABLE REPORTS

The format of each Variance Table report is broadly similar. The report starts with a Common
Header. This header includes the date, the project name, the type of table, the Comparison

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Range, base positions, and options. This is followed by a header containing more pertinent
information, and finally the body of the report.

VARIANCE TABLE REPORT

The Variance Table Report consists of three parts: the Common Header, the Variance Table,
and the Comparison Range Coverage table. This table lists the name of each sequence, the
type of coverage (complete or incomplete), and the range of bases in the comparison (see
Figure 13-19).

Figure 13-18 Review Mode showing Sister Tables

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 Figure 13-19 Extract from Variance Table Report

INDIVIDUAL VARIANCE REPORTS

You can create a separate report for each sample in the table. The Individual Variance Report
lists the variants in a single column. This table is followed by the Comparison Range Coverage
table which lists the name of each sequence, whether coverage is complete or incomplete, and
the range of bases in the comparison (see Figure 13-20).
Note: This can produce a very long report.

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 Figure 13-20 Extract from Individual Variance Table Report

VARIANCE DETAIL REPORT

As its name indicates, the Variance Detail Report contains comprehensive information on the
variants in a Variance Table. Below the Common Header, you will see detailed information for
each of the described variants.
The information for each variant consists of the sample name and comparison range for the
parent sequence. The Detail Table is divided into two parts. The first part contains the
sequence name, orientation, confidence score (where available), base call, primary peak base
call, secondary peak base call, and the height of the secondary peak as a percentage of the
primary peak. The second part of the table contains a tracelet, which is an extract of the
sequence trace of the variant combined with some flanking bases (see Figure 13-21). It only
appears in the report if the sequence has associated chromatogram data.
Note: If the Detail Table displays a gap for a specific base position, this will still be given a
confidence value. The confidence value will be equal to the average confidence ratings of the
bases flanking the gap. Sequencher will append an asterisk to the confidence value and a
footnote will explain how the value was derived.
When the report contains a great deal of data, it may be useful to use the Chromatogram Scale
function which can be accessed by clicking on the Report’s Option button.

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 Figure 13-21 Extract from Variance Detail Report

Note: If the Detail Table displays a gap for a specific base position, this will still be given a
confidence value. The confidence value will be equal to the average confidence ratings of the
bases flanking the gap. Sequencher will append an asterisk to the confidence value and a
footnote will explain how the value was derived.

Figure 13-22 Extract from a Population Report

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WORKING WITH VARIANCE TABLE REPORTS

You now have a number of ways to present the data from your Variance Table. You can use the
table’s Reports function to print your data or to export it. The options vary depending on the
data you have chosen to use in your report.

PRINTING A VARIANCE TABLE REPORT

You can print the data from an entire table, selected columns, or selected rows. The type of
report you can generate will depend on this initial selection (see Table 13-2).
Table 13-2 Variance Table Print Options

Variance Table Individual Variance Variance Detail Population

Report Table Reports Report Report

Entire Table Yes Yes Yes Yes

Selected Yes Yes Yes Yes

Columns Rows
Selected Yes n/a Yes n/a

To print all the data in a table, click on the Reports button. The Variance Table Reports dialog
appears (see Figure 13-23). A radio button called Entire Table will be checked. Below that you
will see a drop-down menu called Report Format:. Choose Variance Table Report, Individual
Variance Table Reports, Variance Detail Report, or Population Report. Click on the Open
Report… button. The Sequencher Report Viewer opens in a new window.

Figure 13-23 Variance Table Reports drop-down menu

Use the Print Preview button from the button bar at the top of the Report Viewer to preview
your report (see Figure 13-24).

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 Figure 13-24 Reports Viewer button bar

The Report View is replaced by the Preview View. Note the Zoom In and Zoom Out buttons
on the button bar (see Figure 13-25). Click on the Report View button to return to the
previous view.

Figure 13-25 Print Preview button bar

Use the Page Setup button to choose paper size. Click on the Print button to send the report
to a printer. You can also save the report in PDF format by clicking on the Save as PDF…
button. If you wish to dismiss the window without performing any of these actions, click on the
Close button.
It is important to note that you can reduce the size of the tracelets within the Variance Detail
Report, this can decrease the number of pages you need to print out for your report. Having
chosen Variance Detail Report as your report format, click on the Options… button on the
button bar. Now either type in the scaling value you wish to use or use the elevator buttons.
You can also limit the number of chromatograms displayed to None or a specific number by
clicking on the Display radio button and inserting a number.

PRINTING SELECTED DATA FROM A VARIANCE TABLE

You can print selected data from your table. You have several options. You can remove sample
sequences from the table using the Remove from Table command. You can print selected
variants by choosing selected rows and you can print selected samples by choosing selected
columns. You may already have restricted the data in the table by using the Comparison
Range command.
If you want to remove sample sequences from your table, you can either select a continuous
range of sequences or a discontinuous range of sequences. For a continuous range of sequences,
select the first sequence in your range by clicking on its column header, and then, holding down
the Shift key, select the last sequence in the same fashion. To select a discontinuous list of
sequences, hold down the Ctrl (Windows) or Cmd (Mac) key while clicking on the sequences.
Then go to the Edit menu and choose the Remove From Table command.
Note: This does not remove sequences from the underlying contig.
If you want to print selected samples from your table, you can choose a continuous or a
discontinuous range of sequences by clicking in the column header as described previously.
Click on the Reports button. The Variance Table Reports dialog appears (see Figure 13-26). A
radio button called Selected Columns will be checked. Below that you will see a drop-down

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menu called Report Format:. Choose Variance Table Report, Individual Variance Table
Reports, Variance Detail Report, or Population Report. Click on the Open Report… button.
The Sequencher Report Viewer opens in a new window.

Figure 13-26 Variance Table Reports dialog

Use the Print Preview button from the button bar at the top of the Report Viewer to preview
your report. Click on the Print button to send the report to a printer.
If you want to print selected variants from your table, you can choose a continuous or a
discontinuous range of base positions by clicking the equivalent exemplar base position as
described previously.
Click on the Reports button. The Variance Table Reports dialog appears (see Figure 13-26). A
radio button called Selected Rows will be checked. Below that you will see a drop-down menu
called Report Format:. Choose either Variance Table Report or Variance Detail Report. Click
on the Open Report… button. The Sequencher Report Viewer opens in a new window.
Use the Print Preview button from the button bar at the top of the Report Viewer to preview
your report. Click on the Print button to send the report to a printer.
Note: Not all reports are available when you print selected rows.

EXPORTING A VARIANCE TABLE REPORT

You can export the data from an entire table, selected columns, or selected rows. The type of
report you can generate will depend on this selection (see Table 13-3).

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 Table 13-3 Variance Table Report export options

Variance Individual Variance Population

Table Report Variance Table Detail Report Report
Reports
Entire Table Yes Yes No No

Selected Yes Yes No No

Columns
Selected Yes n/a No n/a

Rows

You can remove sample sequences from your table before you export your data. Select a
continuous range of sequences. Choose the first sequence by clicking on its column header.
Then, holding down the Shift key, select the last sequence in the same fashion. To select a
discontinuous list of sequences, hold down the Ctrl (Windows) or Cmd (Mac) key while
clicking on the sequences. Go to the Edit menu and choose the Remove From Table
command.
Note: This does not remove sequences from the underlying contig. You can then export the
remaining data as normal.
To export all the data in the table, click on the Reports button. The Variance Table Reports
dialog appears. A radio button called Entire Table will be checked. Below that you will see a
drop-down menu called Report Format:. Choose either Variance Table Report or Individual
Variance Table Reports. If you choose Variance Table, Sequencher will generate a single
table. If you choose Individual Variance Table Reports, each sample will appear in a separate
report.
There are four buttons below the Report Format drop-down menu, Cancel, Reports, Copy as
Text, and Save as Text... Save as Text… is the default setting. If you click on the Save as
Text… button, Sequencher presents you with a dialog to assign the name and location of your
export. The report is saved in a tabbed text format.
Note: You can select and then remove columns from your table using the Remove From Table
command before you export your data.

EXPORTING SELECTED SAMPLE SEQUENCES

Each column in the table represents a sequence or consensus. If you need to export specific
columns in the table, make your selection by using Shift+click for a continuous range or
Ctrl+click (Windows) or Cmd+click (Mac) for a discontinuous range (see Figure 13-27). Then
click on the Reports button. The Variance Table Reports dialog appears, the Selected
Columns radio button is checked. Choose a Report Format from the drop-down menu.

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Select Individual Variance Reports if you want to save each column (sample) as a separate
file. Select Variance Table Report if you want the selected columns in one table. Click on the
Save as Text… button, Sequencher presents you with a dialog to assign the name and location
of your export.
You can copy your Variance Table into another document or application that accepts tabbed
text. To paste the contents of your Variance Table Report in the document of your choice,
click on the Copy as Text button. The data can now be pasted into its new location.

Figure 13-27 Variance Table with discontinuous range of columns selected

EXPORTING DATA FOR SELECTED VARIANTS

Each row in the table represents a variant. If you need to export selected rows, make your
selection by using Shift+click for a continuous range or Ctrl+click ( Windows) or Cmd+click
( Mac) for a discontinuous range. Then click on the Reports button. The Variance Table
Reports dialog appears. The Selected Rows radio button is checked. There are four buttons,
Cancel, Open Report…, Copy as Text, and Save as Text... Save as Text… is the default
setting.
The chosen rows are saved as one table. The options in the Reports Format: drop-down menu
are limited to Variance Table Report and Variance Detail Report. Click on the Save as Text…
button. You will see a dialog prompting you to assign the name and location of your export.
You can type a name for the table in this window. Click on the Save button to save your table
as a tabbed text file.
Although the Selected Rows radio button is checked, you can change your selection to include
all rows by clicking on the Entire Table radio button.

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You can copy your Variance Table into another document or application that accepts tabbed
text. To paste the contents of your Variance Table Report in the document of your choice,
click on the Copy as Text button. The data can now be pasted into its new location.

TRANSLATED VARIANCE TABLE REPORT

TRANSLATED VARIANCE TABLE REPORT

The Translated Variance Table Report consists of three parts: the header information, the
Comparison Range Coverage, and the Translated Variance Table.
The Translated Variance Table contains the name of the sample sequence, the variant codon,
the amino acid residue, and the type of coverage (whether the coverage is complete or
incomplete) for each variant in the table. The Comparison Range Coverage table lists the name
of each sequence and the range of bases in the comparison.

WORKING WITH A TRANSLATED VARIANCE TABLE REPORT

There are a number of ways to present the data from a Translated Variance Table. You can
export the data from an entire table, selected columns, or selected rows. You can also remove
data from the table before you export it.

EXPORTING A TRANSLATED VARIANCE TABLE REPORT

To export all the data in the table, click on the Reports button. The Translated Variance Table
Reports dialog appears (see Figure 13-28). A radio button called Entire Table will be checked.

Figure 13-28 Translated Variance Table Reports dialog

You will see three enabled buttons in the Translate Variance Table Reports dialog, Cancel,
Copy as Text, and Save as Text...

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Save as Text… is the default setting. If you click on the Save as Text… button, Sequencher
presents you with a dialog to assign the name and location of your export. The report is saved
in a tabbed text format.
If you prefer to copy your Translated Variance Table to another document or application that
accepts tabbed text, click on the Copy as Text button. The data can now be pasted into its
new location.

EXPORTING SELECTED SAMPLE SEQUENCES FROM A TRANSLATED VARIANCE TABLE

Each column in the table represents a sequence or consensus. If you need to export specific
columns in the table, make your selection by using Shift+click for a continuous range or
Ctrl+click (Windows) or Cmd+click (Mac) for a discontinuous range (see Figure 13-29). Then
click on the Reports button. The Translated Variance Table Reports dialog appears (see
figure above). A radio button called Selected Columns will be checked.

Figure 13-29 Discontinuous selection of columns

Choose one of the three enabled buttons in the Translated Variance Table Reports dialog,
Cancel, Copy as Text, and Save as Text... Save as Text… is the default setting. If you click on
the Save as Text… button, Sequencher presents you with a dialog to assign the name and
location of your export. The report is saved in a tabbed text format.
Although the Selected Columns radio button is checked, you can change your selection to
include all rows by clicking on the Entire Table radio button.

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You can copy your Translated Variance Table into another document or application that
accepts tabbed text. To copy the contents of your Translated Variance Table Report, click on
the Copy as Text button. The data can now be pasted into its new location.

EXPORTING DATA FOR SELECTED VARIANTS

Each row in the table represents a variant. If you need to export selected rows, make your
selection by using Shift+click for a continuous range or Ctrl+click (Windows) or Cmd+click
(Mac) for a discontinuous range (see Figure 13-30). Then click on the Reports button.

Figure 13-30 Translated Variance Table - selected variants

The Translated Variance Table Reports dialog appears (see Figure 13-31). Although the
Selected Rows radio button is checked, you can change your selection to include all rows by
clicking on the Entire Table radio button.

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 Figure 13-31 Translated Variance Table Reports dialog - Selected Rows

Choose one of three buttons in the Translated Variance Table Reports dialog, Cancel, Copy as
Text, and Save as Text... Save as Text… is the default setting. The rows you have selected will
be saved in one table. Click on the Save as Text… button. A dialog to assign the name and
location of your export appears. You can type a name for the table in this window. Click on the
Save button to save your table as a tabbed text file.
You can copy your Translated Variance Table into another document or application that
accepts tabbed text. To paste the contents of your Translated Variance Table Report, click on
the Copy as Text button.

REMOVING DATA FROM A TABLE BEFORE EXPORT

If you need to reduce the amount of data in your table, you can remove selected sample
sequences.
Make your selection by using Shift+click for a continuous range or Ctrl+click (Windows) or
Cmd+click (Mac) for a discontinuous range.
Go to the Edit menu and choose the Remove From Table command. Your selection will be
removed. You can then export the remaining data as usual.
Note: This command does not remove sequences from the underlying contig.

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 14. THE SUMMARY REPORT

In this chapter, we explain how to use the Summary Report. This is a static report that has a
number of options making it a useful addition to the Variance Table for mutation hunting,
verifying re-sequenced fragments, comparisons to a Reference Sequence, or just as a report for
your lab notebook.

THE SUMMARY REPORT VIEW

VIEW BY SUMMARY

You can display this report by going to the Contig Editor and clicking on the Summary button
in either the Bases View window or the Overview…. Alternatively, you can go to the View
menu, choose Bases, Map, Overview…, and then click on Summary Report.
You will be presented with a new window which shows the assembled sequences within your
chosen contig. There are five buttons arranged on a button bar (see below).
Table 14-1 Button bar

Button Description

Bases This button returns you to the Contig Editor.

Overview This button returns you to the Contig Overview.

Ruler This button brings up a simple text editor-like ruler.

Options This button allows you to control which information

will be displayed.

Find This button brings up a window into which you can

type a substring and perform a search (see Chapter 20
“Finding Items”).

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SUMMARY REPORT DISPLAY OPTIONS

Sequencher has a default format for the Summary Report but you can change it to display
different types of information. Click on the Options button or go to the View menu and
choose View Options…
The Summary Options are divided into those that affect the sequence fragments and those
that affect the display of protein translations. You can change the consensus calculation while
in this view. Any change you make will be reflected immediately in the report.
Sequencher displays the window shown in Figure 14-1 Summary Report Options.

Figure 14-1 Summary Report Options

COMPARE TO CONSENSUS OR REFERENCE SEQUENCE

If you have a Reference Sequence in your contig, you can choose whether to display a
Summary Report compared to the consensus sequence or to the assembled Reference
Sequence. The default setting is Compare To: Consensus but you can change this by clicking
on the Reference radio button. Some of the Display options are context sensitive and will
change to reflect your choice.
The Reference radio button will be grayed out when the selected contig does not contain a
Reference Sequence.

ICONS

If you check the box for the Icons display, you will see the sequence fragment icons positioned
to the left of the sequence name. Unchecking this option will hide the icons.
Note: When you have a Reference Sequence in your contig, its icon is distinguished by the
addition of an “R”.

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BULLETS, PLUSES AND DASHES

Clicking the Bullets, Pluses boxes highlights disagreements and ambiguities in a line below the
consensus. If you also click on & Dashes, you are marking positions where the bases in
individual sequences match so that the disagreements are more visible.
Note: You can display Bullets, Pluses without the & Dashes but you cannot display & Dashes
without the Bullets, Pluses. However, see Matching Bases As Dashes below.

CONSENSUS SEQUENCE

You can choose to hide the consensus sequence by clicking on the Consensus Sequence box.
Figure 14-2 shows a summary line without the consensus line but with Bullets, Pluses and &
Dashes all checked.
Figure 14-2 Summary Report with no consensus line, with bullets, pluses and dashes

FRAGMENT SEQUENCES

You can choose to hide the fragment sequence by clicking on the Fragment Sequences box.
This gives you a very compressed report that can be useful when viewing protein translations.
Figure 14-2 above shows a summary line without the consensus line, but with Bullets, Pluses
and & Dashes all checked.

MATCHING BASES AS DASHES

Matching Bases As Dashes is a very powerful option when you are interested in any kind of
mutation detection. When you check this option, all the bases that match each other are
converted to dashes.
If you have selected Display Color Bases, the dashes will be marked in the color of the original
base call. If you also go to the View menu and chose Colors as Background, each dash will
appear embedded in a small color block. Bases that disagree are displayed as characters and as
such stand out in stark relief. With this option, you can easily see mutation hot spots or
clusters and potential SNPs.

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PROTEIN TRANSLATIONS AND THE SUMMARY REPORT

Sequencher can show protein translations for individual sequences and mark matching amino
acids with dashes. This helps you see whether a particular discrepancy in an alignment has
functional implications or whether it may simply be a silent mutation. If you combine options,
you can turn off the DNA display altogether and just focus on your translations. Alternately,
you can use a mixture of options to tailor the report to your specific needs.

ENABLING THE PROTEIN DISPLAY

To enable the protein display in the summary view, go to the View menu and click on
Translation. You can then choose from the following list of menu suboptions: Single Stranded,
Double Stranded, Protein 1st Frame, Protein 2nd Frame, Protein 3rd Frame and, Protein
All 3 Frames.
To see the protein translations for the opposite strand, go the View menu and select Reverse
& Comp. Then select the desired option. Where the assembly algorithm has inserted a gap in a
sequence, Sequencher will translate the codon correctly.
Figure 14-3 Insert Summary Report displaying translation across a sequence gap

PROTEIN TRANSLATIONS FOR THE CONSENSUS OR REFERENCE SEQUENCE

This option is context sensitive. If you do not have a Reference Sequence or you have selected
the Consensus radio button, you will be able to show protein translations for the consensus.
Click on the Consensus checkbox at the bottom of the Summary Options dialog.
If you have a Reference Sequence in your contig and you have selected the Reference radio
button, you will be able to show translations for the Reference Sequence. Click on the
Reference checkbox at the bottom of the Summary Options dialog.

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PROTEIN TRANSLATIONS FOR FRAGMENTS

To show protein translations for fragments, click on the Fragment checkbox in the Summary
Options dialog.

MATCHING PROTEINS AS DASHES

To show Matching Proteins As Dashes, click on that checkbox in the Summary Options
dialog.

USING THE FORMATTING RULER

You can use the formatting ruler to change the blocking (for example, to groups of 3s instead
of 10s) or to show protein translations. (See Chapter 8 “The Sequence Editor” for more
information on the formatting ruler.)
Click on the Ruler button in the button bar or go to the View menu and choose Display
Format Ruler. You can use the smaller font options in conjunction with the landscape page
format to obtain full-width displays of your assembled sequences for use in posters and
figures.

REVERSE AND COMPLEMENT A CONTIG

You can change the orientation of the contig while displaying any of its views by going to the
View menu and choosing Reverse & Comp. You can display the contig’s original orientation
by choosing Reverse & Comp again.

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 15. CHROMATOGRAMS

In this chapter, you will learn how to use the chromatograms from an automated sequencer to
help you when editing your contigs. We discuss how Sequencher displays traces and secondary
peaks to find heterozygotes and how you can edit bases from traces or revert to experimental
data.

WORKING WITH AUTOMATED SEQUENCER DATA

When you import a trace file from an automated sequencer, Sequencher imports the
chromatograms and the confidence values if these are available.
Note: If you import a text file containing just ASCII characters, you will not have imported
traces. To import traces, you must import the trace file. Text files from an automated
sequencer will be only a couple of kilobytes in size, whereas trace files will be well over 50
kilobytes.
When you copy a Sequencher project file from one machine to another, you don’t need to
copy the trace files separately because Sequencher stores the trace information along with
everything else in the project file. Sequencher compresses the imported traces to conserve
disk space, so don’t be alarmed if a project file containing several sequences from an
automated sequencer is smaller than any one of the trace files.

VIEWING CHROMATOGRAMS FROM A SEQUENCE EDITOR

Sequencher’s chromatogram display is tightly integrated with its other editors. You can view
the entire original trace for a sequence by opening the Sequence Editor for that sequence.
Click on the Show Chromatogram button on the button bar or go to the Window menu and
choose Chromatogram. You can scroll either vertically (the default) or horizontally. To change
orientation, click the appropriate button in the left bottom corner of the sequence
chromatogram window.
Note: Remember that, even if a sequence is incorporated into a contig, you can still open its
editor by double-clicking the sequence name in the list at the left of the Contig Editor.

VIEWING CHROMATOGRAMS FROM A CONTIG EDITOR

The Sequencher Contig Editor allows you to view simultaneously all chromatogram data
relevant to a consensus base call. If you are at the 5’ end of the contig, Sequencher selects the
most 5’ base in the consensus and displays all the associated traces. If you are elsewhere in the
contig, Sequencher selects the center-most consensus base in the contig display and displays
the associated traces. This allows you to check the signal strength of any peak in conjunction
with its base call and edit your data accordingly.

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Select a contig column by clicking on its base in the consensus line (Figure 15-1). Then click on
the Show Chromatograms button or go to the Window menu and choose Chromatogram.
Sequencher opens a window to display the traces (Figure 15-1). The bases you highlighted in
the Contig Editor will also be highlighted in the trace windows.
Figure 15-1 Selected column chromatograms

When you click the Show Chromatograms button in a region without trace data, you will see
a “No Chromatogram Data” message in the Contig Chromatogram window.
When you move the cursor back into a region where there are sequences with chromatogram
data, the traces will reappear.

UNDERSTANDING THE TRACE DISPLAY

Above the traces are two lines of bases. The upper line displays the sequence as it currently
appears in the editor, including any edits you may have made. The lower line displays the base
calls as originally imported, including the trimmed data. The arrows to the left show the
orientation and relative lengths of the trimmed and untrimmed data.
Below the arrows is a slider for changing the height of the trace. Below the slider are four
buttons that let you hide the lanes for any or all of the traces. Click on the button to hide any

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lane. Click it again to display the lane. Figure 15-2 shows a contig with adjusted scales and
hidden lanes.
Scrolling through a single chromatogram is simple. Position your cursor on the small
rectangular slider button to the right of the trace. With the mouse button held down, you can
move the slider down or up, depending on your current position within the sequence.
Alternately, you can use the left arrow and right arrow keys to step through the sequence
trace base by base.
When you are working in a contig, if you highlight a base in the consensus line you can use the
arrow keys to move through the consensus sequence. Note that, as you do so, all the contig
chromatograms will move left or right, in step with the highlighted base.
Figure 15-2 Trace with hidden lanes and adjusted scales

You can specify the settings for trace display by going to the Window menu and choosing User
Preferences and selecting Chromatogram. You can specify the height of the trace, the width
from peak to peak, the way the lanes are identified, and various screen display attributes. (See
Chapter 23 on “Customizing Sequencher and User Preferences” for more information.)
If you select the Contig Chromatogram section, you can specify how many columns of traces
you want to view at once, whether Sequencher should scroll to the selected column when you
make a new selection in the contig, and where the window should appear.
Note: If you hold down the Ctrl (Windows) or Command (Mac) key while changing the scale,
the new scale will apply to all the traces in that window. If you hold down the Ctrl (Windows)
or Command (Mac) key while clicking any of the buttons to hide a lane, the lane(s) will be
hidden for all the traces in the window. (See Figure 15-2 above.)

DISPLAY SECONDARY PEAK

You can identify heterozygotes by identifying the second-highest peak beneath the primary
peak. If you have a particular candidate for a heterozygote, first open the chromatogram for
the individual sequence then click on the base call(s) as shown in Figure 15-3.

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 Figure 15-3 Trace with base selected

Go to the Sequence menu and choose Call Secondary Peaks…. A dialog lets you specify how
the secondary peak should be called (Figure 15-4).
The slider lets you specify how significant the second-highest peak must be to generate a
change. In Figure 15-4 above, the slider is set so the secondary peak has to be at least 75% as
high as the highest peak.
If you want Ns to be replaced, then select the Allow Ns to be replaced checkbox.

Figure 15-4 Secondary peak parameters

If you’ve already edited bases by hand and don't want them changed, you must deselect the
checkbox called Allow edited bases to be replaced.
If you select Only make changes that result in an ambiguity, Sequencher may change an
ambiguous base call to an A, C, G, or T if it does not meet the secondary peak criteria. If you
prefer it to remain ambiguous, do not select this option.
If you wish to search just a range of bases, highlight them and check the Search Selection Only
checkbox.
When you click OK to make the changes, a dialog notes how many bases will be changed; you
can choose to Cancel or Continue at that point. If you continue, the changes take effect. Once
they have taken effect, unlike normal edits to a base, they cannot be undone.

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Once you have pressed the Continue button, you can find all the changes made during the
session by selecting the first base in the sequence (you can click on it right on the
chromatogram) and going to the Select menu and clicking on Next Edited Base. After you
execute any Select Next command, Sequencher creates a shortcut for that command on the
space bar so you can continue to search for your edits, using the same function over and over,
by simply pressing the space bar.

SELECT NEXT COMMANDS

The Select menu provides you with several commands for navigating to bases which need
further review or attention (see Table 15-1).
Figure 15-5 Base change generated by secondary peak

As you move your selection from each position that requires scrutiny to the next, you are likely
to need to repeat these commands frequently. Therefore, Sequencher creates a shortcut for
that command in the space bar after the first time you invoke a Next command. Press the
space bar to move from one selected base to the next candidate.
To change any Next command to search in the reverse direction, combine the command with
the Shift key.
If you use the Next Met To Stop (>0bp), you can highlight the next pair of start and stop
codons in one of the three forward reading frames. The number of bases shown in this
command depends on whether you specified a preference for a minimum length in the User
Preferences.

EDITING BASES FROM TRACES

Now that you can easily locate the bases that need attention, you can edit your data in either
the Contig Editor window or the Chromatogram display. As you edit, both windows update
automatically. (Refer to Chapter 12 “Editing Contigs” for more information on editing.)
To edit a base while viewing a chromatogram, click on the current base call (upper line of text)
you want to edit, then type the new base call (
Figure 15-6). You can also select multiple bases for deletion by dragging the cursor across
them.

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 Table 15-1 Summary of Select commands

Select Command Contig

Next Ambiguous Base Ambiguous bases, Disagreements, First base in large gap
run

Next Contig Disagree Disagreements

Next Edited Base Edited bases

Next Low Confidence Base Any consensus base that is derived from a low
confidence base

Next Met To Stop (>0bp) Any pair of Start and Stop codons in one of forward ORFs

Next Inadequate Coverage Finds consensus base that does not have a forward and
reverse base that agree and are below low quality
threshold

Figure 15-6 Clicking on a base displayed in a trace

REVERTING TO EXPERIMENTAL DATA

If you have deleted too much of a sequence or have made edits you want to remove, you can
copy the original base calls back into the current sequence. From within the chromatogram,
select the original bases (lower line of text) you want to restore (Figure 15-7).

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 Figure 15-7 Original base calls selected

Go to the Sequence menu and choose Revert To Experimental Data. Figure 15-8 shows the
original bases restored.
Figure 15-8 Bases restored

Revert To Experimental Data is a powerful feature. It allows you to recover original data very
precisely, as described above, or it can be used to globally revert an entire sequence or
selection of sequences.
You can revert unassembled sequences to original data by selecting the sequences in the
Project Window and executing the Revert To Experimental Data command.

REVERTING ASSEMBLED SEQUENCES BACK TO EXPERIMENTAL DATA

If you need to revert a sequence that has been assembled in a contig, select its icon from the
left-hand side of the Contig Editor. Go to the Contig menu and use the Remove Selected
Sequence… command. Now you can use the Revert To Experimental Data command as
described above.
If your sequence has trace data, select all of the original bases. Go to the Sequence menu and
choose Revert To Experimental Data.
Note: The Extend Selection command allows you to select all of the bases in a chromatogram
without scrolling.

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ADJUSTING TRACE POSITIONS

As the four dyes migrate in different lanes on certain automated sequencers, the
synchronization among the four traces can deteriorate in output files. Sequencher allows for
adjusting or “re-tracking” the position of one trace relative to the others. When you are
looking at a chromatogram, specify which of the four traces you want to shift by selecting a
base in the original data line (the lower of the two lines of sequence characters).
Go to the Edit menu and choose Shift to display a submenu that lets you shift the bases line
left or right by a few pixels. The adjustments made are to aid you in reviewing the original
data; they will not be saved to the file when you save the project.

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 16. NGS FOR DNA AND RNA-SEQ

In this chapter, we explain how to align or assemble Next-Generation sequences using Maq,
GSNAP, BWA, or Velvet. We will show you how to use FastQ Quality Reports to check the
quality of your data. If you are working with reference-guided alignment, then you only need
to choose a Reference Sequence, choose your reads files, and begin the alignment. If you are
working with de novo assembly, then you simply choose your reads files and begin the
assembly. We also discuss viewing your results in an external viewer. You will learn how to
hunt for SNPs, analyze methylation data, and perform RNA editing-tolerant alignments. You
will also learn how to work with Multiplex ID data and use advanced settings.
In the second half of the chapter, you will learn how to use the programs in the Cufflinks suite
for analysing RNA-Seq NGS data, normalizing your data, quantifying your data, and performing
expression and differential expression analysis. You will also learn how to visualize your results
using the RNA-Seq plots and charts and export the results in PNG (Portable Network Graphic)
format.
For information on installing the External Alignment or RNA-Seq tools, please refer to the
installation instructions for the tools that can be found on our website at
http://genecodes.com/support.

SEQUENCE FILE FORMATS

Sequencher accepts many sequence formats. For Next-Generation sequencing, the most
relevant formats are FastA and FastQ. Although most sequencers have their own native
formats, as long as the data can be converted into FastA or FastQ format, Sequencher will be
able to align or assemble those reads.
When performing reference-guided alignment, you will be using a Reference Sequence that
will have been imported into Sequencher. It can be either a FastA file or a GenBank file. The
advantage of using a GenBank sequence is that it will usually carry annotations, although you
can mark up any sequence with annotations using Sequencher’s Mark Selection as Feature
command.

FASTA AND FASTQ FORMATS

FastA is a simple format which contains a sequence name preceded by the > character and
then followed by a sequence. The format can accommodate more than one sequence per file
(concatenated FastA). The FastQ format contains the sequence name preceded by the @
character, the sequence, and the PHRED scores associated with the sequence. Again, the files
can contain more than one sequence per file.
One thing to note, although it is possible to extract FastQ format files from Illumina and 454
sequencing runs, the formats may differ slightly. 454 conforms to what is popularly known as
Sanger format. Illumina has several versions of the FastQ format. If your Illumina data has been
produced by Casava 1.8 or later, then it will conform to Sanger FastQ.

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PAIRED-END DATA

If you are dealing with paired-end data in FastQ format, then your data will need to be in two
files. The read pairs must be in the same order. For example, in the image below you can see
the data files. In each file, the pairs end in /1 in the first file and /2 in the second file.
Figure 16-1 Format of Paired-Ends reads files

In some instances, there may be other special formatting/layout of your files, ensure that this
is done before submitting your sequences for alignment or the alignment will fail.

GSNAP LIST OF KNOWN SNPS FILE

If you plan to work with GSNAP, you will need to prepare a file containing a list of known SNPs.
The file resembles a FastA file in that each line is prefixed with the > character.

Figure 16-2 Format of known SNPs file

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GSNAP FASTA FORMAT FILE

If you plan to work with FastA files and wish to use the GSNAP algorithm, then you must
format the paired-end reads files as follows. First you need to merge the two reads files into
one file such that the paired-end reads are sequential. Then you need to remove the name of
the second read.

SAM FORMAT

SAM is the acronym for Sequence Alignment Map. The SAM file is a text format file but is
capable of holding a rich set of information that can be used by a number of programs. It
stores read alignments against Reference Sequences. Of interest to Sequencher users is that it
is the resultant format for GSNAP and can be read by the Tablet program, which is an external
Next-Generation assembly viewer.

AFG FORMAT

AFG is a text format file produced by Velvet that holds layout information relating to contigs,
reads, and paired-ends. An AFG file can be viewed using the Tablet viewer.
Figure 16-3 Steps for creating paired-end FastA file

LOCATION OF RESULTS FILES – THE EXTERNAL DATA HOME

The results may be saved as a contig in your project and/or a set of files in the
Documents/Gene Codes/Sequencher/ folder. This location is referred to as the Home
Directory. In the Sequencher directory, you will find a series of folders containing the results of

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any alignments and analyses performed using those external algorithms. In these folders, you
will find a folder for each run you perform. The alignment and analyses run folders have a
unique name assigned to them ensuring that previous runs are never overwritten. The GSNAP
database/BWA index folders will be given the name you assign to them.
If you wish, you may change the location of this folder by going to the External Data pane in
User Preferences.
Click on the Browse… button, browse to the desired location, and click the OK (Windows) or
Choose (Mac) button. You may even create a new folder at your chosen location. Click the
Make New Folder (Windows) or New Folder (Mac) button, then give it a name and click the OK
(Windows) or Choose (Mac) button. The User Preferences pane will remember the new
location of the Gene Codes Home Directory.
Table 16-1 Location of results in External Data Home

Algorithm Type Folder in External Data Home

Maq Reference-guided aligner Maq
BWA Index Reference sequence indexes BWA_Indexes
BWA-MEM Reference-guided aligner BWA
GSNAP Databases Reference sequence database GSNAP_Databases
GSNAP Reference-guided aligner GSNAP
Velvet De novo assembler Velvet
Cufflinks RNA-Seq Cufflinks
Cuffmerge RNA-Seq merge of transcript.gtf Cuffmerge
Cuffdiff RNA-Seq differential expression Cuffdiff
Cuffquant RNA-Seq quantification Cuffquant
Cuffnorm RNA-Seq normalisation Cuffnorm
MUSCLE DNA multiple-sequence alignment MUSCLE

Figure 16-4 Setting the Home directory

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Note: If you have already generated some results in the old location, you will need to move
their corresponding Run folders from their current location to the new location so that
referential integrity between the project, the contigs within it, and their Run folders is
maintained.

STRATEGIES

Before you start, review how to use the External Data Browser. This is your access point to all
the analyses you perform whether you are creating reference sequence databases/indexes or
are using DNA-Seq or RNA-Seq tools. You can monitor and annotate your runs, view log files,
delete old runs, or launch Tablet (where appropriate) to view alignments. For more
information, see the next section entitled “External Data Browser”.
No matter how well prepared your samples were, it is always a good idea to check the quality
of your reads data. Look at the section “FastQ Quality Control Reports” for more information
on how to do this with Sequencher.
The alignment strategy itself can be divided into several parts. The first part consists of
choosing whether you are going to perform reference-guided alignment or de novo assembly.
If you are performing reference-guided alignment, then you will have a choice of algorithm.
You also need to choose a reference sequence, and one or two reads files (in FastA or FastQ
format), depending on whether you are working with single-end or paired-end data. If you
want to, you can also perform SNP, methylation analysis, or RNA-tolerant editing analysis at
the same time as the reference-guided alignment (GSNAP only). If you are working with de
novo assembly, then you only need to know whether you are working with single-end or
paired-end data (in FastA or FastQ format).
Sequencher can load your aligned results into the Tablet genome browser automatically.
Whether you are performing an alignment or an assembly, you will therefore need to decide
whether you want to view the alignment or assembly result immediately after the alignment
has completed, using Maqview (Maq only) or Tablet, or to view the results later.
If you choose to perform any of the additional analyses, you can also view the results of your
analysis later, using your web browser (the default viewer) or a text editor since the results are
saved in the associated Run folder in both formats.
If the results are brought into the Project Window, you can also look at the results within your
Sequencher project. You can use Sequencher’s features to examine the Overview or produce a
Summary Report.

EXTERNAL DATA BROWSER

You can launch the External Data Browser using the Window>Open External Data Browser
menu item. This opens the Sequencher External Data Browser dialog which consists of a
button bar, a top pane divided into a number of columns, a lower pane, and finally some
additional buttons. However, most of the time, you will not need to use this command as the
External Data Browser opens automatically whenever you use any of the DNA-Seq or RNA-Seq
tools such as GSNAP or Cufflinks.

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USING THE BUTTON BAR

The button bar contains two types of controls; the first set is a set of buttons that perform
specific functions such as Open Run Folders or View Using Tablet, while the second set is a set
of drop-down menus that act to filter out specific algorithms from the Runs folder list. Clicking
on an algorithm name in the drop-down menu toggles between removing and restoring the
runs for that algorithm in the Runs list.
Figure 16-5 The External Data Browser

RUNS PANE

This part of the dialog contains six columns. The first column lists all of the runs in your
Sequencher External Data Folder (unless you have filtered one or more algorithms). The
header of this column lists the number of runs available for viewing. This can be less than the
total number of runs if you have already filtered out a specific algorithm. The second column
contains the date and time the run was initiated. The third column lists the algorithm used for
that particular run. The fourth column gives the size of the run folder and its contents. Column
five is called Final Run Status and shows whether the run was a SUCCESS or if it FAILED. The
final column contains any notes you may have added to the run information. Clicking the
header name of any column will sort that column in ascending or descending order.

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LOG FILE/NOTES PANE

The lower pane will list the contents of the log file for any selected run. To alternate between
the log file view and the editable notes area, click the Log File button or the Notes button.
When you want to add notes, click on the row of interest and then click on the Notes button
below the pane. A blank pane is revealed in place of the Log File view and a new Save button
appears. You can type text notes into this pane. Clicking the Save button saves the notes and
clicking the Refresh button refreshes the overall view. The Auto Refresh On control is checked
by default, refreshes will happen automatically. The notes will be listed in the Run row for
which they were entered.
Runs which generate a database/index will have a note automatically added. This Note
contains the name of the reference sequence used to build the database/index.
You can always leave the External Data Browser by clicking on the Close button.

PERFORMING REFERENCE-GUIDED ALIGNMENTS

Reference-guided alignment differs from de novo assembly in that the reads are only
compared to the reference sequence and not each other or nascent contigs. The actual
alignment process forms part of a workflow which starts with the choice of reference sequence
and algorithms. Figure 16-6 A generalized reference-guided alignment workflow gives an
overview of the reference-guided workflow starting from the choice of a Reference Sequence.

FASTQ QUALITY CONTROL REPORTS

Before you start to work with your reads, it is always a good idea to check their quality.
Sequencher makes use of the FastQC application to provide a series of reports on your FastQ
data.
Choose Sequence>Analyses>FastQ Quality Control Report…. A new window appears. Click on
the Add File button and browse to the file(s) you want to analyse.

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 Figure 16-6 A generalized reference-guided alignment workflow

Select the file you want to add and click on the Open button. The file will now be listed in the
Quality Control Using FastQC dialog. If you want to remove a file, highlight it and then click on
the Remove File button. The results are saved automatically in HTML format in the FastQC
Reports folder within your Documents folder. You can change the default location for your
results by clicking on the Change Location… button and browsing to a new location. To view
the results immediately after the analysis, click on the Yes radio button in the View Results
groupbox. If you don't want to view the results immediately, then click on the No radio button,
otherwise there are no other options to be set.
Note: If you have added 4 or fewer than 4 files to be analyzed, their reports will be opened
automatically once the analysis is complete. If you have added greater than 4 files to analyze,
and have specified to view them immediately after the analysis, you’ll be presented with the
Report Selection dialog asking you to select the reports you want to view at the end of the
analysis.

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 Figure 16-7 Quality Control Using FastQC

If you have previously created results you want to see, use the View>Display FastQC Report…
menu item. A new window opens, choose the reports you want to open and click on the Open
button. Whether you choose to see the analyses immediately or review them later, each
report will open in its own window. On the left-hand side of the window is a clickable list of the
analyses performed. The status of each test is indicated by a traffic lights system with a green
check for pass, amber exclamation point for a warning, and a red cross for a failed test. You
can find out more about each test by consulting the online documentation - FastQC Analysis
Modules.

CREATING A REFERENCE SEQUENCE DATABASE OR INDEX

As the cost of sequencing has dropped and our ability to produce both longer reads and
sequence longer genomes has increased, scientists have looked for ways to decrease the
amount of time it takes to align each read on a reference sequence. With modern reference-
guided aligners, the first step is to create a reference sequence database or index. The
principle behind this is to enable the actual mapping of reads to the reference sequence to be
speeded up. There are many techniques for doing this and each aligner will have its own.
From Sequencher 5.4.1 and later, you can now create databases/indexes that can be re-used.
If you choose a sequence from your Project Window, you can choose to create a permanent
database/index or just have Sequencher delete the database/index for you at the end of the
alignment. For large genomes, having the database/index saved is especially useful since the
time taken to build can be in the order of an hour (depending on the speed of the hardware
used and the available RAM). Moreover, once a database/index has been built, you can share it
with other Sequencher users.

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 Figure 16-8 FastQC Report

You can use a single sequence in FastA format or a series of sequences in concatenated FastA
format (for example a series of chromosomes). If you plan to perform an in silico experiment
such as comparing the BWA-MEM algorithm to the GSNAP algorithm, you will need to build a
database/index for both aligners.
The files for the database/index are placed in the External Data Home folder (the default
location for this is your Documents folder but you may change this through a User Preference).
You will be able to give the database/index a name of your own choosing. In addition,
information about the database/index run will be visible in the External Data Browser window.
Choose Assemble>Build Reference Database or Index>GSNAP Database… to use a reference
sequence with the GSNAP algorithm. If you want to use the reference sequence with BWA-
MEM, choose Assemble>Build Reference Database or Index>BWA Index…. The External Data
Browser window opens automatically with either command.

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 Figure 16-9 Build Reference Database or Index menu item

A new dialog appears. The look of the dialog is similar in both cases but the title of the dialog
is dictated by the algorithm being used to generate the database/index. Click on the Reference
FASTA button and browse to the FastA file containing your reference sequence(s). Click on the
Open button. Give your database or index a name and click on the Build button.
Figure 16-10 Build BWA Index dialog

If using a reference sequence from a selection in the Project Window, the following dialog will
appear instead. In this case, as you can see, the reference sequence name replaces the
Reference FASTA button.

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 Figure 16-11 Build BWA Index with selection from Project Window

When you are using the External Data Browser to monitor the progress of the build (especially
useful with large genomes), note that the log file pane refreshes automatically if the Auto
Refresh On control is checked.
Once Sequencher has finished the creation of the database or index, you will see a green
SUCCESS status in the Final Run Status column. If there was a problem with the creation of the
database or index, you may see a red FAILED status. Consulting the log file will often give you a
clue as to the reason for the failure. Very rarely, there will be no status message at all. This is
also an indicator of problems with the creation/build run.
The Notes field allows you to add pertinent information about your reference sequence. This is
a good place to record information such as the build version for complete or partial genomes.
Sequencher will automatically add the name of the sequence file used to create the database
or index to the Notes field.
Once the database/index is built, you will see it listed in a drop-down menu the next time you
use GSNAP or BWA-MEM.

REFERENCE-GUIDED ALIGNMENT USING THE MAQ ALGORITHM

The Maq reference-guided aligner is an older aligner but is kept for legacy purposes. Compared
to other aligners, it has limitations. The major limitation is the number of reads (two million) it
can deal with before its performance seriously degrades. This makes it impractical for large
projects.
Launch Sequencher and import your Reference Sequence into a new project. You could use
any sequence in your Sequencher project to act as the reference. It does not have to be
marked as a Reference Sequence using the Sequence>Reference Sequence command first.

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 Figure 16-12 GSNAP Database and BWA Index listings in the External Data Browser

Select the Reference Sequence and choose Assemble>Align Data Files to Ref Using>Maq...

Figure 16-13 Choosing a sequence to act as the reference from the Project Window

Choose your first reads file by clicking on the Select File 1 button to browse to the file you want
to use. Select the file and click on the Open button. Now choose the second reads file by
clicking on the Select File 2 button and click on the Open button in the same manner as for the
first set of reads.
Select the viewer you want to use by clicking on its radio button. With Maq, you have the
choice of the Maqview or Tablet viewers.

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 Figure 16-14 Choosing a viewer

Once you have clicked on the Align button, the alignment will take place. The consensus
sequence that results is then formed into a contig with the reference sequence. If you chose to
view your alignment immediately after the algorithm was finished, then the viewer you
selected will launch automatically with the contig already loaded. If you prefer to view it later,
then choose Contig>Show NGS Data Using>Maqview.
If you choose Maqview, then a Terminal window will open and display the reads. Maqview has
different views, to move between these views, use the function keys on your keyboard. F1
switches to the bases mode, F2 switches to the colored blocks mode, and F3 switches to the
overview mode. In the F2 and F3 views, you can zoom in and out using + and – keys.

Figure 16-15 Maqview reads directionality

REFERENCE-GUIDED ALIGNMENT USING GSNAP OR BWA

When working with large NGS projects, you should use either BWA-MEM or GSNAP. There are
two ways that you can provide a reference sequence, the first is to use a sequence from your
current project (see Figure 16-13 Choosing a sequence to act as the reference from the Project
Window) or you can use a prebuilt database/index. In most cases, these will be large
sequence(s)/genome(s) which have been used to create the prebuilt database/index. Choose
the database/index you want to use from the BWA-MEM or GSNAP dialog.
If you are going to use a sequence from your project, simply select it and then choose
Assemble>Align Data Files to Ref Using>GSNAP... or choose Assemble>Align Data Files to Ref
Using>BWA-MEM.... The name of the reference sequence will appear automatically in the
Current reference seq or index (for BWA) or Current reference seq or db (for GSNAP) drop-
down menu of the aligner’s graphical user interface. If you are working with a prebuilt
database, then choose it now from the Current reference seq or db or Current reference seq
or index drop-down menu.

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 Figure 16-16 Choosing the GSNAP alignment algorithm

Once the dialog has appeared, you will be able to choose the reads files and make changes to
the various options and settings. If you want to use a prebuilt database/index, then choose
Assemble>Align Data Files to Ref Using>GSNAP... or choose Assemble>Align Data Files to Ref
Using>BWA -MEM... and then pick a reference sequence database/index from the drop-down
menu at the top of the dialog. All databases or indexes for your chosen algorithm will be listed
in this drop-down. The External Data Browser launches automatically with either of these
commands.
Figure 16-17 Choosing reads files – BWA-MEM with chosen index

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 Figure 16-18 Choosing reads files – GSNAP with menu showing available databases

Note: If you have created databases/indexes in a different location than your current External
Data Home setting, they will not be listed.
Once you have chosen your database/index, you can go on to choose your reads files and
change any settings or options. When you are satisfied with your selections, click on the Align
button to initiate the alignment.

LOOKING AT REFERENCE-GUIDED ALIGNMENT RESULTS

If you chose to view your alignment at some other time by choosing the None option, you can
view it at any time by choosing Contig>Show NGS Data Using>Tablet (which can be used with
BWA-MEM, GSNAP, or Velvet).
If you chose Tablet as your view option, your results will be displayed in Tablet. You view a
contig by clicking on its name in the contig list at the left-hand side of the window. You can
move about and change the look of the view. For example, using the Zoom slider increases and
decreases the size of the reads bases.
Using the Variants slider increases or decreases the background grey on which the bases are
displayed so that moving the slider to the extreme right will effectively mask normal bases
while potential SNPs will be highlighted. Clicking on the Direction button in the Color Schemes
tab changes the color of the bases from their normal individual base color to dark blue or khaki

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depending on the direction of the read. The Classic view shows the bases without any
background coloration. In order to return to the bases with colored backgrounds, click on the
Nucleotide button in the Color Schemes tab.
Figure 16-19 Tablet viewer

ADVANCED PARAMETERS FOR GSNAP

When you send your reads to GSNAP, Sequencher is protecting you from having to work from
the command line.

Figure 16-20 Example of a command line

GSNAP has many parameters that can be set providing you use the command line. Sequencher
has a novel GUI interface that allows you to choose advanced parameters and change their
settings.
In order to see what the advanced parameters are, click on the Advanced (Edit) button. The
GSNAP Advanced Options dialog appears. This is in the form of a table with 3 columns. The
first column contains a checkbox and parameter, the second column contains the value of that
parameter, and the third column contains its description.

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 Figure 16-21 GSNAP Advanced Options dialog

In order to use a parameter, simply click on the checkbox so that a check appears. If you want
to change the value, then click in the Value cell for that parameter and edit the value. If you
are unable to edit the value or the value cell is empty initially, it may be that this parameter is
a flag. That is to say, it has no value but is used in an on or off state.
Figure 16-22 Enabled parameters

Below this table are two buttons, a + (plus) and a – (minus). These are used to add or remove
parameters, values, and their descriptions.
Beneath this is a text field titled Current Parameters. Any changes to the checkbox will cause a
parameter to appear or disappear from this text field. Items in this text field will become part
of the command line that Sequencher composes on your behalf. Together with the values that
Sequencher controls, these values are sent to the GSNAP program.

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If you find that you have removed a parameter by mistake or wish to restore all the settings to
their original values, click on the Restore Defaults button.

GSNAP AND INDELS

The GSNAP algorithm allows you to control the appearance of insertions and deletions using
several different parameters. These parameters can be set using the Advanced Options. The
parameters you can set include --indel-penalty and --min-coverage.
Figure 16-23 Enabled parameters allowing indels in alignment

In addition to setting these penalties, you also need to set a value in the Insert Threshold text
field in the Options group box on the Align Using GSNAP dialog. The default value is 10 which
is the same as the Tablet value. This means that at least 10 reads containing this insertion are
required in order to see the insert in Tablet. If you change the value in Sequencher, there is a
possibility that the views will diverge.
Note: In order to change the insertion threshold in Tablet, visit its home page at
http://bioinf.scri.ac.uk/tablet/

Figure 16-24 Sequencher Insertion Threshold in GSNAP dialog

ADVANCED PARAMETERS FOR BWA-MEM

BWA-MEM is able to align reads between 70bp and 1Mbp. While BWA-MEM does not have
quite the same sophistication when it comes to controlling indels as GSNAP, it does have some
advanced options which are worthy of note. To see what the advanced parameters are, click
on the Advanced (Edit) button on the Align Using BWA-MEM dialog. The BWA Advanced
Options dialog appears. This is in the form of a table with 3 columns with a pane below it. The

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first column contains a checkbox and parameter, the second column contains the value of that
parameter, and the third column contains its description.
In order to use a parameter, simply click the checkbox so that the check mark appears. If you
want to change the value, then click in the Value cell for that parameter and edit the value. If
you are unable to edit the value or the value cell is empty initially, it may be that this
parameter is a flag. That is to say, it has no value but is used in an on or off state. Below this
table are two buttons, a + (plus) and a – (minus). These are used to add or remove parameters,
values, and their associated descriptions.
One example of controlling BWA-MEM using the Advanced Options is the Band Width, when
this parameter is chosen and a value given, the BWA-MEM algorithm will ignore gaps longer
than this value although other aspects of the algorithm also have some effect.
If you are working with paired-end (PE) data, you can use the Paired End Mode to use a more
rigorous algorithm to rescue missing hits, that is, reads that have not already been aligned and
properly paired.
If you prefer not to find too many unmatched reads pairs, you can choose an option to
penalize unpaired read pairs. A default value is provided but you can change this if you prefer.
Beneath the table containing the Advanced Options is a text field entitled Current Parameters.
A change to one of the checkboxes in the table will cause a parameter, and its value if it has
one, to appear or disappear from this text field. Items in this text field will become part of the
command line that Sequencher composes on your behalf. Together with the values that
Sequencher controls, these values are sent to the BWA-MEM program. If you find that you
have removed a parameter by mistake or wish to restore all the settings to their original
values, click on the Restore Defaults button.

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 Figure 16-25 BWA Advanced options

FURTHER ANALYSES WITH REFERENCE-GUIDED ALIGNMENTS

SNP HUNTING WITH MAQ

To perform this analysis, select your Reference Sequence from the Project Window. Now
choose the command to perform SNP analysis with Maq, Assemble>Align Data Files to Ref
Using>Maq… . The Align Using Maq dialog will appear.

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From this dialog, click on the Select File 1 button and browse to the reads file you want to use.
If you are working with paired-end data, then click on the Select File 2 button and browse to
the second reads file you want to use.
Figure 16-26 Align Using Maq dialog

Choose the SNP Analysis Report option by clicking on the SNP Analysis Report checkbox.
Finally, choose your viewing options. The results are saved as a contig in your project and a
SNP report in the Gene Codes/Sequencher/Maq folder. If you chose Tablet as your view
option, follow the instructions for using Tablet above. If you chose None, then you can view
the contig assembly in Tablet (Contig>Show NGS Data Using>Tablet) or in Maqview
(Contig>Show NGS Data Using>Maqview) at a later time.
To view the SNP report results, select the contig of interest and choose the
Sequence>Analyses>Maq SNP Report command. The results will be displayed in your default
web browser. The first column is the name of the Reference Sequence, the second column is
the position of the reference base, next is the reference base itself followed by variant base.
Column 5 contains a Phred-like quality value which will assist in determining the reliability of
the SNP. Column 6 gives you the read depth. When the table reports 1.00 in column 7, this
means that the region is near enough unique. The final columns relate to the second best and
third best base calls at this position.

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 Figure 16-27 Explanation of MAQ SNP report fields

SNP TOLERANT ALIGNMENT WITH GSNAP

GSNAP performs SNP hunting in a different way than Maq. You need to supply a file that
contains a list of known SNPs, this has to be in a specific format which will be described below.

Figure 16-28 Format of known SNPs file

The file is a text file and each SNP is placed on a separate line. Look at the image above.
Each line must begin with the > character. This is followed by an identifier, in this case it is a
number preceded by the word Mycoplasma_5’. Next comes the Reference Sequence name
and positional information in the format name:xxx..xxx. If there are spaces in the name, these
should be replaced by underscores. The position information in this file always assumes that
the first base of the Reference Sequence in Sequencher is 1, no matter its actual numbering
relative to its chromosomal or contig position.
To perform the analysis, you need to select a Reference Sequence from the Project Window or
a prebuilt database/index from the drop-down menu on the GSNAP dialog. Choose
Assemble>Align Data Files to Ref Using>GSNAP…. The Align Using GSNAP and External Data
Browser dialogs will appear. Click Select Reads File 1 and browse to the first reads file you
want to use. Click on Select Reads File 2 if you are using paired-end data. Now choose SNP-
Tolerant alignment from the Additional Analysis groupbox. The Known SNPs File button is
now enabled. Click on this button and browse to the file containing your list of known SNPs.

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 Figure 16-29 Setting GSNAP SNP-tolerant alignment

Choose also whether or not you want to see the results file in Tablet immediately after the
alignment has finished. If you choose to see the results immediately, the results file will be
opened in Tablet if it is installed. The results file will also be saved in the Run folder. Now click
on the Align button to initiate the analysis. You can review the results later by choosing the
contig of interest in the Project Window and choosing the Sequence>Analyses>GSNAP SNP
Analysis command.

METHYLATION STUDIES WITH GSNAP

Studies of methylated parts of the genome involve using bisulfite treatment of the DNA
followed by sequencing of the regions of interest. The bisulfite treatment chemically converts
unmethylated Cs to Ts. GSNAP is capable of aligning the sequenced reads to the genomic
(untreated) sequence.

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To perform the analysis, you need to select a Reference Sequence from the Project Window or
a prebuilt database/index from the Align Using GSNAP dialog and one or two (if paired-end)
FastA or FastQ files containing reads from bisulfite treated DNA.
Load the Reference Sequence into Sequencher and ensure it is highlighted in the Project
Window. Choose Assemble>Align Data Files to Ref Using>GSNAP…. The Align Using GSNAP
and External Data Browser dialogs will appear. If you are working with a prebuilt database,
choose it now from the Current reference seq or db or index drop-down menu.
Next, click on the Select Reads File 1 button and browse to the first reads file you want to use.
Click on the Select Reads File 2 button if you are using paired-end data. Two protocols are
supported, referred to as stranded (for protocols which allow only 5’ -> 3’ RNA) and non-
stranded (both sense and antisense reads are allowed). In Sequencher, you choose the
stranded mode by clicking on the Methylation stranded. 5’ -> 3’ both strands, no reverse
complement radio button. To choose the non-stranded protocol, click on the Methylation
non-stranded. 5’ -> 3’ both strands, with reverse complement radio button.

Figure 16-30 GSNAP methylation settings

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You can review the results later by choosing the contig of interest and choosing the relevant
Sequence>Analyses>GSNAP Methylation Analysis drop-down menu item. The results will
appear in your default browser.
Figure 16-31 Results File for Methylation Analysis

The image above explains the various parts of the report. The most informative part of the
report is the character circled in red and called unmethylated. The . (period) character
indicates a C in the Reference Sequence which occurs opposite a T in the query read. This
indicates that this C was unmethylated and hence open to modification by the bisulfite
reagent.

RNA A-TO-I TOLERANT ALIGNMENT WITH GSNAP

RNA editing is a process where some adenosines in mRNA are converted to inosine by
adenosine deaminase acting on RNA (ADAR). The mutations caused by this process have been
associated with diversification of the transcriptome. Aberrant activity is associated with a wide
variety of human diseases, regulation of splicing and disease pathologies. The changes caused
by ADAR activity may be detected using NGS and looking for adenosines that have been
converted to guanosines.
To perform the analysis, you need to select a Reference Sequence from the Project Window or
a prebuilt database/index from the Align Using GSNAP dialog and one or two (if paired-end)
FastA or FastQ files. Load the Reference Sequence into Sequencher and ensure it is
highlighted in the Project Window. Choose Assemble>Align Data Files to Ref Using>GSNAP….
The Align Using GSNAP and External Data Browser dialogs will appear. If you are working with
a prebuilt database, then choose it now from the Current reference seq db or index drop-
down menu. From the dialog, click on the Select Reads File 1 button and browse to the first
reads file you want to use. Click on the Select Reads File 2 button if you are using paired-end
data.
In order to use the alignment mode that is tolerant to the A-to-G changes correctly, you need
to know which laboratory protocol was used in preparing your reads. Two protocols are
supported, you may see these referred to as stranded and non-stranded. In Sequencher, you
choose the stranded mode (for protocols which allow only 5’ -> 3’ RNA) by clicking on the RNA-
Tolerant stranded. 5’ -> 3’ both strands, no reverse complement radio button. To choose the
non-stranded protocol (both sense and antisense reads are allowed), click on the RNA-
Tolerant stranded. 5’ -> 3’ both strands, with reverse complement radio button.
After you have set any other options or advanced options, you launch the analysis by clicking
on the Align button.

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You can review the results later by choosing the contig of interest in the Project Window and
choosing the Sequence>Analyses>GSNAP RNA-Tolerant Analysis menu item. The results will
appear in your default browser.
Note: If you are in Viewer mode and you request an HTML report for a newly run GSNAP SNP
Tolerant alignment, Methylation Analysis, or RNA-Tolerant alignment, you will see an example
report.
Figure 16-32 RNA-Tolerant stranded alignment

VARIANT CALLING WITH SAMTOOLS

You can now use SAMtools to search for variants in any alignment where the results have been
saved in SAM or BAM format. SAMtools does this by changing the mapped data to genome or
chromosome numbering (based on your reference). It then calculates the Bayesian prior
probability from the data. Next, SAMtools uses the prior probability distribution and the data
to produce a genotype which is filtered and written out to a VCF file. You can view the VCF file
and your aligned data in any genome browser that reads SAM and VCF formats.

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There are two paths you can take for analysing your SAM/BAM files. In the first path, open a
project containing contigs which have been created from aligned NGS data. Highlight a single
contig, then choose Sequence>Analyses>SAMtools Variant Calling… A dialog opens. Notice
that the path information for the SAM file and the reference sequence are already filled in. The
second method is to choose Sequence>Analyses>SAMtools Variant Calling… and when the
dialog opens, use the buttons to browse to your SAM/BAM file and then browse to your FastA
reference sequence.
There are three options for you to set, the Minimum Read Depth, the Maximum Read Depth,
and the Minimum Number of Alternate Reads Supporting Alternate Bases.
The Minimum Read Depth is self-explanatory. The Maximum Read Depth is used to provide a
threshold since SNPs with very high coverage might represent variation from variable copy
number repeats. The parameter should be altered to suit the coverage in your data. The
Minimum Number of Alternate Reads Supporting Alternate Bases parameter is designed to
set a lower threshold for the alternate reads supporting each alternative allele. Once you are
happy with your selections, click on the Analyze button. A VCF (Variant Calling Format) file will
be created in the same folder as the SAM/BAM file you have just analyzed.
Figure 16-33 Variant Calling with SAMtools dialog

VIEWING VCF FILES IN TABLET

You can view the results using the Tablet genome browser. If you are working in a project and
have aligned your reads and called the variants as described above, you can review the
alignments as follows, highlight one contig in your project and choose Contig>Show NGS Data
Using>Tablet. The Tablet genome browser will launch and the aligned reads will be loaded into
the browser. You now load the VCF file into the browser by clicking on the Import Features
button. Click on the second tab to see a list of the variants and their locations. Click on a
variant to jump to its location in the contig. You can see an example of this in the image below.
Note: You can also load other VCF files and GTF files using this same method. This will give you
a rich representation of known features in your assembled reads.

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 Figure 16-34 Example of VCF file import in Tablet with features from GTF file

REFERENCE-GUIDED ALIGNERS AND MULTIPLEX IDS

Multiplexing reads is a way of maximizing reagent and sequencer use by mixing a number of
separate samples together in one flowcell (for example) and sequencing them at the same
time. The DNA from each sample is tagged with a unique DNA identifier. After the sequencing
run, the reads are separated using the unique DNA identifier.
Sequencher can perform this separation or demultiplexing of the reads automatically for you.
This is known as binning and the resultant files are called bins. Sequencher then takes each bin
and aligns the reads it contains against the chosen Reference Sequence.
You need to supply a Reference Sequence, one FastA/FastQ file containing reads from a
Multiplex ID experiment if you are working with single-end sequences, or two FastA/FastQ files
containing reads if you are working with paired-end sequences.

FORMAT OF BARCODE FILE

You will also need to supply a text file containing the barcodes used in this Multiplex ID
experiment. This file is called the barcodes file and it contains one barcode name and

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sequence per line. The file can accommodate a single comment line where the comment is
preceded by a # mark.

Figure 16-35 Format of barcodes file

Note: The sequence should consist of the tag and barcode if the tag has not been removed by
your sequencing pipeline.

ENABLING MULTIPLEX ID MODE

First you will need to enable the Multiplex ID mode by choosing Multiplex ID from the drop-
down menu on the Project Window button bar.

Figure 16-36 Drop-down Mode menu

Once you have enabled this mode, a new column appears in the Project Window, this is called
MID and will contain the name of the barcode that originally formed part of the reads aligned
to create this contig.

Figure 16-37 Project Window view showing MID column

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If the Multiplex ID mode is still enabled when you save and close your project, Sequencher will
remember this setting for you.

RUNNING GSNAP WITH MULTIPLEX DATA

Load the Reference Sequence into Sequencher and ensure it is highlighted in the Project
Window or choose a database/index from the Align Using GSNAP dialog. Choose
Assemble>Align Data Files to Ref Using>GSNAP…. The Align Using GSNAP with MID and
External Data Browser dialogs will appear. Now click on the Select Reads File 1 button (if using
single-end reads) and browse to the reads file you want to use. Click on the Select Barcodes
File button and browse to your prepared barcodes file. Once you are ready, click on the Align
button.

Figure 16-38 Align Using GSNAP with MID dialog

A new dialog appears (below). This dialog is divided into two panes. The top pane shows a
constantly updated status line for the overall process. This pane also contains a Cancel button,
if you click this button, you will cancel the current and all subsequent operations for the
current MID run. The lower pane is a table that is divided into a number of columns (see figure

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below) showing the name of the Barcode, the number of reads in the bin, the % of the total
reads sequenced that this represents, the status of the specific bin alignment, and finally a link
to the log file. Click on this link to open the log file in the default viewer.

Figure 16-39 Multiplex ID with GSNAP status report

There is also a Close button that becomes enabled when the entire process has completed.
Click on the Report button to obtain a formatted Multiplex Status Report that will open in a
Report Preview window. You can save or print this report.
When you return to the Project Window, you will see the consensus sequence for each bin
that has been returned to Sequencher and assembled to the Reference Sequence in order to
create a contig. In the case of the above image, if 14 files containing reads were created during
de-multiplexing, then 14 contigs will be created within Sequencher.

RUNNING BWA-MEM WITH MULTIPLEX DATA

Load the Reference Sequence into Sequencher and ensure it is highlighted in the Project
Window or choose a database/index from the Align Using BWA-MEM dialog. Choose
Assemble>Align Data Files to Ref Using>BWA-MEM…. The Align Using BWA-MEM with MID
and External Data Browser dialogs will appear. Now click on the Select Reads File 1 button (if
using single-end reads) and browse to the reads file you want to use. Click on the Select
Barcodes File button and browse to your prepared barcodes file. Once you are ready, click the
Align button.

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 Figure 16-40 Align Using BWA-MEM with MID dialog

A new dialog appears (see similar figure above, Figure 16-39 Multiplex ID with GSNAP status
report).
This dialog is divided into two panes. The top pane shows a constantly updated status line for
the overall process. This pane also contains a Cancel button, if you click this button you will
cancel the current and all subsequent operations for the current MID run. The lower pane is a
table that is divided into a number of columns showing the name of the Barcode, the number
of reads in the bin, the % of the total reads sequenced that this represents, the status of the
specific bin alignment, and finally a link to the log file. Click on this link to open the log file in
the default viewer.
There is also a Close button that becomes enabled when the entire process has completed.
Click on the Report button to obtain a formatted Multiplex Status Report that will open in a
Report Preview window. You can save or print this report.
When you return to the Project Window, you will see the consensus sequence for each bin
that has been returned to Sequencher and assembled to the Reference Sequence in order to
create a contig. In the case of the above image, if 14 files containing reads were created during
de-multiplexing, then 14 contigs will be created within Sequencher.

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PERFORMING DE NOVO ASSEMBLIES

GSNAP and BWA-MEM are great programs for reference-guided alignment, that is to say you
have a sequence that is identical or closely related to the region you are sequencing. If you are
sequencing an entirely new organism for example, or sequencing a new region that has not
been sequenced before, you may not have a reference sequence available. In this case, you
will need to perform a De novo assembly. Sequencher allows you to perform a De novo
assembly using the Velvet algorithm.

Figure 16-41 Velvet de novo assembly workflow

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ASSEMBLING SINGLE-END DATA WITH VELVET

Since this is a De novo assembly, you do not need a Reference Sequence. Choose
Assemble>Assemble Data Files Using>Velvet…. The Assemble Using Velvet and External Data
Browser dialogs will appear. Click on the Select File 1 button and browse to the reads file you
want to use. Hash Length is also known as k-mer or word length. If a DNA string can be
considered to consist of a series of characters (with an alphabet of four letters), then a word/k-
mer/hash length will be a string of characters of a specified length.
The hash length is one of the key parameters for Velvet and is set in the Assemble Using
Velvet dialog. The hash length must be an odd integer (23, 35, or 47 for example) and this
value should be shorter than most of the reads in the reads file. Anything shorter than this
value will be ignored. Anything equal to or longer than this value will be used in the assembly.
The longer the hash length, the more specific it is. The shorter the hash length, the more
sensitive it is. Finding the best hash length will require a balance between specificity and
sensitivity. Enter the new value in the Hash Length input field and then run the assembly by
clicking on the Assemble button.
Once the assembly is complete, the dialog is automatically dismissed and a number of
sequences will appear in the Project Window. Each sequence will represent the consensus of a
contig created by the Velvet application, of which there may be many. It may be worth using
Sequencher’s built-in assembly algorithms to form contigs from some or all of these consensus
sequences.
If you want to review the contigs from which these consensus sequences were derived, then
highlight one consensus sequence and choose Contig>Show NGS Data Using>Tablet.
Figure 16-42 Setting the Hash Length for Velvet in the Velvet dialog

ASSEMBLING PAIRED-END DATA WITH VELVET

The Velvet assembler also works with paired-end data. Select menu command
Assemble>Assemble Data Files Using>Velvet…, then choose the two paired-ends reads files by

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clicking on the Select File 1 button and then the Select File 2 button. Next, choose whether you
want to view the results after assembly by selecting the Tablet or None radio button. Finally,
click on the Assemble button.
Figure 16-43 Choosing to view the reads of a highlighted consensus in Tablet

If you know that your paired files have already been merged into a single file, then follow these
steps instead. Click on the Select File 1 button and locate the merged file. Next click in the File
1 is paired reads checkbox. The hash length is also known as k-mer or word length. If a DNA
string can be considered to consist of a series of characters (with an alphabet of four letters),
then a word/k-mer/hash length will be a string of characters of a specified length.
The hash length is one of the key parameters for Velvet and is set in the Assemble Using
Velvet dialog. It must be an odd integer (23, 35, or 47 for example) and this value should be
shorter than most of the reads in the reads file. Anything shorter than this value will be
ignored. Anything equal to or longer than this value will be used in the assembly. The longer
the hash length, the more specific it is. The shorter the hash length, the more sensitive it is.
Finding the best hash length will require a balance between specificity and sensitivity.
Enter the new value in the Hash Length input field. Choose your viewing options and then click
on the Assemble button.

LOOKING AT VELVET RESULTS IN TABLET

Once you have submitted your data, the results come back into Sequencher as a series of
consensus sequences representing each contig constructed by Velvet. If you want to explore
the underlying data contributing to the consensus, then you need to use the Tablet browser.
To do this, you need to select the consensus whose contig you want to explore, then choose
menu command Contig>Show NGS Data Using>Tablet. The contig will open in the Tablet
browser.

ADVANCED PARAMETERS FOR VELVET

In order to see what the advanced parameters are, click on the Advanced (Edit) button. The
Velvet Advanced Options dialog appears. This is in the form of a table with 3 columns. The first
column contains a checkbox and parameter, the second column contains the value of that
parameter, and the third column its description. These parameters are specific to Velvetg. In
order to use a parameter, simply click on the checkbox so that a check appears. If you want to
change the value, then click in the Value cell for that parameter and edit the value. If you are
unable to edit the value or the value cell is empty initially, it may be that this parameter is a
flag. That is to say, it has no value but is used in an on or off state.

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 Figure 16-44 Enabling parameters and setting values in Velvet Advanced dialog

Below this table are two buttons, a + (plus) and a – (minus). These are used to add or remove
parameters, values, and their descriptions.
Beneath this is a pane titled Current Parameters. Any changes to the states of checkboxes will
cause a parameter to appear or disappear from this pane. Items in this pane will become part
of the command line that Sequencher composes on your behalf. Together with the values that
Sequencher controls, these values are sent to the Velveth program.

Figure 16-45 Add and Remove parameters with Preview window

If you find that you have removed a parameter by mistake or wish to restore all the settings to
their original values, then click on the Restore Defaults button. This will restore the
parameters to the values that were set before you started editing.

USING SEQUENCHER TO IMPROVE A VELVET ALIGNMENT

To do this, you will need to select the consensus sequences you want to merge. Then choose
an assembly algorithm and assembly parameters by clicking on the Assembly Parameters
button that you can find on the Project Window button bar. Now click the Assemble
Automatically button. Sequencher will use the parameters you set and try to assemble the
reads. You will generally find some improvement in your results. However, setting the

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parameters to be too relaxed would not result in an improvement. The overlaps would
probably contain too many gaps or mismatches.

VELVET AND MULTIPLEX IDS

Multiplexing reads is a way of maximizing reagent and sequencer use by mixing a number of
separate samples together in one flowcell, for example, and sequencing them at the same
time. The DNA from each sample is tagged with a unique DNA identifier. After the sequencing
run, the reads are separated using the unique DNA identifier.
Sequencher can perform this separation or demultiplexing of the reads automatically for you.
This is known as binning and each reads file is called a bin.
You need to supply a FastA or FastQ file containing reads from a Multiplex ID experiment, and
a text file containing the barcodes used in this Multiplex ID experiment. This file is called the
barcodes file and it contains one barcode name and sequence per line. The file can
accommodate a single comment line, where the comment is preceded by a # mark.

FORMAT OF BARCODE FILE

Figure 16-46 Format of barcode file

Note: The sequence should consist of the tag and barcode if the tag has not been removed by
your sequencing pipeline.

ENABLING MULTIPLEX ID MODE

First you will need to enable the Multiplex ID mode by choosing Multiplex ID from the drop-
down menu on the Project Window button bar.

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 Figure 16-47 Drop-down Mode menu

Once you have enabled this mode, a new MID column appears in the Project Window that will
contain the name of the barcode that originally formed part of the reads aligned to create the
consensus sequence. Depending on the settings you have chosen, there may be a number of
consensus sequences that had the same barcode.

Figure 16-48 Project Window view showing MID column

If this mode is still enabled when you save and close your project, Sequencher will remember
this setting for you.

RUNNING VELVET WITH MULTIPLEX DATA

The first step is to click on the Assembly Mode drop-down menu which is found on the Project
Window button bar. Next select the Multiplex ID menu item. Choose Assemble>Align Data
Files Using>Velvet…. The Assemble Using Velvet with MID and External Data Browser dialogs
will appear. Click on the Select Reads File 1 button and browse to the reads file you want to
use. Click on the Select Barcodes File button and browse to your prepared barcodes file. Once
you are ready, click the Assemble button.
The Assembling MID Data with Velvet dialog appears. This dialog is divided into two panes.
The top pane shows a constantly updated status line for the overall process. This pane also
contains a Cancel button. If you click on this button, you will cancel the current and all
subsequent operations for the current MID run.

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 Figure 16-49 Assembling MID data with Velvet Status Report

The lower pane is a table that is divided into a number of columns (see figure above) showing
the name of the Barcode, the number of reads in the bin, the % of the total reads sequenced
that this represents, the status of the specific bin alignment, and finally a link to the log file.
Click on this link to open the log file in the default viewer.
There is also a Close button that becomes enabled when the entire process has completed.
Click on the Report button to obtain a formatted Multiplex Status Report that will open in a
Report Preview window. You can save or print this report.
When you return to the Project Window, you will see that one or more consensus sequences
for each bin have been brought into Sequencher. Check the MID column, this holds the name
of the barcode for each consensus sequence. You can click on the MID column header to sort
the column by barcode name so all consensus sequences belonging to a particular barcode
name will be grouped together.
You can review your results in Tablet. To do this, you need to select the consensus in the
Project Window whose contig you want to explore, then choose menu command
Contig>Show NGS Data Using>Tablet. The contig will open in the Tablet viewer.

RNA-SEQ AND DIFFERENTIAL EXPRESSION

RNA-Seq is an alternative to microarray analysis for analyzing gene expression. RNA is

extracted from the tissue or sample under examination and then sequenced using NGS
technology. This produces a snapshot of the RNA in the sample. The millions of reads produced
means a greater depth of coverage is achievable using NGS which in turn may enable a user to
find rare transcripts.

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We have added the Cufflinks1 suite of programs enabling you to perform RNA-Seq analysis on
your own computer. The Cufflinks suite is a series of command line programs. We have added
a graphical user interface to these programs and their options, as well as adding plots and
charts so that you can avoid using the command line altogether. You will need to align your
RNA-Seq read data using GSNAP or BWA-MEM or another aligner that can produce results in
SAM or BAM format and then perform the RNA-Seq analysis on these files.
In the simplest workflow for analyzing RNA-Seq data to test for differential expression, using
the Cufflinks suite of programs is a three-stage process with the first step being repeated. The
reason for this repetition is two-fold. First, assembly is more computationally expensive as we
get increases in read numbers. So the first step, which requires a SAM/BAM file and a GTF
annotation file, is performed on one set of data at a time. The second reason is that, with a
pooled mixture of samples, Cufflinks faces the problem of trying to sort out and select from a
complex mixture of spliced isoforms. It is easier to do this on one data set at a time.

RNA-SEQ WORKFLOW

In the first step of the RNA-Seq workflow, the Cufflinks program quantifies the expression level
of a single sample. This first stage is then repeated for each sample in your experiment. In the
second stage, the transcript annotation results from analyzing each sample are merged.
Finally, in the third stage, differential expression is analysed and reported. This final step
consists of testing the statistical significance of each observed change in expression between
the samples. The model evaluates changes assuming that the number of reads produced by
each transcript is proportional to its abundance. The final output contains information
including log2(fold_change), p_values, and q_values, and attributes such as gene_id and
chromosomal location.
This is the simplest form of the workflow. However, it is possible to split this workflow so that
the quantification of the number of reads aligned per transcript is undertaken by Cuffquant.
This reduces the computational burden normally imposed by having the Cuffdiff program
perform this step. This is also a necessary step if, instead of performing differential expression
tests, you are simply looking at transcription levels. In this case, you would use Cuffnorm to
normalize expression values after the quantification step.

1
Trapnell C, Williams BA, Pertea G, Mortazavi AM, Kwan G, van Baren MJ, Salzberg SL, Wold B, Pachter L. Transcript assembly
and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation Nature
Biotechnology doi:10.1038/nbt.1621

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 Figure 16-50 RNA-Seq workflow using Cufflinks suite

WHAT IS A GTF FILE

GTF stands for Gene Transfer Format. It is a type of annotation file with information in a
tabbed text format. Unlike the more familiar GenBank format, each line contains one feature
and its ancillary information.
Table 16-2 GTF file fields and description

Column Description

Seqname Name of the chromosome or scaffold chromosome names can be

given with or without the 'chr' prefix.

Source Name of the program or data source that generated this feature.

Feature Feature type name, e.g. Gene

Start Start position of the feature

End End position of the feature

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 Score A floating point value

Strand Defined as + (forward) or - (reverse)

Frame One of '0', '1', or '2'. '0' indicates that the first base of the feature is
the first base of a codon and so on.

Attribute A semicolon-separated list of tag-value pairs of additional information

The image below shows a few lines from a GTF file. This was obtained from UCSC and is part of
the annotation for human chromosome 1. It shows four feature types, exon, start codon, stop
codon, and CDS. Columns four and five contain the start and end positions of each feature.
Column seven indicates that these features are all on the forward (+) strand. Column eight
indicates that, for only two of the three CDS features, the first base of the position is also the
first base of the codon.

Figure 16-51 Example of GTF file for human genome

OBTAINING GTF FILES FROM UCSC

We will only talk about the procedure for obtaining a GTF file from the UCSC browser but there
are other resources from which you can obtain this type of annotation file, such as ENSEMBL.
Go to the UCSC browser website at http://genome.ucsc.edu/. You will see a menu bar just
below the words USCS Genome Bioinformatics. Click on the item called Tables. This will take
you to a new web page with the options you need to set for obtaining your GTF file.
Choose the correct Clade, Genome, and Assembly you want to use from the drop-down menus
provided. Set the Group and Track types if required. Now set the Region. If you are choosing a
position on a chromosome, you will need to specify it in the following manner: chrX:1-
150000000, for example. Ensure the Output format is GTF - gene transfer format. Do not click
in any of the checkboxes to the right of this menu as you will be downloading the file, not
sending it to another website.
If you are not setting any other values on the Table Browser, then give your file a name by
typing in the Output file input field. If the file is likely to be large, then also click on the gzip

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compressed radio button. Most computers are able to open this type of file. Finally, click on
the Get Output button. The file will be downloaded to your default downloads location.
Figure 16-52 UCSC Table Browser

OBTAINING A FASTA FILE OF TRANSCRIPTS

The process is similar to that for obtaining the GTF file. Follow all the steps until you get to the
Output format, this time choose sequence as your format. When you click on the Get Output
button, you will be directed to a new webpage. Here you will be asked to choose between
genomic, protein, or mRNA. Finally, click on the Submit button. The file will be downloaded to
your default downloads location.
Figure 16-53 Choosing an export format for the UCSC browser

MANAGING YOUR RNA-SEQ ANALYSES WITH THE EXTERNAL DATA BROWSER

The results for each step of the RNA-Seq workflow can be found in individual run folders in
your External Data Home folder. The default location is in your Documents folder. Contained
within the Documents folder is another folder called Gene Codes. Inside that folder you will
find the Sequencher folder which holds the Cufflinks, Cuffmerge, Cuffdiff, Cuffquant, and
Cuffnorm folders.
The External Data Browser is an important tool when managing any of your NGS or RNA-Seq
analyses and results. You can track the progress of a current run by viewing its log file in the
External Data Browser log file pane. You can add notes to remind you of important details
relating to the data files you used or even experimental details such as the source of the data.
All Run folders are assigned names containing random alphanumeric characters to prevent you
from accidentally over-writing results which may have taken hours to gather. The notes you

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add will enable you to distinguish which results should be merged together during the RNA-Seq
workflow as well as allowing you to review your results at a later date.
The Extenal Data Browser is launched automatically whenever you use any of the RNA-Seq
tools and also by going to the Window menu and choosing Open External Data Browser. The
dialog that opens contains a button bar, a table of your current NGS and RNA-Seq runs, and a
lower pane which can be used to display the log file for the chosen run or any notes you have
made. The drop-down menus labeled DNA-Seq, RNA-Seq, and MSA are used to filter out Run
folders associated with the different analysis types. If you click on the DNA-Seq drop-down
menu, you can click on any item to remove or include a specific set of runs in the browser.
They are not removed from your computer. If you want to remove a Run from your computer,
click on the Run name and then click Delete Run.
Figure 16-54 The External Data Browser

Once you have started a new analysis, you can see its progress in the External Data Browser.
The log file pane updates automatically when the Auto Refresh On control is checked or by
periodically clicking on the Refresh button. This will update the log file view so you can see the
exact progress of your analysis.

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 Figure 16-55 The External Data Browser displaying log from running Cufflinks algorithm

The log file may accumulate many rows of information so be aware that you might need to
scroll down to the bottom of the file in order to see the most recent information.
Figure 16-56 External Data Browser viewing a log file

To add a note to your run, click on the Notes tab and enter the information. Click on the Save
button and then click on the Refresh button. The new Notes information will now appear next
to the Run information in the Notes Preview column. If you have entered several lines of
information, it may not all appear in the default table view but it will be saved in the body of

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the Note and will be seen in a tooltip for the note as you hover over the note field. You can
also drag the window out to change its size or proportions.
Figure 16-57 The External Data Browser - adding a note

CUFFLINKS

A requirement of quantifying expression levels is to identify accurately which reads are

produced by a given isoform. Cufflinks uses RNA-Seq reads previously aligned to a genome and
assembles individual transcripts from that data. The program attempts to infer the splicing
structure for each gene. However, many alternate splicing events are possible, so Cufflinks
takes a frugal approach when assembling the transcriptome assembly. It reports as few
transcript fragments as possible when trying to satisfy the explanation of the possible splicing
events in the original data.
In order to run Cufflinks, a reference file is required. This reference will be a GTF format file,
not the more familiar GenBank file. This is used to guide the initial re-alignment of your data.

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This type of alignment is called Reference-Annotation Based Transcript Assembly2. The GTF file
provides information on all reference transcripts as well as any novel genes and isoforms that
are assembled.
Alternatively, you can use a GTF file simply to estimate isoform expression only. Transcripts
that cannot be explained by the reference transcripts file are ignored.
It is also possible to tell the program to ignore certain transcripts, such as mitochondrial
transcripts, or other transcripts you wish to ignore in your analysis. This is called the mask file
and is also in GTF format.
Cufflinks includes an option to detect and correct any bias and this can improve overall
estimates of transcript abundance. This file is a FastA format file and generally contains
multiple sequences.
This first step, aligning a SAM and GTF file, needs to be repeated one or more times for each of
your sample data sets. The output from this step is taken forward to the next stage. This
strategy is adopted over a pooled read method because it reduces the computational cost, and
additionally it lowers the complexity of splice events that need to be accounted for, reducing
the probability of incorrect transcript assembly.
To run Cufflinks, go to the Assemble menu and choose RNA-Seq Using Cufflinks…. The dialog
in the following figure appears along with the External Data Browser. Click on the Select SAM
or BAM File button and navigate to the first of the files that you are going to analyse. You will
also need to provide a GTF file and optionally a FastA file if you are performing fragment bias
correction. You will need to decide whether you are performing a reference-guided alignment
looking for all transcripts including novel ones, estimating isoform expression, or masking out
certain transcripts such as those emanating from mitochondria or highly abundant genes such
as actin. Then choose the appropriate button. You will also need to choose a library type from
that drop-down menu.

2
Roberts A, Pimentel H, Trapnell C, Pachter L. Identification of novel transcripts in annotated genomes using RNA-Seq
Bioinformatics doi:10.1093/bioinformatics/btr355

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 Figure 16-58 Cufflinks dialog

Each run will produce a transcripts.gtf file amongst its output files, it is these files that are
required for the next step with Cuffmerge. Each Cufflinks run automatically creates a separate
run folder with a unique name. You will find the transcripts.gtf files in the Run folders situated
within the Cufflinks folder of your External Data Home folder.

ADVANCED OPTIONS FOR CUFFLINKS

Cufflinks is provided with a rich set of options. These can be found by clicking on the Advanced
(Edit) button. You will see a new dialog (see below). To select an option, click on the checkbox
adjacent to the option you wish to use. The parameter will be added to the Current
Parameters window at the bottom of the dialog.
Some parameters require you to change a value. For example, the default value for –frag-len-
mean is 200 but, in this example, the user has changed it to 300. Others are on/off switches. In
this example, the user has specified the use of multi-read-correct which is used when reads
map to more than one location in the genome and a more accurate method of assigning reads
to locations is required. Once you have chosen all the Advanced Options you wish to apply,
click on the OK button. The Advanced Options dialog closes. Click on the Analyze button on
the RNA-Seq Using Cufflinks dialog to start the analysis of your data

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 Figure 16-59 Cufflinks Advanced Options

VIEWING CUFFLINKS RESULTS FILES

Cufflinks produces four main results files. These are called skipped.gtf, transcripts.gtf,
isoforms.fpkm_tracking, and genes.fpkm_tracking. The skipped.gtf file contains any skipped
loci, this depends on the maximum number of fragments a locus may have, which is set in the
Advanced (Edit) options. The transcripts.gtf file contains isoforms detected by Cufflinks and is
used in the Cuffmerge step. The isoforms.fpkm_tracking file contains the estimated isoform
expression values. The genes.fpkm_tracking file contains the estimated gene expression
values.
If you are not studying differential expression or you want to view the intermediate steps in
the differential expression pipeline, you can view the contents of the .fpkm_tracking files and
explore the results as a table and bar chart. To view the contents of the two fpkm_tracking
files, use Sequencher’s View>Display RNA-Seq Data and Plots… menu command.
When you hold the cursor over a bar in the bar chart, the FPKM value will be displayed.
Clicking on the bar will display and highlight the data row linked to that bar. We have also
added a colored background to the column from which the data is taken. As well as a gene ID,
the row also contains the gene short name, its locus, the length of the transcript, and FPKM
value.

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 Figure 16-60 Cufflinks Bar chart

CUFFMERGE

In this stage, the program takes the output files from the repeated Cufflinks runs and merges
them. You provide Cuffmerge with the transcripts.gtf files from each of your Cufflinks runs
and a GTF reference file. You can optionally provide Cuffmerge with a FASTA file for excluding
artefacts and classifying transcript fragments. Cuffmerge then produces a ‘consensus’ GTF file
that can be used by Cuffdiff to estimate differential expression.
Cuffmerge performs a Reference-Annotation Based Transcript assembly, producing a single
merged.gtf file from the transcripts.gtf files and the reference GTF file you provided.
Choose the Merge Cufflinks Alignments with Cuffmerge… menu item from the Assemble
menu. The following dialog appears along with the External Data Browser. Click on the Add
File button and navigate to the Cufflinks folder and choose a Run folder, double-click on the
Run folder to open it and select the transcripts.gtf file. Click on the Open button. Repeat these
steps for each transcripts.gtf file you want to merge.

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Choose the reference GTF file you intend to use by clicking on the Select GTF Reference File
button. Browse to your reference GTF file and, once you have selected it, click on the Open
button. Set any other options and then click on the Merge button.
This step produces the merged.gtf file that you will use in the final step with Cuffdiff. You will
find these files in the Cuffmerge folder in your External Data Home folder. Each Cuffmerge run
automatically creates a separate Run folder with a unique name.
Figure 16-61 Cuffmerge dialog

CUFFDIFF

In the final stage, the merged.gtf file and the original SAM/BAM or CXB files are analysed to
find genes that are differentially expressed. The results files contain statistical information such
as p-values and log2(fold change) in expression between the samples. The outputs (.diff files
and .fpkm_tracking files) from this stage may be viewed as tables or charts.
Cufflinks has the ability to filter out transcripts that are very low in abundance. The authors
explain that this is due to the fact that analyzing expression, at such low levels, is not reliable.
You can change the threshold for filtering out these low-abundance transcripts by altering the
value of the minimum isoform fraction.
Cuffdiff supports different library types and you need to know which protocol was used to
generate the data you are analyzing. The standard Illumina protocol is specified by fr-

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unstranded and the standard Solid protocol is specified by fr-secondstrand. You can find more
details in the Cufflinks online manual3.
Go to the Assemble menu and choose the RNA-Seq Differential Expression Using Cuffdiff…
menu item.
Figure 16-62 Cuffdiff dialog

You will need to tell Sequencher which SAM/BAM or CXB files you used in the Cufflinks or
Cuffquant steps. Click on the Add/Remove Input Files button. The Add Conditions and
Replicates dialog opens. Click on the Add Input File button and browse to the location of the
file you want to add. The files you select with the Add Input File button will appear in the Input
File Name list. If you chose the wrong files, simply highlight them and click on the Remove
Input File button to delete them from the list. Initially, each file will have a default condition
label of Condition 1. You can edit this as you add files or change the label after you have added
all your files. Change a Condition Label by double-clicking on it and entering a new name in the

3
http://cufflinks.cbcb.umd.edu/manual.html

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Edit Condition Label dialog. Click on the OK button to dismiss the dialog. To dismiss the Add
Conditions and Replicates dialog, click on its OK button.
Figure 16-63 Add Conditions and Replicates editing a conditions label

You will notice that the status of a number of items in Differential Expression Using Cuffdiff
dialog have changed. These status changes display the list of files you have added for analysis,
whether you have fulfilled the requirements for differential expression (at least two
conditions), and whether your data includes replicates. Click on the Select Merged GTF File
button and navigate to the location of the merged.gtf file for your data. You will find it in the
Cuffmerge Run folder that was created from the corresponding Cuffmerge run. These Run
folders are in your External Data Home folder. If you are providing a FASTA file for fragment
bias correction, click on the Select Reference FASTA File button, browse to the location of the
FASTA file, and click on the Open button. Cuffdiff tries to correct4 for positional fragment bias
(a local effect) where reads are located preferentially at the beginning and end of a transcript,
as well as sequence-specific bias (a global effect).
Ensure that you have the correct values set for Library Type, Dispersion Method, and Library
Normalization Method by making selections from the drop-down menus. If your data is from a
time-series, then click on the Treat As Time Series button. Now click on the Analyze button.

4
Adam Roberts, Cole Trapnell, Julie Donaghey, John L. Rinn and Lior Pachter, Improving RNA-
Seq expression estimates by correcting for fragment bias Genome Biology, Volume 12, R22
(2011)

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ADVANCED OPTIONS FOR CUFFDIFF

Cuffdiff is provided with a rich set of advanced options. These can be found by clicking on the
Advanced (Edit) button on the Differential Expression Using Cuffdiff dialog.
You will see a new dialog (see below). The table in the dialog contains a list of the options,
descriptions, and checkboxes. To select an option, click on the checkbox adjacent to the option
you wish to use. The parameter will be added to the Current Parameters window at the
bottom of the dialog.
Some parameters require you to change a value. For example, the default value for --frag-len-
mean (fragment mean length for unpaired reads) is 200 but in this example the user has
changed it to 300.

Figure 16-64 Status changes on Cuffdiff dialog

Other options are on/off switches. In the example below, the user has specified the use of --
multi-read-correct which is used when reads map to more than one location in the genome
and a more accurate method of assigning reads to locations is required. Once you have chosen
all the Advanced Options you wish to apply, click on the OK button.

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The Cuffdiff Advanced Options dialog closes. Click on the Analyze button on the Differential
Expression Using Cuffdiff dialog to start the analysis of your data.
Figure 16-65 Cuffdiff Advanced Options

VIEWING RNA-SEQ DIFFERENTIAL EXPRESSION RESULTS

Once Cuffdiff has completed its run, you will find a new Run folder has been created within the
Cuffdiff folder located in your External Data Home folder. This run folder contains 23 files.
These files contain information on FPKM tracking, Count tracking, Read group tracking,
Differential expression, Differential splicing, Differential coding sequence output, Differential
promoter use, Read group info, and Run info. The files that contain information on differential
expression testing all have the file extension .diff.

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 Table 16-3 Differential Expression results files

Diff Read FPKM Count Info

group tracking tracking

CDS ✔ ✔ ✔ ✔

Isoform ✔ ✔ ✔ ✔

Gene ✔ ✔ ✔ ✔

Tss ✔ ✔ ✔ ✔

Promotors ✔

Splicing ✔

Bias params ✔

Run ✔

Read groups ✔

Var model ✔

Note: Some files may not contain any results. This does not mean the analysis has failed and is
dependant on the initial analysis choices or data.
You can view data from files with a .diff file extension by going to the View menu and selecting
the Display RNA-Seq Data & Plots… menu item.

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 Figure 16-66 Display RNA-Seq plots & data in view menu

A file picker dialog appears, browse to a .diff file such as gene_exp.diff or isoform_exp.diff and
click on the Open button. A new window opens with a table of your data in the top pane and a
plot in the lower pane. Note that if there is no differential expression reported, then the plot
will be empty. Check by scrolling down through the table of data.

THE VOLCANO AND SCATTER PLOT

There are several plots you can view. The Volcano Plot is a type of scatter plot where the
log2(fold-change) is plotted on the x axis and the –log(p-value) is plotted on the y axis. Spots
that are further left or right of the origin have a greater fold-change. Spots that have a higher
value on the y-axis are likely to have a more significant p-value.
To assist in locating the data used to create the plot, we have added colored backgrounds to
those columns. When your cursor moves over a spot, it changes from an arrow to a hand, if
you click on it the spot turns red. The hand-shaped cursor also indicates that, when you click
on the spot, its data row will be highlighted in the table. Notice also that when you click on a
data row, if the data has been plotted it will be highlighted with a red spot.

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 Figure 16-67 Differential Expression Volcano Plot

Another plot you can use is the standard Scatter Plot, this takes the log10 FPKM values from
the two samples in a .diff file and plots them against each other. When your cursor moves over
a spot, it changes from an arrow to a hand, if you click on it the spot turns red. The hand-
shaped cursor also indicates that, when you click on the spot, its data row will be highlighted in
the table. Notice also that when you click on a data row, if the data has been plotted, it will be
highlighted with a red spot.

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 Figure 16-68 Differential Expression Scatter Plot

Note: The title bar of each plot is the name of the file used to create it.

THE BAR CHART

There is also a Bar Chart. This plot displays changes in expression based on the FPKM metric.
This is defined as Fragments Per Kilobase of exon per Million fragments mapped. You can
switch to this by clicking on the Bar Chart tab. The axes displayed in this chart are log10(FPKM)
vs gene_id or gene.
As you move your cursor over a bar, its FPKM value is displayed in a tool tip. If there are two
bars plotted for a particular gene or locus, by moving the cursor over each bar, you can
compare the FPKM values. When you click on a bar, its data row is highlighted in the table and
it is marked with a red arrow. Likewise, if you click on a data row and its data has been
plotted, it will be highlighted with a red arrow. Note that we have added colored backgrounds
to those columns whose data is plotted in the chart.

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 Figure 16-69 Differential Expression Bar Chart

SEARCHING FOR SPECIFIC GENES IN A RESULTS TABLE

Depending on your experiment, the results table could contain tens of thousands of rows. You
can search for specific genes in your results table by typing a few characters into the Search by
Gene input field. As you type a few characters, you will see that a status message reports the
number of items matching those characters. You can navigate the list of items using the two
buttons to the right of the Search by Gene search field. The status message indicates which hit
is currently selected from the hit list.

SORTING AND PLOTTING SELECTED RESULTS

While you may want to view all your results in the Volcano or Scatter Plot, you may want to
plot a subset of results in the Bar Chart. As a prelude to plotting only some of your results, you
can sort the data table simply by clicking in the header row of your chosen column. You can
now select the rows that you want to include or exclude from the plot by using Shift+click or
Ctrl/Cmd+click. Once you are satisfied with the selection, choose Selected or Unselected from

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the Plot menu. You can restore all the plottable items to the plots by clicking on the Plot All
button.
When mixed letters and numbers are sorted on a computer, the ASCII sort order is used.
Suppose you had three items called A1, A2, and A11. These would be sorted A1, A11, A2 by a
computer although you might consider the correct order to be A1, A2, A11. Clicking on the
locus column in the results table will cause it to be sorted in this fashion. To avoid this
problem, we have created the Chromosome Order button. Click this button when you want to
restore the original chromosomal locus sort order.
Figure 16-70 Plot All and Chromosome Order buttons

THE ALTERNATE ROUTE TO ANALYSING YOUR RNA-SEQ DATA

Performing RNA-Seq analysis, especially expression or differential expression with large data
sets and replicates, can be very compute intensive. It is not unusual for runs to take tens of
hours.
The Cufflinks suite contains two programs, Cuffquant and Cuffnorm, which can help to reduce
the analysis time-scale. Cuffquant performs some of the elements of Cuffdiff by quantifying
gene and transcript expression for a SAM/BAM file. The results are saved in the CXB format
which is a binary format like BAM files and not human readable like SAM files. The results can
then be sent to Cuffdiff and its run is both sped up and requires less memory.
Cuffnorm can also use the output from Cuffquant. Its function is to calculate normalized
expression values for genes and transcripts. It is not used for differential expression but is
designed to be used in the preparation of expression values for genes, transcripts, CDS groups,
or TSS groups.

CUFFQUANT

Go to the Assemble menu and choose the Quantify RNA-Seq Data Using Cuffquant… menu
item. For each Cuffquant run, you will need to tell Sequencher which SAM/BAM files you
analyzed in the Cufflinks step. Click on the Select SAM or BAM File button and browse to the
location of the SAM/BAM file. Click on the Open button. You will also need to provide a GTF
file for the reference annotation, and optionally a GTF Mask file for abundant transcripts or
FastA file if you are performing fragment bias correction, choosing the appropriate button(s).
You will also need to choose a library type from its drop-down menu.
Each run will produce a CXB file. The result of a Cuffquant analysis cannot be directly viewed,
instead it must be further analysed by Cuffdiff or Cuffnorm. Each Cuffquant run automatically
creates a uniquely named run folder within the Cuffquant folder of your External Data Home
folder.

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ADVANCED OPTIONS FOR CUFFQUANT

Cufflquant is provided with a set of Advanced Options. These can be found by clicking on the
Advanced (Edit) button. You will see a new dialog. To select an option, click on the checkbox
adjacent to the option you wish to use. The parameter will be added to the Current
Parameters window at the bottom of the dialog.
Some parameters require you to change a value. For example, the default value for –frag-len-
mean is 200. Others are on/off switches. Once you have chosen all the Advanced Options you
wish to apply, click on the OK button. The Advanced Options dialog closes. Click on the
Analyze button on the Quantify RNA-Seq Data Using Cuffquant dialog to start the analysis of
your data.

CUFFNORM

Go to the Assemble menu and choose the Normalization Using Cuffnorm… menu item.
You will need to tell Sequencher which SAM/BAM files you used in the Cufflinks step. Click on
the Add/Remove Input Files button. The Add Conditions and Replicates dialog opens. Click on
the Add Input File button and browse to the location of the SAM, BAM, or CXB file you want to
add. The files you select with the Add Input File button will appear in the Input File Name list.
If you chose the wrong files, simply highlight them and click on the Remove Input File button
to delete them from the list. Initially, each file will have a default condition label of Condition 1.
You can edit this as you add files or change the label after you have added all your files.
Change a Condition Label by double-clicking on it and entering a new name in the Edit
Condition Label dialog. Click on the OK button to dismiss the dialog. To dismiss the Add
Conditions and Replicates dialog, click on its OK button.

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 Figure 16-71 Cuffnorm dialog

You will notice that the status of a number of items in the Normalization Using Cuffnorm
dialog have changed. These status changes display the list of files you have added for analysis,
whether you have fulfilled the requirements for normalization (at least two conditions), and
whether your data includes replicates. Click on the Select Merged GTF File button and navigate
to the location of the merged.gtf file for your data. You will find it in the Cuffmerge Run folder
that was created from the corresponding Cuffmerge run. These Run folders are in your
Cuffmerge External Data Home folder. If you are providing a spike-in file to assist in
normalizing your data, click on the Select Spike-In File button, browse to the location of the
spike-in file, and click on the Open button.

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 Figure 16-72 Add Conditions and Replicates editing a conditions label

Ensure that you have the correct values set for Library Type and Library Normalization
Method by making selections from the drop-down menus. Now click on the Analyze button.

ADVANCED OPTIONS FOR CUFFNORM

There are only three advanced options for Cuffnorm, unlike the other members of the
Cufflinks suite. Probably the most important options are –compatible-hits-norm, which only
counts the fragments compatible with a reference transcript toward the number of mapped
fragments in the FPKM denominator, and –total-hits-norm, which counts all the fragments.

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 17. RESTRICTION MAPS

In this chapter, we discuss restriction maps and how to work with them. We discuss choosing
Restriction Map display options, selecting and changing selected enzymes, saving new or
modified enzymes, and copying the map.

DISPLAYING A RESTRICTION MAP

You can view a Restriction Map for a sequence or a contig by selecting its icon in the Project
Window and opening an editor. Go to the button bar of the editor and click on the Cut Map
button. Alternately, from the editor, go the View menu and choose Bases, Map, Overview, …,
then choose Restriction Map from the submenu.
Figure 17-1 shows a restriction map display. The number in parentheses shows the cut
location.
Figure 17-1 A single restriction map

In this type of restriction map, clicking the name of an enzyme highlights all the locations
where that enzyme cuts.

RESTRICTION MAP DISPLAY OPTIONS

HOW TO SELECT ENZYMES

To change the restriction map display, click on the Options button in the button bar or go the
View menu and choose View Options…
A dialog (Figure 17-2) lets you change several attributes of the restriction map.

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 Figure 17-2 Restriction Map Options

Use the Cutters: checkboxes to display enzymes according to the number of times they would
cut. For example, you could select just 2 Cutters, that is, only those enzymes that cut in two
locations. If you select the All button, all of the checkboxes are turned on.

HOW TO SHOW CUT POSITIONS OR FRAGMENT SIZES

Use the Show options to choose the Fragment Sizes or the Cut Positions display by clicking on
one of the radio buttons.

HOW TO SET THE MAP STYLE

Use a radio button from the Style: pane to choose one of the three basic map styles. The
Single Line map is the default display.
Choose Multiple Lines map (Figure 17-3) if you want to show each enzyme on its own line.

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 Figure 17-3 Multiple line Restriction Map

Choose the Text representation (Figure 17-4) to list the cut sites in tabular form. In this view,
the number in parentheses following each enzyme shows the number of times that particular
enzyme cuts.
Figure 17-4 Text Restriction Map

SETTING THE MAP WIDTH

The restriction map normally scales to the width of the window. When you print a restriction
map, it scales to the size of the printed page.
To set the width of the diagram in inches, turn off the Scale to window checkbox and use the
elevator button to set the width in inches (see Figure 17-5). This option is particularly useful
when copying a map to paste into a drawing or presentation program.

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 Figure 17-5 Map options showing width in inches

SETTING THE MAP CAPTION

Use the checkbox List enzymes names at bottom of map to toggle the caption at the bottom
of the restriction map. It lists all of the enzymes used to make the map (see Figure 17-5 above).
The caption also lists as “Non-Cutters” enzymes that were not mapped. For example, if you
were displaying only unique cutters, then enzymes that cut more than once would be
excluded. They would be listed as “Cutters that are not mapped.” Enzymes that did not cut at
all would be listed as “Non-Cutters.”

GETTING MORE INFORMATION ABOUT AN ENZYME

You can get more information on the enzymes in your restriction map or edit the enzymes
included in the map. In Figure 17-6, where the Multiple Lines style is being used, the SacI
enzyme is selected.
Figure 17-6 SacI enzyme selected

If you click on an enzyme label in the map and go the File menu and then choose Get Info…,
you can bring up the Restriction Enzymes dialog. You can also use the keyboard shortcut
Ctrl+I (Windows) or Cmd+I (Mac). Available enzymes appear in the scrolling list on the left side

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of the window. Sequencher highlights the enzyme you have selected and puts the appropriate
information in the Enzyme Name: and Recognition Sequence: fields.

CHANGING THE SELECTED ENZYME

You can also bring up the dialog by clicking on the Select Enzymes… button in the button bar
of any cut map. Alternately, you can go to the Window menu and choose Specify Restriction
Enzymes. The enzyme editor dialog is shown in Figure 17-7.
Figure 17-7 Restriction Enzymes Editor

Available enzymes appear in the scrolling list on the left side of the window. To view
information about a particular enzyme, click on its name. Sequencher highlights the enzyme
and puts the appropriate information in the Enzyme Name and Recognition Sequence fields.
If an enzyme is currently selected for display in the map, the bullet to the left of the name on
Mac is filled in or the checkbox to the left of the name on Windows will be filled in with an x.
An enzyme not selected for the map has an open bullet ‘o’ on Mac or an empty checkbox on
Windows. When you move the cursor to the bullet/checkbox column, it changes to a check
mark. To change your map selections, click on the bullets (Mac) or checkboxes (Windows) to
toggle them on or off. Figure 17-8 shows the bulleted list in detail.

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 Figure 17-8 Bulleted Enzyme list

If you are interested in enzymes with special characteristics, such as blunt ends or 3’
overhangs, explore the commands in the Select menu within the dialog.
Start by choosing Select None to clear all selections. Each menu item you choose after that
will add all of the enzymes with the specified characteristic. Selections are cumulative—no
command (except Select None) removes any enzymes from the list.
If, for example, you want all the enzymes that recognize sequences either 4 or 5 bases in
length, choose Select None followed by 4-base and then 5-base.
When you are finished selecting enzymes, click OK in the lower right corner of the dialog or
press the Return key. Sequencher redraws your restriction map with the newly selected
enzymes.

CHANGING THE DEFAULT ENZYMES

To change the default enzyme selections for items in a project, select the sequences or contigs
you want to change. Go to the Window menu and choose Specify Restriction Enzymes while
viewing the Project Window. Sequencher will warn you if no sequences are chosen. The
enzymes you choose at this time are now the default set for any selected sequences or contigs
in your project.

ADDING AND EDITING ENZYMES

Sequencher lets you add new enzymes to the list. Go to the top of the enzyme editor and
select Allow Changes. Click on New Enzyme to open a blank entry in the enzyme editor. Note
that the OK button changes to Done.
You can now enter information in the Enzyme Name and Recognition Sequence fields. When
you click Done, Sequencher adds the new enzyme to the list in alphabetical order.

CHANGING THE RECOGNITION SEQUENCE

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The recognition sequence field shows a double-stranded sequence. If you type in the sense
strand, the anti-sense strand will be calculated automatically.
Sliders above and below the recognition sequence window are used to move the lines which
indicate the cleavage positions. You can use the mouse to move the sliders left and right.
Note: The maximum length for a recognition sequence is fifteen bases. If you exceed this
length, the right end will be truncated.

SALT CONCENTRATION EFFECTS

You can record buffer sensitivity with the Effect of Salt Concentration: slider controls.
The default is an unspecified value for all concentrations of NaCI. Use the slider controls to
indicate the percentage activity for a given NaCl concentration.

SAVING NEW OR MODIFIED ENZYMES

If you enter an enzyme that you will want to use later, click Save Enzymes to save the enzyme
set as a file. Click Load Enzymes to retrieve a saved file.

SETTING THE SEQUENCE SELECTION IN A SEQUENCE EDITOR

When you click on the Cut Map button, a full-page cut-map is displayed. If you click between
two cut sites, Sequencher highlights the resulting restriction fragment. Hold down the Shift
key and click in the rectangle between other cut sites. Your selection is extended from the
original site you selected. When you toggle back to the Bases View, you will see that the
sequence representing the chosen region also has been highlighted.
If you wish to display the sequence and the cut map at the same time, click on the button
showing the staggered enzyme cut icon. A window with the restriction map appears. You can
adjust the size of the panes by dragging the splitter bar that separates the scroll bars for each
pane.
Like the full-page cut map, this window is linked to the Sequence Editor. If you click on the
name of an enzyme, Sequencher boxes the recognition sites. If you click between two cut
sites, Sequencher highlights the resulting restriction fragment.
Hold down the Shift key and click in the rectangle between other cut sites. Your selection is
extended from the original site you selected. You can copy the bases between the two cut sites
by going to the Edit menu and choosing Copy Selection.
You can use this command to excise a particular restriction fragment by clicking on it in the cut
map and switching back to the Bases View. Once you are in the Bases View, you can remove
the highlighted fragment by going to the Edit menu and using the Cut Selection command.

SETTING THE SEQUENCE SELECTION IN A CONTIG EDITOR

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When you are working in the Bases View of a Contig Editor, you can display a restriction
enzyme map by clicking on the Cut Map button in the editor’s button bar. You can also display
this view by going to the View menu, selecting Bases, Map, Overview, ..., and then choosing
Restriction Map from the submenu.
If you click between two cut sites, Sequencher highlights the resulting restriction fragment.
Hold down the Shift key and click in the rectangle between other cut sites.
Your selection is extended from the original site you selected. When you return to the Bases
View, you will see that the sequence representing the chosen region has been highlighted also.
(See Figure 17-9.)

COPYING THE MAP

After you have made a selection in the restriction map, as in Figure 17-9, you can copy the
bases between the two cut sites by going to the Edit menu and choosing Copy Selection.
You can also copy the entire diagram for use in an illustration program by going to the Edit
menu and choosing Copy As and Picture from the submenu.
Figure 17-9 Fragment selected in map view and its equivalent bases highlighted

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 18. SIMPLE BLAST

In this chapter, we describe how to use the built-in Blast feature to verify your sequence.
Depending on the speed of your internet connection, you will be able to query and verify short
sequences rapidly, locate features, check primers, and so on, whilst still working on your
project.

HOW TO USE BLAST WITH A SEQUENCE

In the image below, a sequence icon has been highlighted (indicated by red arrow). The entire
sequence will be BLASTed. To initiate the search, choose the Sequence menu and then select
the NCBI Blast Search menu item.
Figure 18-1 NCBI Blast menu command

HOW TO USE BLAST WITH A RANGE OF BASES

Instead of selecting a whole sequence, you may also choose to send part of a sequence. In this
image, hovering the mouse over a feature in the Overview gives the location of the feature
(yellow pop-up). You can then specify these coordinates by choosing the Select menu and then
clicking on the Bases By Number… menu item. Next choose the Sequence menu and then
select the NCBI Blast Search menu item.

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 Figure 18-2 Selecting bases for a BLAST search

HOW TO USE BLAST WITH A SPECIFIC SEGMENT OF SEQUENCE

You may also choose a subset of a sequence by highlighting bases in the Sequence Editor or
Contig Editor. Next choose the Sequence menu and then select the NCBI Blast Search menu
item to initiate the search.
This image shows this action (highlighted in blue) together with the BLAST Format Request
page which will be shown in your default web browser. Click on the View Report button in the
web page to see the actual results.
Figure 18-3 Selecting bases in a Sequence Editor for a BLAST search

In this image, you can see that a segment from a sequence in the Contig Editor has been
highlighted and sent to NCBI BLAST.

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 Figure 18-4 Selecting bases in a Contig Editor for a BLAST search

HOW TO USE BLAST WITH A CONTIG CONSENSUS

If you choose a contig icon from the Project Window and then choose the Sequence menu
and select the NCBI Blast Search menu item, then only the consensus of the contig will be
used as the search query.

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 19. MOTIFS AND FEATURES

In this chapter, we describe Sequencher’s ability to locate and highlight subsequences that you
specify as motifs. You will learn how to create, edit, and display features and list a sequence’s
features.

MOTIFS

Sequencher lets you define up to 12 patterns of 50 or fewer bases as motifs.

ENTERING MOTIFS

You define new motifs by going to the Window menu and clicking on the Motif Definitions…
command.
Figure 19-1 Motif entry window

The default setting shows exact matches only. To show the reverse complement for a motif,
click on the Find Reverse Complement checkbox. Use the radio buttons to specify whether
you want to highlight the motif using the exact bases you entered or using ambiguous
matches.
To specify how you would like the motif you are entering to be highlighted, click on the Display
Style pull-down menu (Figure 19-2) to choose color, case, and underlining while the cursor is
still blinking in the entry field.
In the example (Figure 19-2), every instance of ‘AAAAA’ will be underlined and every start
codon (ATG) will be shown in lower-case letters. You can save the motifs in a file on your hard
disk by clicking on the Save Motifs button. Enter a suitable name for your motif file in the Save
dialog.

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 Figure 19-2 Display style drop-down menu

QUICK MOTIF ENTRY

Select the bases in a Sequence Editor, then go to the Edit menu and click on Enter Selection
As Motif. If you choose this command, the default style will be used (see Chapter 23
“Customizing Sequencher and User Preferences”).

DISPLAYING MOTIFS

To use the motifs you have entered, go the View menu and choose Display Motifs.
Figure 19-3 shows how motifs are displayed in the Contig Overview: they are the small
downward facing colored blocks on the arrows representing the sequences. The colors are
those you set in the Motif Default Style menu (see Chapter 23 “Customizing Sequencher and
User Preferences”).
Figure 19-3 Overview showing motifs

In the Bases View of a sequence, motifs will appear highlighted in the manner you specified.
You may need to deselect Display Color Bases, Colors As Backgrounds, or Display Base
Confidences to see the Motifs more clearly. In Figure 19-4, for example, all start codons are in
lower case type and blue with only Colors as Backgrounds on.

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 Figure 19-4 Sequence Editor showing motifs

If you have a certain set of motifs you want to use for several projects (for example, if you use
motif definitions to highlight certain restriction sites), you can save your definitions to a file. To
do so, click Save Motifs at the top of the Motifs dialog and follow the instructions. Then click
on the Load Motifs button to use those definitions in other projects.

HIGHLIGHTING

The Highlighting aspect of Motifs gives you extended functionality where you will obtain a
colored base only if it meets certain criteria. To access Highlighting, you must first click on the
Highlighting button at the bottom of the Motifs dialog. Unlike Motifs which are restricted to a
palette of colors, with highlighting, you can use the system colors which greatly extends the
range of colors available to you.
To enable Highlighting, you must check the Enable Highlighting checkbox. Then choose
whether you wish to have any of the criteria or all of the criteria you choose met. Choose the
value from the Highlight bases that meet drop-down menu. Click on the color picker box to
assign your color. Now choose the criteria you want. For example, you may want to see how
many bases have a Phred score of 50 or above. Check the Confidence between checkbox and
then assign the confidence range values. Finally, decide whether you want the coloring to
override any other type of decoration, such as a feature, by clicking on the appropriate radio
button.

FEATURES

WHAT IS A FEATURE

A feature represents a subsequence or region of a sequence which has some biological

significance. Assigning a feature is a method of describing and naming such a region.
Sequencher supports both personal features and GenBank standardized features. The Feature
Editor enables you to name and highlight sequence regions of special interest.
Note: Features will not appear unless you go the View menu and select Display Features.

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CREATING AND EDITING FEATURES

To create or edit features in a sequence, double-click on the sequence icon. This will open a
Sequence Editor for your selected sequence. Go to the Sequence menu and choose Edit
Features… to display the Feature Editor (Figure 19-5).
Figure 19-5 Feature Editor dialog

To create a new feature, click on the Add button in the dialog. From the Feature Key: menu,
choose Sequencher if you are creating personal annotations, or choose one of the GenBank
keys.
If you have chosen a GenBank Feature Key:, Sequencher will insert a default name in the
Feature Name: field using the Feature Key and Feature Qualifier. You may edit this if you wish.
Enter the starting and ending base numbers for the feature in the Feature Location input
fields. The default strand is 5’-> 3’but you can change this using the Complement checkbox.
Use the Display Style pull down menu to specify strand type, color, case and underlining. You
may select one attribute from each of the four style categories. When you have finished
entering or editing your features, click Done.
Check the Display Feature in Editors checkbox and then go to the View menu and click on
Display Features to have your feature appear in the Sequence or Contig Editor and the
associated overviews. When you set a feature to include Protein or DNA-RNA, this will only be
displayed in appropriate views.

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 Figure 19-6 The Display Style menu

EDITING AN EXISTING FEATURE

To edit an existing feature, go to the Sequence menu and choose Edit Features… To display
the Feature Editor, click on the name of the feature in the feature list. Use the Display Style
pull-down menu to specify new display attributes. You can also change the Feature Key, edit
the Feature Name, and the Feature Location. For example, you could change a Sequencher
annotation into a GenBank feature.
If you want to remove a feature, select its name from the list and click on the Remove button.
When you have finished entering or editing your features, click Done. The features will appear
in the Sequence Editor if you have checked the Display Features in Editors checkbox and
Display Features in the View menu.

QUICK FEATURE CREATION

To create a feature quickly, select a region in the Sequence or Contig Editor and then go to
the Sequence menu and choose Mark Selection As Feature. Sequencher displays a dialog
called Mark Selection As Feature that lets you assign a Feature Key, name the feature, and
specify display attributes. When you have finished assigning the feature attributes click the OK
button.

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If you create a feature while selecting bases in the consensus of a contig, you will actually
create a feature for each of the sequences that contribute to the consensus at the selected
bases.
Note: If a sequence spans only a portion of the bases selected for a feature, that sequence will
not carry the new feature.
If you do not define display properties, the display attributes will default to the ones you
specified in the Feature, Motif section of the User Preference settings. (See Chapter 23
“Customizing Sequencher and User Preferences” for more information.)
Figure 19-7 The Mark Selection As Feature dialog

SHOWING AND HIDING FEATURES

Go to the View menu and use Display Features to toggle feature display on and off. For each
individual feature, you must also enable or disable its display in User Preferences. (See Chapter
23 “Customizing Sequencher and User Preferences” for more information.)

LISTING A SEQUENCE’S FEATURES

If you want to get a complete list of all your sequence’s features, go to the Sequence menu
and choose Feature Listing. This will display a text list of each feature giving its name, Feature
Key, range, style, and any Feature Qualifiers. You can print out this listing for your records.

LISTING SEVERAL SEQUENCE’S FEATURES

You can also view the listings for several sequences at once by selecting the sequences using
Ctrl+click (Windows) or Cmd+click (Mac). Then go to the Sequence menu and choose Feature

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Listing. A new dialog will appear asking if you want to open a feature listing window for each
of the selected sequences. Click on the OK button to see the listings.
Note: If you have imported a GenBank sequence and Sequencher does not recognize any
Feature Keys, this list may differ.

HOW FEATURES ARE DISPLAYED

When you annotate your sequence with features or if you have imported a sequence
containing a GenBank Feature Table, Sequencher will present a graphical representation of the
features under the display of overviews and restriction maps.
The graphical feature map has two parts. Above the sequences (red/green lines), the features
are presented on one line; in this example, it is the Reference Sequence which has been
marked up. Below the coverage map, the feature may be presented over several lines. You can
see this at the five prime end of the sequence in the image below, where there are three
features in blue, red, and green which overlap. In the bottom map, the features are displayed
on separate lines.
As you hold your cursor over a feature, a tooltip appears with information about that feature.
In the image above the cursor was left over the pink colored feature. The tool lets you know
that it is called AltIII and its range is from base position 16, 127 to 16, 208. Note also the green
features around base position 16, 642. Here the same feature exists on all the reads which
overlap at this position.
Figure 19-8 Overlapping Features on the Overview

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 20. FINDING ITEMS

In this chapter, you will learn how to find items according to specific criteria. It also discusses
how to refine your subsequence search and search for and find particular items in the Project
Window. This chapter also discusses how to find open windows and editors.

FINDING THE PROJECT WINDOW

You can always bring the Project Window to the front by going to the Window menu and
choosing Project Window.

FINDING OPEN WINDOWS WITH MENU COMMANDS

Sequencher has a number of commands in the Window menu for finding open windows
hidden behind other windows—Project Window, Variance Table, Translated Variance Table,
Chromatograms, Contig Editors, and Sequence Editors. Choose the appropriate command to
bring the window you want to the front of your screen. If one or another of the commands is
dimmed, you do not currently have those editors open. If you have several windows of a type
open, these will be listed as submenus of that type.

FINDING OPEN WINDOWS IN THE PROJECT VIEW

If you are looking at a Project Window, any icons that already have open editors are dimmed.
To move an open editor to the front, just double-click the dimmed icon, for example, the icon
for ABV shown in Figure 20-1 A dimmed icon.

FINDING THE CURRENT SELECTION

In a Sequence or Contig Editor, you can always scroll to the currently selected bases by using
the Enter key on the number keypad.
Figure 20-1 A dimmed icon

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SEARCHING IN OPEN WINDOWS

FINDING A SUBSEQUENCE

You can search for a specific subsequence in an open Sequence or Contig Editor. Go to the
Select menu and choose Find Bases…; Sequencher displays the Find dialog shown in Figure
20-2. In the Find What: input field, type the string of bases you want to find. Use the drop-
down menu to specify the recognition sequence of one of a subset of restriction enzymes
available. Use the Other option in the drop-down menu to type in the name of your enzyme of
interest. If it is in the database, the recognition sequence will be placed in the Find What:
input field.
Figure 20-2 Find Bases dialog

Find locates the first occurrence of the search string anywhere and then selects the string in
the editor. Find Next finds the next occurrence after the current selection, whether it is a
match found by Sequencher or a selection that you marked.
The Find Bases dialog stays in front until you click Done. If you want to repeat the same
subsequence search but without invoking the Find Bases dialog, you can go to the Select
menu and use the Find Bases Again command.

FIND AMINO ACIDS

Find Amino Acids is a protein identity search feature. You can use it to search in three or six
frames in a Sequence Editor or Contig Editor. You need to use the single letter amino acid
codes. As well as the standard set of codes, you can also use ? (question mark) or . (dot
character). The ? (question mark) is used when an amino acid cannot be determined from the
codon. The . (dot) is used to represent a stop codon. To search from the beginning of your
sequence(s), use the Find button. To search from the position of the cursor, use the Find Next
button. Click on the Done button when you have finished searching. If you later find that you
wish to repeat the search, you can use the Select>Find Again command. The results of the
search are shown in the Sequence or Contig Editor as a highlighted region.

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 Figure 20-3 Find Amino Acids dialog

REFINING A SUBSEQUENCE SEARCH

You can refine any of your searches by using one of the radio buttons. The default setting is
Exact Matches.

EXACT MATCHING

If you click Exact Matches, the search is based on exact character matches and disregards
matches that occur due to ambiguities. For example, AGGTW would only match AGGTW.

MATCHING AMBIGUOUS BASES

If you click Specified Bases, the search finds only matches that are identical or more specific
than the sequence you entered in the Find What: input field. For example, the string AGGTW
would match AGGTT or AGGTA, since W is the ambiguity code for T or A.

ANY AMBIGUOUS MATCH

If you click Any Ambiguous Match, the search will find any degenerate DNA that may match
the sequence you have entered, including matches consisting of all unknown bases. For
example, AGGTW would match NNGTN or even NNNNN.

INCLUDE REFERENCE SEQUENCE IN FIND

If you have a Reference Sequence assembled into your contig, then you have the option of
including it in your Find Bases search. Open the contig of interest to the Bases View and
choose Select>Find Bases. The Find Bases dialog opens, but with an additional checkbox called
Include Reference Sequence in Find. Click on this checkbox to include the Reference Sequence.

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FINDING BASES BY NUMBER

To select a range of bases quickly, go to the Select menu and choose Bases by Number. A
dialog lets you enter the range of bases you want to highlight.

EXTEND SELECTION

You can extend the current selection to the beginning or end of your sequence by going to the
Select menu and choosing Extend Selection. Then choose the suboption To Left End or To
Right End.

Note: In a Contig Chromatogram, the Extend Selection command can extend your selection
across the entire sequence in one click.

OTHER SEARCH OPTIONS

You can also search for ambiguous bases by going to the Select menu and using the Next
Ambiguous Base command. You can search for edited bases using the Next Edited Base
option. (These are described in more detail in Chapter 8 “The Sequence Editor”.) You can also
search for potential open reading frames using the Next Met to Stop (> 0b) option. (This is
described in more detail in Chapter 23 “Customizing Sequencher and User Preferences.”)

PROJECT WINDOW SELECTION

FINDING AN ITEM BY NAME

To locate a specific sequence or contig, click in the Project Window and type the name of the
item you are trying to find, for example, ‘Sequence C.’ As you type, Sequencher starts looking
in the Project Window for an icon with the name you are typing and highlights a match. If it
finds more than one, it highlights the first one.
Note: If you pause between characters for more than a few seconds, the program assumes
you’ve started typing a different name and looks for the new name.
Another way to locate a sequence is to go to the Select menu and choose Item Named.... This
brings up the dialog shown below.
Figure 20-4 Item Named… dialog

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Type the name of the sequence you want to find and click Find. Sequencher searches through
all the sequences displayed in the Project Window for a name that matches the name you
entered.

• When it finds a sequence, the sequence is displayed and highlighted in the

Project Window.

• If Sequencher does not find a sequence with the exact name you typed, it looks
for a sequence with a name starting with the first few letters you typed.

• If Sequencher still does not find a match, it looks at all the sequences contained
in contigs. If it finds a matching sequence within a contig, it highlights the contig
that contains the matching sequence.

• If Sequencher can find no matches anywhere, it stops searching and informs

you that the item was not found.

• If there is more than one sequence with a matching name, Sequencher selects
and highlights the first one. If that is not the one you want, click Find Next. This
continues the search in alphabetical order.

Note: The Find window is movable.

SELECTING ALL ITEMS THAT…

The Select menu’s All Items That command provides a series of suboptions based on different
criteria. For example, you can choose sequences that have chromatograms or that include a
particular annotation in the comments field. The menus are context sensitive so not all options
may be available for a particular view.
Most options will have a dialog containing a number of important buttons that control the
results of your search. The Select All Matches button will select matches based on your search
criteria. If you change your criteria and search again, these results will be added to your initial
search. If you don’t want this information added, you must choose Select None.
A status line will indicate how many items matched out of the total number of items available.
You can dismiss the window by clicking on Done or by simply clicking on the Project Window
or performing another action.
If you want to select all the items that do not contain the specified string, go to Select All
Items That, click on the All Items That command, and click on the Done button. Then go to
the Select menu and choose Invert Selection.

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CONTAIN A SUBSEQUENCE

To select all the sequences and contigs in the Project Window that contain a specific string of
bases, you can use the Contain Subsequence command. Go to the Select menu, click on the
All Items That command, and then choose the Contain Subsequence menu item to display a
dialog (Figure 20-5).
Note: You can do a Boolean ‘and’ search using two specifiers for the subsequence. Enter the
first specifier in the Contains: field and the second in the And: field. There are also two drop-
down menus to assist you in the search for specific restriction enzyme sites.
Figure 20-5 Contain subsequence… dialog

CONTAIN ITEMS NAMED

If you want to find which contig contains a specific sequence, go to the Select menu and
choose All Items That and then Contain Items Named… to display a dialog (Figure 20-6). This
lets you select items in the Project Window that contain a sequence bearing the specified
name.
Figure 20-6 Contain Items Named… dialog

CONTAIN ITEMS WITH NAMES CONTAINING

If you are looking for several sequences with related or similar names and need to check which
contig or refrigerator they are in, go to the Select menu and choose All Items That. Click on
the submenu option Contain Items With Names Containing… to display a dialog (Figure 20-
7). This lets you select items in the Project Window where the search string is based on only
part of the complete name.

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 Figure 20-7 Contain Items With Names Containing… dialog

HAVE CHROMATOGRAMS

If you want to find out which sequences have a chromatogram or which contigs contain
sequences with chromatograms, go to the Select menu and choose All Items That and then
Have Chromatograms.

HAVE COMMENTS CONTAINING…

If you want to select all the sequences in the Project Window whose comments contain a
specific text string, go to the Select menu and choose All Items That and then Have
Comments Containing… to display a dialog (Figure 20-8).
Figure 20-8 Have Comments Containing… dialog

HAVE LABELS CONTAINING…

If you want to select all the sequences in the Project Window whose label contains a specific
text string, go to the Select menu and choose All Items That and then Have Labels
Containing… to display a dialog (Figure 20-9).
Figure 20-9 Have Labels Containing… dialog

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HAVE NAMES CONTAINING…

If you want to select all the sequences in the Project Window whose names contain a specific
text string, go to the Select menu and choose All Items That and then Have Names
Containing… to display a dialog (Figure 20-10).
Figure 20-10 Have Names Containing… dialog

WERE EDITED ON OR SINCE…

If you want to select all the sequences and contigs in the Project Window that were edited on
a specified day, go to the Select menu and choose All Items That and then Were Edited On
Or Since… to display a dialog. You can use the drop-down menu to specify a preset day such as
today, yesterday, 1 week ago, or even 2 months ago. You can also type a date in the Find
What: box.
Note: You can also include items edited on all the days since a specific date by checking the
And All Days Since checkbox.

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 21. EXPORTING DATA

In this chapter, you will learn how to export your work. This chapter discusses how to export
sequences, consensuses, and contigs, and also how to export defined data such as selected
bases and subprojects.

EXPORTING DATA FROM SEQUENCHER

Sequencher has a rich set of export formats. You can export sequence(s), contig(s), or
consensus sequence(s), subsets of your data, the Variance Table, the Summary Report and
even protein translations.
Sequencher offers you a number of options depending on the type of data you choose to
export. For each data type, you can select a choice of format and the location for the export.
For some data types, there are additional options. Sequencher may offer a default for the
export. Unless you deselect the option to use default names, Sequencher saves the file(s) in
the default name. The Export dialog will default to the last location and choice of format used.
All of the export commands are accessible from the File menu.

EXPORTING SEQUENCES

To export a sequence or a number of sequences, click on the item(s) you want to export. Go to
the File menu and click on Export and choose Sequence(s)… from the submenu. Sequencher
provides a context-sensitive dialog for changing the export options.
The Browse… button opens a dialog called Choose a Folder (Mac) or Browse for Folder
(Windows). From this dialog, you can choose where to store the new text file(s). You may
create a new folder in which to store your sequences by clicking on the Make New Folder
(Windows) or New Folder (Mac) button at the bottom left of the window. Click on the OK
(Windows) or Choose (Mac) button to record your chosen location.
Figure 21-1 Export Options dialog

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The Format: drop-down menu allows you to choose a format type for your exported
sequence. You may set the case of the exported sequence, the sequence strand to export, and
the treatment of ambiguities and gaps. To choose these settings, click on the Options…
button, select the options you want, and click OK (see Figure 21-2). Finally, click on the Export
button to execute the command.
Figure 21-2 Export Sequences drop-down menu

Note: If you have added features to a sequence and chosen GenBank as your export format,
these will be exported as a GenBank Feature Table.

EXPORTING A CONSENSUS

To export a consensus sequence, click on the contig icon(s) in the Project Window. Then go to
the File menu and choose Export and Consensus… from the submenu. The Format: drop-
down menu allows you to choose the format type for your exported sequence. Clicking on the
Options… button displays the window where you turn options on or off. In most cases, you
will want to just click on Export and use the original consensus name.
The Browse… button opens a window called Choose a Folder (Mac) or Browse for Folder
(Windows). From this window, you can choose where to store the new text file(s). You may
create a new folder in which to store your sequences by clicking on the Make New Folder
(Windows) or New Folder (Mac) button at the bottom left of the window. Click on the OK
(Windows) or Choose (Mac) button to record your chosen location.
Uncheck the Use Default Names checkbox if you wish to set a specific export name.

EXPORTING CONTIGS

To export a contig, click on the item you want to export. Go to the File menu and click on
Export and Contig… from the submenu. Sequencher displays the export dialog. The browse
button allows you to choose a disk and folder to store the new text file(s). The format button
allows you to choose the format type for your exported contig. You can choose from MSF,
Nexus Interleaved, Nexus Sequential, Aligned FastA, and CAF. You may also set a number of
options. Clicking on the Options… button displays the window where you turn options on or
off. Finally, clicking on the Export button executes the command.

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The Use Default Names option gives each export the name of its contig. Uncheck the Use
Default Names checkbox if you wish to set a specific export name.
The Browse… button opens a window called Choose a Folder (Mac) or Browse for Folder
(Windows). From this window you can choose where to store the new text file(s). You may
create a new folder in which to store your sequences by clicking on the Make New Folder
(Windows) or New Folder (Mac) button at the bottom left of the window. Click on the OK
(Windows) or Choose (Mac) button to record your chosen location.
If you execute the Export command with the Sequence… suboption while a contig is selected,
Sequencher will create a folder with the name of the contig. The folder contains all of the
sequences in the contig.

EXPORTING SELECTION AS SUBPROJECT

If you wish to share your data with colleagues who may have older versions of Sequencher or
another program, choose Export from the File menu and then Selection As Subproject…,
then select the appropriate version or type from the Format drop-down menu.
In certain cases, you may want to move some but not all items from one Sequencher project
into another. Working in the Project Window, select the sequences and contigs you want to
export. Go to the File menu and choose Export and then choose Selection As Subproject.
Sequencher creates a new project containing only the items you selected. Now you can import
the newly created sub-project into the chosen Sequencher project.
You should remember to provide a relevant name to define the new project because you have
derived it from a selection of multiple items.
Note: Unlike other Export commands which may create a number of files on your hard disk,
this command will create only one new project.

EXPORT SELECTED BASES

You may want to export selected bases rather than an entire sequence. To do so, drag the
mouse to highlight your selected bases or go to the Select menu and use the Bases by
Number command. Then go to the File menu and choose Export and click on Selected
Bases… from the submenu. You will then see the Save As window which allows you to choose
your format options and preferred location.

EXPORT OPTIONS

If you click on the Options button in an export dialog, Sequencher provides a context-sensitive
dialog for changing the export options. Select the options you want and click Export.

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EXPORT FORMATS

When you export a file, you may want to specify a file format so other software programs can
access it. Click on the Format: drop-down menu in the export dialog. The menu will display a
list of the available formats.
The Sequencher Export menu is context sensitive, which means that it varies based on
whether your selection contains a sequence, a contig, or both. For each kind of export, you will
be prompted to apply appropriate formats. See the Table below for more information.
Note: SCF, Standard Chromatogram File format, is a file format for exchanging data between
different sequencing systems that use four color chromatograms to show sequencing data.
Most current sequencing hardware supports this file standard.

EXPORTING PROTEIN TRANSLATIONS

Sequencher can automatically translate bases into proteins as it copies items to the clipboard.
These protein sequences can then be pasted in to another file or program.
To perform this, select a portion or all of the sequence and then go to the Edit menu and
choose Copy As and Protein Translation… from the submenu. Sequencher asks you whether
you want to translate only the selected bases or whether you want the entire sequence but in
a specific frame. The default setting is to translate only the bases you selected. The Selected
Bases Translated radio button is only available if you have selected some bases.
Figure 21-3 Copy As Protein Translation dialog

You can also export the translation. Go to the File menu. Click on the Export command and
choose Selected as Protein… from the submenu. Pick from the following options:

Figure 21-4 Selected As Protein options

The Selected Bases Translated radio button is only available if you have selected some
bases. You will then be prompted with a Save As: dialog.

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 Table 21-1

Export format type Description

Sequence & Consensus ASCII plain, Plain text

formats Unformatted,

AFDIL, Fitch, Genentech, Specialist and legacy formats

IG, Strider

GenBank, NBRF, EMBL Database formats

FASTA, concatenated Commonly used formats

FASTA, FASTQ

Nexus/PAUP Phylogenetic program format

interleaved,
Nexus/PAUP sequential,
Phylip, Phylip 3, Phylip4

SCF 2.0, SCF 3.0 Standard Chromatogram Format

Contig formats CAF Common Assembly Format

MSF Multiple Sequence Format

FASTA aligned Phylogenetic program format

Nexus/Paup Phylogenetic program format

Subproject formats CAF Common Assembly Format

FASTA Commonly used formats

Sequencher Older project file formats

Special import formats ACE, CAF, CEP, FASTA Project formats from other
aligned, GCG programs such as Contig Express

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EXPORT OVERVIEWS

If you are looking at the schematic view of a contig, you can generate a text version map of the
sequences, as shown in Figure 21-5, for viewing in other programs or word processors. There
are two ways to do this.
1. Go to the File menu and choose Export, then Overview as Text from the submenu
.Sequencher will treat the map as any other exported file.
2. Alternatively, you can copy the overview as a picture and paste it into other programs.
Select the overview window by clicking on it, then go to the Edit menu and choose Copy As
and Picture from the submenu.
Note: When viewing the output, you must specify a fixed-width font to preserve the scale of
the arrows in relation to each other and the sequence names.
Figure 21-5 ASCII map of sequence

EXPORTING CONTIG SUMMARIES

When you are displaying the Summary view of a contig (see “Summary Report View” in
Chapter 12, “Editing Contigs”), you can export the contents of the report to a text file. Go to
the File menu and choose Export then the Summary… submenu. This is particularly useful if
you wish to add annotations to a report of your assembly in standard page format.

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 22. OUTPUT

In this chapter, we discuss printing options. We will explain how to set up pages, copy pictures,
print, and use different report formats.

COPY PICTURE

Sequencher can copy the images from many of its windows, including text windows, to a
picture on the clipboard. Select the window you want copied, go to the Edit menu and choose
Copy As, then click on Picture.

PAGE SETUP

Before printing, go to the File menu and choose Print Setup… (Windows) or Page Setup…
(Mac) to change the options that are specific to your printer. Some options that may be
available on your printer include portrait or landscape printing (vertical or horizontal
orientation), paper size, reduction, enlargement, or special handling for color printing. (See
Chapter 23, “Customizing Sequencher and User Preferences” for more information.)

PRINT TRACE IN ONE PAGE

This command appears within the File menu and allows you to print an individual trace so that
the entire sequence fits on one page.

PRINTING IN DETAIL

Most windows that contain data can be printed. To print, select a window by bringing it to the
front. Then go to the File menu and choose Print and enter your print specifications.

SETTING HEADER, FOOTER AND MARGIN OPTIONS

Specify header, footer, and margins for any printout by going to the File menu and choosing
Set Header & Footer…. You enter the text of the header and footer and a code style for
elements into a dialog (Figure 22-1) that Sequencher will automatically increase incrementally.
The code elements are [Date], [Time], [Page], [Total Pages], [Project Name], and [User Name].
You can also add your own free text in either the header or the footer. Headers and footers are
supported in all reports.
Set the margins by typing appropriate numbers into the Left, Right, Top, Bottom, and Gutter
boxes in the Margin pane. Remember not to set margins smaller than those your printer can
handle.

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 Figure 22-1 Setting Margins for printouts

REPORT FORMATS

Special report formats are used for printing the contents of the Contig Editor. Specify the
report format you want by clicking on the Report, Book, or Poster radio buttons from the
Style pane of the Set Header, Footer, & Margins dialog. Sequencher then adjusts the report
accordingly.
The Report option is for reports printed on single-sided pieces of paper that need to be
displayed one page at a time. Each page is fully labeled.
The Book option is for two-sided printouts when the user can look at a left and a right page
simultaneously. Only the left-hand page of each pair will have all the labels for each sequence
printed in a report; the right page uses the additional space for more data.
The Poster option is for displaying all your data together. It minimizes the amount of labeling
and assumes that the entire report is to be laid out as one big rectangle for paste-up and
display.

PAGE BREAKS

Page breaks, which are shown on screen as in Figure 22-2, appear in the Sequence Editor and
in the Summary Report view of a contig.

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 Figure 22-2 A page break in the Summary Report

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 23. CUSTOMIZING SEQUENCHER & USER PREFERENCES

In this chapter, we discuss how to set your view preferences such as colors, custom codes,
features and motifs, and the genetic codes you want to use. We also discuss how to set your
user preferences such as confidence scores, labels and names, assembly parameters, and how
you want to display chromatograms. You will learn that all of these preferences can be saved
as individual or group preferences.

VIEW PREFERENCES

Sequencher offers you a number of options for specifying how you want to view your data in
the Sequence and Contig Editors. These options can provide visual cues which help to
differentiate your data.

AMBIGUOUS BASES

Sequencher can underline all ambiguous bases in the Sequence or Contig Editors. Go to the
View menu and choose Base Ambiguities As. Go to the submenu and choose Underlined to
mark ambiguities or Not Highlighted for unmarked ambiguities.

EDITED BASES

Sequencher can highlight bases that have been edited, which helps you audit the integrity of
your data. Go to the View menu and choose Base Edits As. Select Not Highlighted for
unmarked edits, Bold Magenta for colored edits, and bOLD & cASE cHANGE for bold and a
changed case.

DISPLAY COLOR BASES

Sequencher can display bases in color. To turn on this feature for either a Sequence or Contig
Editor display, choose Display Color Bases from the View menu. (For information on how to
change the color assignments, see the section “Ambiguity Codes” in this chapter.)

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COLORS AS BACKGROUNDS

Sometimes, displaying bases in color does not differentiate them enough. You can further
enhance the colored bases by going to the View menu and choosing Colors As Backgrounds.
Sequencher then displays the base as a colored rectangle with the base character printed over
it.

DISPLAY BASE CONFIDENCES

Sequencher can represent imported confidence scores as a colored background. In order to

view base confidence values as colored backgrounds, go to the View menu and click on
Display Base Confidences. Sequencher will only display these backgrounds if the original data
included base quality scores. (For information on how to change the threshold values, see the
section “User Preferences” in this chapter.) You may need to turn off Colors As Backgrounds.

DISPLAY FEATURES

Sequencher can display features you have entered. Go to the View menu and click on Display
Features to toggle this view on and off. For each individual feature, you must also enable or
disable its display in User Preferences. (See also Chapter 19, “Motifs and Features”, for more
information.)

DISPLAY MOTIFS

Sequencher can display motifs you have entered. Go to the View menu and click on Display
Motifs to toggle this view on and off. (See also Chapter 19, “Motifs and Features”, for more
information.)

SETTING UP CUSTOM CODES

STANDARD CODING SYSTEM

The IUPAC-IUB ambiguity set is the most common coding system for representing DNA.
Sequencher uses that coding system. See Appendix 28 for a table of the IUPAC-IUB code
system.

CONFIGURING AMBIGUITY CODES

If you wish to configure Sequencher’s ambiguity coding system yourself, go to the Window
menu and choose Ambiguity/Key Codes...

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 Figure 23-1 Ambiguity Editor

Each base and each ambiguity has a display character which is drawn on the screen and sent to
the printer to represent that base or ambiguity. The display character interprets ASCII text files
imported into Sequencher. Each code is also assigned a color which can be displayed in
sequences or contigs by going to the View menu and clicking on Display Color Bases.
Select a base or ambiguity from the scrolling list at the left by clicking on its name. When the
item is highlighted, information about it appears on the right side of the dialog.
To change the display character, click on the box to highlight it.

REPLACING AN EXISTING CHARACTER KEYSTROKE CODE

Each base code can be assigned up to two alternate keys from the keyboard. Using either of
these keys generates the desired base code when using the Sequence or Contig Editors. These
keystroke settings do not affect importing of data.
To change a keystroke setting for a base code, select the base code from the list on the left of
the window, click on one of the keyboard keys to highlight it, and then press the key you want
to assign to that base code.
To change a color setting for a base code, select the base code from the list on the left of the
window and then select a different color from the Color drop-down menu.

SAVING AND LOADING CUSTOM CODES

You can load and save your customized ambiguity settings by going to the button bar at the
top of the Ambiguity Editor and clicking on Load Ambiguities and then Save Ambiguities.

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SHOW AND HIDE AMBIGUITY HELPER

Sequencher can show a small quick-reference window that lists the current ambiguity
symbols. To bring up this window, go to the Window menu and click on Show Ambiguity
Helper. Close the window with the close box in the upper left corner.

CHOOSING A GENETIC CODE

You will find that Sequencher already has the major genetic codes built in. You can choose
from among these predefined coding systems by going to the Window menu and choosing
Genetic Code…. Select the code you want from Reset Code To pull down menu. The Genetic
Code dialog is shown in Figure 23-2. Use the buttons at the bottom of the box to choose three
letter or one-letter abbreviations. If you chose one-letter abbreviations, the letter will be
aligned with the first (left) base of the codon.
Figure 23-2 Genetic Code Editor

EDITING A GENETIC CODE

You can take any one of the pre-existing genetic codes and edit it in the Genetic Code Editor to
change the codon translations. The Genetic Code Editor has two main parts: Abbreviations
and 3-Letters or 1-Letter Left codes.
Go to the Window menu and choose Genetic Code. Choose the code you want to edit and
then click on the table entry you want to change. Finally, click on the abbreviation you want in
that position.
To undo any changes, select the appropriate genetic code from the Reset Code pull-down
menu.

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ABBREVIATIONS

To edit the abbreviations, click on the Abbrvs… button in the bottom-right corner of the
dialog.
Another dialog (Figure 23-3) then lets you change both the three-letter abbreviations for the
amino acid names and the corresponding one-letter codes. The standard abbreviations and
one-letter codes are available as the default cases.
Figure 23-3 Abbreviations editor

REMEMBER WINDOW LAYOUT

The Remember Window Layout command lets you define default positions for the Contig
Editor and Contig Chromatogram when you are editing in the Bases View. First you must
organize the windows. Then go to the Window menu and choose the command Remember
Window Layout.
Select the submenu option called Contig Chromatogram. Every time you select the Show
Chromatograms button, Sequencher will open the windows in your layout positions.

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 Figure 23-4 Remember Window Layout

You can also use the Remember Window Layout command to define default positions for the
Contig Editor, Contig Chromatogram, Variance Table, or Translated Variance Table when you
are in Review Mode. First you must organize the windows. Then go to the Window menu and
choose the command Remember Window Layout. Select the submenu option Single
Variance Table Review if you have one Variance Table open and Double Variance Table
Review if both are open. Every time you select the Review button from the Variance Table,
Sequencher will open the windows in your layout positions.

USER PREFERENCES

You can specify your preferences for many program settings in Sequencher. To set these, go to
the Window menu and choose User Preferences….
The User Preferences window contains a hierarchical list of preference settings. The main
categories of settings are General, Display and Input/Output. Click on the triangle icon next
to a category name to expand the list and show the various preference topics within that
category. When a category list is expanded, the triangle changes to point downwards.
When you click a preference topic name, a Preference Pane appears on the right side of the
window and enables you to specify your preferences for that topic.
Note: Each preference setting you select will be stored when you quit the program.

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GENERAL

SETTINGS

On the File menu, you will find a command called Open Recent. The submenu for this
command is a list of projects. You can specify the number of recent projects Sequencher will
remember in this submenu. The maximum number is 99.
Sequencher is installed with a set of default values. As you work with the program, it replaces
the default values with your preferences. However, if you want to restore the original settings,
you will find a command button at the bottom of the General > Settings user preference pane
called Reset user preferences to factory defaults. This option resets all of the user
preferences to their original status.
Figure 23-5 Settings preferences

AUTO-SAVE

Auto-Save preferences control the timing of automatic saving of project files. To use this
preference, you must have saved your data and given your project a name at least once.
To activate Auto-Save, click on the Auto-Save Project Files checkbox. Click on the Ask Before
Each Auto-Save checkbox if you want to confirm the save each time.
Use the Time Between Saves slider to set how long Sequencher should wait between each
Auto-Save. Use the Idle Time Before Auto Save slider to set how long your computer should
be idle before Sequencher executes an Auto-Save. The latter setting will prevent Sequencher
from performing an Auto-Save while you are typing.

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 Figure 23-6 AutoSave preference settings

CONFIDENCE

Many of the new base callers generate confidence values associated with each base.
Sequencher supports confidence values from PHRED, ACE, Trace Tuner, SCF, ABI, ESD and
compatible files. The Confidence preferences control the display of base calls and confidence
values from these files.
Sequencher’s default settings show confidences below 20 as dark blue, the midrange values as
a medium blue, and high confidences, that is above 40, as a light blue. Change the threshold
values by altering the numbers in either of the Confidence Ranges boxes. If you have a
Sequence or Contig Editor visible behind the User Preference pane, you will notice that the
colors change as you set the new confidence ranges.
Figure 23-7 Confidence Ranges settings

EXTERNAL DATA

Results from external algorithms such as Clustal, Maq, and GSNAP are located in individual run
folders each with a unique Run number. The default location for the Home Directory which
contains these folders is in your Documents folder. Using the External Data User Preference,

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you can browse to a new location and use the Choose (Mac) or OK (Windows) buttons to
confirm your choice.
Figure 23-8 External Data preference settings

LABEL & NAME

The Label & Name preferences control the labels and descriptions used to mark sequences
and contigs. Use the boxes grouped under Available Labels to choose label colors (use the
pull-down color menus at the left of the label name) and descriptions for marking individual
sequences.
Once you have set your preferences here, you can apply them to sequences, contigs, and
refrigerators while you are working in a project by selecting the icon(s) in the Project Window.
Go to the Edit menu and choose the Label submenu.
Under the Default Names section of the Label & Name pane, you can enter default names for
the new contigs and sequenes. A bracketed asterisk tells Sequencher to add a number to the
name and increase it for each new contig or sequence you add to the project.
Figure 23-9 Label and Names preference settings

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MENU

The Menu preferences control command options that let you optimize the way you work in
your own lab. Use the buttons under Use Ctrl+O For (Windows) or Use Cmd+O For (Mac) to
program the Ctrl+O (Windows) or Cmd+O (Mac) key combination to open a project or open a
window.
Use the pull-down menus grouped under User-Defined Command Keys to create keyboard
shortcuts for any of the menu commands. After you have assigned a keyboard shortcut to a
menu item, the shortcut will be listed to the right of the menu command on the drop-down
menus.
Figure 23-10 Menu preference settings

NEW PROJECT

This User Preference determines what type of project will be opened by the New Project
command.
Click on the Use Blank Project radio button if you want your new projecy to be blank. Click on
the Use Project Template radio button if you want the project settings to be controlled by a
template. Specify the template you want by clicking on a name in the drop-down menu.
Templates are stored in a folder on your system. If you click onthe Open Templates Folder
button, Sequencher will open a window displaying the templates. You can store up to 1,000
templates on your system. (For more information about templates, see Chapter 3, “The Project
Window.”)

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 Figure 23-11 New Project User preference settings

SOUND

Sequencher provides audio feedback to help you maintain the quality of data input.
When you select the Sound preference, you will see the preference pane shown in Figure
23-12. The slider controls how quickly Sequencher reads bases to you.
Click on the Play Audible Welcome at Startup checkbox if you want Sequencher to say
“hello” at startup.
Figure 23-12 Sound preference settings

Note: You have to select a voice file to get audio output. (For more details, see Chapter 8 “The
Sequence Editor” and review the section on “Voice Verification.”)

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DISPLAY

CHROMATOGRAM

Chromatogram preferences control the display of chromatograms. You can specify the Height
Of Chromatograms by clicking one of the radio buttons from Tiny to Tall. If you use a black
and white monitor, click on the Pattern radio button for Lane Identification By.
You can set lanes to be identified by color or pattern by selecting the appropriate radio button.
When viewing multiple sequences, you will notice that a reversed and complemented
sequence displays the lane colors according to the original data. Click on the Colors Match
Current Bases checkbox to display all current base calls in the same color. Click on the Hide
Original Base Calls checkbox if you do not want them to appear in your chromatogram
windows.
Figure 23-13 Chromatogram preference settings

If you click on the Print Scale Factor checkbox and you used the slider shown at the left in
Figure 23-12, the scale factor will appear on any printout of that chromatogram.
If you click on the Rev & Comp Displayed as Lower Case checkbox, the original base calls for
a reversed and complemented sequence are shown in lower case (as opposed to backwards) as
the second of two lines of bases above the traces (as in Figure 23-14).

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 Figure 23-14 Reversed and Complemented sequence

Under Width Peak to Peak, you can stretch your traces horizontally by selecting the Wide
button.

CONTIG CHROMATOGRAM

The Contig Chromatogram preferences control the display of chromatograms of sequences

from a contig. When you have aligned several sequences from a contig and displayed the
chromatograms, several traces appear in a single window, one above the other across the
width of the window.
Use the Columns for Multiple Traces radio buttons to tile the data into columns so you can
look at more traces at once. For example, if you select 3, you’ll see three columns of traces. If
your screen and window size allow you to see three rows of traces at once, you will be able to
view a total of up to nine traces without having to scroll the window.
Click on the Always Scroll to Selected Column checkbox to have Sequencher automatically
update the Chromatogram window whenever you select a new column of data in the Contig
Editor window.
You can have Sequencher remember your window position. Sequencher can also compress
SCF chromatogram displays.
Figure 23-15 Contig Chromatogram preference settings

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CONTIG

The Contig preferences control the vertical sorting of the sequences when a new contig is
created or when sequences are added to an existing contig.
You can use the Contig Font option to change both the font and the font size. This allows you
to use fonts other than the default Monaco 9 point font in the Contig Editor. You can see a
sample of your current font selection below the font Size: drop-down menu in the Sample
portion of the preference pane.
You can increase the Minimum Overlap range up to 500 by clicking on the Increase Minimum
Overlap to 500 checkbox.
Figure 23-16 Contig preference settings

FEATURE, MOTIF

The Feature, Motif preferences control the default settings for new features and motifs.
When you import a sequence from GenBank into Sequencher, these settings determine the
name and appearance of the feature within both the Overview and Sequencher’s editor
windows. (For more information on GenBank Feature tables, refer to the Appendix 30 “Feature
Keys and Qualifiers”.)
When you click on the Define Feature Key Default Styles… button, a new window, also called
Define Feature Key Default Styles, appears. On the left-hand side of the window is a pane
listing Feature Keys.
To set new attributes, select the appropriate Feature Key. On the right-hand side, you will see
a pane where you can set attributes such as the Default Name, Display Style and Color. You
can restore the default settings at any time by clicking on the Reset Style Defaults button.
Click on the Done button when you have finished.
When you click on an item in the left hand Feature Key: pane, you will see the same text
appear in the Default Name: field. In most instances, there will be extra text enclosed in
square brackets. The enclosed text is the default feature qualifier. GenBank have defined
several Feature Qualifiers for each Feature Key. You can replace the text within the square
brackets with your preferred Feature Qualifier. (For more on Feature Qualifiers, see Appendix

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30 “Feature Keys and Qualifiers” and, for a listing of Sequencher’s default Qualifiers, see
Appendix 29 “Default Feature Qualifiers”.)
Note: If a Label (indicated by /label= “some text”) is used in a Feature Table, it takes
precedence over any default keys or qualifiers since it unambiguously identifies a feature or
item.
Click on the Display Feature in Editors checkbox if you want a specific feature to be displayed
in the Sequence or Contig Editors.
If you are making changes to the way Feature Keys are currently displayed, you can click on
the Update Project… button to update your project. You can decide whether to apply the
current styles, name, or both to All features in your project. If you do not want to do so, just
limit your choices to Selected features in your project by clicking on the appropriate button.
When you are finished, click on the Done button to return to User Preferences.
Use one or more of the three Display Feature Numbering For checkboxes in the Feature,
Motif pane to indicate which of the second-line features Complement Bases, RNA Bases, or
Amino Acids should have its own numbering.
Use the Motif Default Style pull-down menu to set the color, case, and underlining style for
new motifs. You can see a preview of the motif style in a box on the right-hand side of the
Motif Default Style groupbox.
Figure 23-17 Features and Motif preference settings

FORMAT RULER

The Format Ruler preferences control the display defaults for any view which has a format
ruler. These are just defaults; you can change these settings later from within a specific editor.
When you click an icon in the sets of icons above the ruler, your selection is explained and
confirmed in the panel below the ruler (Figure 23-18). For example, use the Preferred Bases
per Line counter to increase or decrease the preferred number of bases in each line. Note
that, if you request a line length that will not fit on the printed page, Sequencher uses the
maximum line length that will fit.

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If you want the margin to be automatically adjusted when you increase the size of a window,
check the Auto-Adjust Margin if Window is Resized checkbox.
Figure 23-18 Format Ruler preference settings

START-STOP

The Start-Stop preferences control the locating of open reading frames from an editor. Use
the buttons grouped under On Codon Maps Highlight to indicate whether you want None,
which gives you the plain map of start and stop codons, Any Start to Stop >=, or Any
Unstopped Run >= to give you a shaded box showing open frames of a length you specify.
Figure 23-19 shows selections in the Start-Stop preference pane that will make Sequencher
highlight ORFs beginning with a MET and longer than 60 bases.
Figure 23-19 Start-stop codon preference settings

Under Select - Next MET to Stop, you can define the minimum length of ORF selections in a
Sequence Editor. If you specify a minimum length here, for example 30 bases, the Next MET
to Stop command under the Select menu in the Sequence Editor will change to read Next
MET To Stop (>30b).

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VARIANCE TABLE

The Variance Table preferences control how ambiguous matches and large gaps will be
treated. If you wish to include ambiguous matches in your table, click on the Include
Ambiguous Matches checkbox. If you wish to exclude large gaps from your table, click on the
Exclude Large Gaps checkbox.
When you generate a Variance Table, you would expect to have blank cells where the base at
that position matches the exemplar. If you would like to see all bases across the comparison
range, whether they match or are different to the exemplar, then click on the Populate All
Cells checkbox (see Figure 23-20).
Figure 23-20 Variance Table preference settings

INPUT/OUTPUT

FILE IMPORT

File Import preferences control how Sequencher applies the automatic trim function to
imported files.
Use the checkboxes grouped under Add Imported Sequences To Trim Window to specify
which sequences Sequencher should examine for poor quality ends and then trim.
You can also specify how you want to resolve conflicts between the names of imported files
and names stored with particular sequences. If you click on the Prefer File Names to Data
Names checkbox, Sequencher uses the file name.
Clicking on the Review Trim Parameters… button displays the Ends Trimming dialog in which
you can set trim criteria. (See Chapter 6, “Preparing your Data for Assembly,” for details.)
Information about sequences which have been trimmed is recorded in a special file. This
happens automatically. If you want to prevent the file from growing too large, then click on the
Limit Trim Log File Size to checkbox. Then enter a value in the input field.

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 Figure 23-21 File Import preference settings

REPORT

The Report preferences control the default page setup for report printouts. Use the fields in
the Page Margins groupbox to set the margins and gutter. Click on Page Setup… to open the
printer Page Setup dialog. The page setup settings will apply to all windows you open
thereafter.
You can specify headers and footers on reports generated from any window in which you are
working. Any text you type into the field above the page layout will appear in the header and
text you type in the field below the layout will appear in the footer.
For variable elements (date, time, page, and total number of pages), enclose the word— typed
with an initial capital—in square brackets, as shown in Figure 23-22.
Figure 23-22 Report Margin preference settings

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 24. FORENSIC FEATURES

If you have the forensic version of Sequencher, you have access to special functions for DNA-
based identification. In this chapter, we describe how you can validate mtDNA profiles, create
reports, and export your results.

WORKING WITH MTDNA PROFILES

VALIDATING MITOCHONDRIAL DNA PROFILES

The Validate mtDNA Profiles command allows you to compare the results of separate
analysts. Select two contigs that have been assembled to the same Reference Sequence. Go to
the Contig menu and click on the Validate mtDNA Profiles item. This command creates a
report in a new window. The title bar includes a date and time stamp as shown in Figure 24-1
below. The report includes the name of the Reference Sequence used, the name of the contigs
being examined, and the HV1 and HV2 ranges. Underneath the main body of the report is a
window pane that displays pertinent warnings.
In the report shown in Figure 24-1, the two mtDNA profiles disagree. The bases in conflict are
highlighted in yellow. Also, there are positions where there is data for one mtDNA profile but
not for the other. The base position is distinguished by a yellow highlight, and the cell with the
missing data has a pink X though it. The cells are unshaded where the two profiles both agree
and confirm each other.

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 Figure 24-1 The Validate mtDNA profiles window

CREATE REPORTS

At the top of the Validate mtDNA Profiles window is a button bar. Click on the Create Report
button. A new window called Analyst Report opens. This window contains a version of the
Validate mtDNA report that you can send to a printer by clicking on the Print button. You may
also save this report by clicking on the Save button.
Note: The report includes spaces for the analysts’ and reviewers’ signatures.

EXPORT CMF

You can create a CMF Report by clicking on the Export CMF button. A new window called
Export CMF will open. Choose the Export Sample: and Specimen Category: from the relevant
drop-down menus. The Fragment Owner Date, Fragment Owner Time, and Specimen ID
fields are filled in automatically. You will need to enter information into the remaining text
fields.
There are a number of optional fields. The Source Identified field is a drop-down menu. There
are two text fields for NCIC ID and ViCAP ID. There is also a free text Comment field. When
you are finished filling in the form, click on the Export button. If you have made an error, you
can click on the Clear All button to reset the form, or you can click on the Cancel button to
return to the Validate mtDNA Profiles window.
Note: If you move your cursor to pause over a field, you will see a brief description
summarizing the information required for that field.

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 Figure 24-2 Export CMF report

FURTHER COMMANDS OF INTEREST

NEW PROJECT FROM TEMPLATE

The New Project From Template command allows you to open a new project containing all
the sequences, settings, and preferences associated with your chosen template. Go to the File
menu and click on New Project From Template. Select a template from the submenu. A new
blank Project Window will open.
Note: A template called rCRS is provided. This template contains the Revised Cambridge
Reference Sequence and is the default template for this command.
For more information on Templates, see Chapter 3 “The Project Window.” There is a tutorial
on analyzing mtDNA with Sequencher in the Tutorials folder.

CONSENSUS TO FORENSIC STANDARDS

This calculation is based on the coverage of sequences at a given position and is taken from
work at the U.S. Armed Forces DNA Identification Laboratory (AFDIL) in Rockville, Maryland.

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 • All positions with coverage of only one sequence are marked as N (ambiguous).

• All IUPAC codes apart from ACG and T are treated as N.

• If any positions disagree where the coverage is between two and four, this position
is marked as N. If the coverage is between five and seven, any position with a
disagreement will be marked as ambiguous.

• If there are two or more disagreeing bases at a position, this will be called an N
regardless of the coverage.

Figure 24-3 Consensus to Forensic Standards example

For more information on other types of consensus calculations, see Chapter 11, “The Contig
Editor”.

DISPLAY SECONDARY PEAK

You can identify heterozygotes by identifying the second-highest peak beneath the primary
peak. If you have a particular candidate for a heterozygote, first open the Chromatogram for
the individual sequence and then click on the base call(s).
Go to the Sequence menu and choose Call Secondary Peaks…. A dialog lets you specify how
the secondary peak should be called.
The slider lets you specify how significant the second-highest peak must be to generate a
change.
If you want Ns to be replaced, then select the Allow Ns to be replaced checkbox.
If you’ve already edited bases by hand and don't want them changed, you must deselect the
Allow edited bases to be replaced checkbox.

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If you select Only make changes that result in an ambiguity, Sequencher may change an
ambiguous base call to an A, C, G, or T if it does not meet the secondary peak criteria. If you
prefer it to remain ambiguous, do not select this option.
If you wish to search just a range of bases, highlight them and check the Search Selection Only
checkbox.
For more information, see Chapter 15 “Chromatograms”.

SET CIRCULAR GENOME SIZE

The Set Circular Genome Size… command allows you to set the number of bases in your DNA
circle.
Select a sequence that has already been defined as a Reference Sequence. Then go to the
Sequence menu and choose the Set Circular Genome Size… command. The dialog below
appears. Enable the circular number by clicking on the Enable For This Fragment checkbox. If
you have enabled the Cambridge Reference Sequence as your Reference Sequence, the number
of base pairs in your sequence will automatically be set to 16,569. Dismiss the dialog by
clicking on the OK button.
Note: Circular numbering can only be used in conjunction with a Reference Sequence. You
must designate your sequence as a Reference Sequence before you can enable circular
numbering.
Figure 24-4 The Circular Genome Size dialog

HOW TO MARK A SEQUENCE AS A REFERENCE SEQUENCE

To designate a sequence as a Reference Sequence, select the sequence icon in the Project
Window and, under the Sequence menu, select the Reference Sequence command. From
now on, that sequence icon will include a small letter "R" to remind you that it has been
marked as a Reference.

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For more information on Reference Sequence properties and how to work with a Reference
Sequence, see Chapter 7 “The Reference Sequence”.

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 25. SEQUENCHER CONNECTIONS

Sequencher Connections heralds an entirely new way of working with your data. Previously,
you were able to work within Sequencher using its internal algorithms, then we introduced
Plugins. These allowed you to choose and work with NGS algorithms such as Maq, GSNAP,
BWA, and Velvet from Sequencher. These algorithms were still located on your local
computer. Now you are able to use remote services such as BLAST and Primer-BLAST or local
algorithms such as MUSCLE or Local-BLAST using the Connections extensible system.
In this chapter, you will learn about Sequencher Connections and how to apply the algorithms
and services. You will also learn how to apply these to single or multiple sequences using Blast
and Primer-BLAST. In addition, you will learn how to save primers to your project. You will also
learn how to work with a group of Sequences using the MUSCLE algorithm. Finally you will
learn how to view previous sessions.

SESSIONS

A session is a coordinated set of data and the analyses that operate on that data. Grouped
sessions operate on sequences as a whole, for example when they are multiply aligned. In an
Individual session, each analysis operates on a single sequence so that many single sequences
can be analyzed in parallel.
Sequencher remembers sessions you have created and you can open them again and review
results you obtained previously. Or run new analyses on the data in the session.
The data is a snapshot of its state as it was when added to the session. If there is a problem
with data in an existing session, for example it has been corrupted, any existing results for that
data will not be affected. However, you will not be able perform any new analyses and
Sequencher will warn you that the data is invalid. This warning is only displayed for the
duration of the session.
Note: Since the data in a session is a snapshot, you may edit the sequence copy in your
Sequencher project without affecting the copy in Connections. Additionally, if data in the
session becomes invalid, the data in your project is unharmed and you can use this in a new
session.
Figure 25-1 Invalid Data warning

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CHANNELS

Each analysis takes place in what is termed a Channel and may act on a single sequence or
multiple sequences. Since you can modify analyses with different options, you can set up a
series of Channels with each representing a new analysis or collection of settings. In the case of
BLAST for example, this means that you can set up several searches against different databases
or use different search settings against a single database. This will let you compare searches
against say, the human EST database, the mouse EST database, and dbsts, while comparing
results between blastn and megablast.

INVOKING A SEQUENCHER CONNECTIONS SESSION FOR THE FIRST TIME

In order to launch Sequencher Connections, you will need to select one or more items from
the Project Window. You may send any number of sequences or even contigs to Sequencher
Connections. If you want to send large numbers of sequences, be aware that the Blast server
imposes restrictions to ensure that everyone may use the service without problems.
Sequencher Connections has been written to take these regulations into account.
Go to the Window menu and choose Add to Connections Session...
You will see the Sequencher Connections Session Launcher dialog. Decide whether you want
to add your sequences to a session for individual sequences (BLAST, Primer-BLAST, or Local-
BLAST if you have installed it on your computer) or whether you want to add your sequences
to a session for grouped sequences (MUSCLE). Click on the radio button next to the session
type you want to launch. Sequencher gives your session a name automatically. You will
probably want to replace this with something more meaningful. Do this by typing the desired
name into the New Session Name: input field. Finally, click on the OK button.

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 Figure 25-2 The Session Launcher

The session now opens in a new window. The upper half of the window contains a grid with
the data you have selected and two default analysis Channels. Each row represents a sequence
you are submitting for analysis and each column represents a “Channel” (described above) that
handles a different kind of analysis. The default Channels are BLAST and Primer-BLAST.
The lower half of the window is where you see the results of those analyses in a series of tabs.
(Hint: If you hover your mouse just above the tabs in the data section, your cursor will turn to a
splitter allowing you to adjust the size of the two parts of the window.)
Initially your sequences will show in the grid as Queued. Click on one of the cells containing the
word Queued. The default tab is the Web View, which opens to the Gene Codes website, but if
you click on the Sequence, tab you will see the sequence you are about to submit. The Text
and the XML views are blank at this time since there are no results to display.

USING GENBANK ACCESSION NUMBERS

Once you have some sequences in the Sequencher Connections dialog, you may add further
sequences if you know their GenBank accession numbers. The accession number is a unique
identifier for a sequence record and is usually formed by two text characters followed by a
series of numbers. Accession numbers don’t change, but if the record changes, you may see a
version number added as in the example below. (The GI number will change.) Connections will
only allow you to add a sequence once to a specific session. If you need to have another copy
of a sequence, you will need to add it to a different session.

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Click on the button and type the accession number into the Add NCBI
Accession Number dialog. To dismiss the dialog, click on the OK button.
In the image below, the sequence with accession number JQ431947.1 has already been added
(row 5 in the table) and the user is about to add another accession number. If you click in any
cell of an Accession Number sequence row, a new tab labeled GenBank appears, this tab
contains the feature table for the sequence.
Figure 25-3 Using the Accession Number dialog to add sequences to a session

Sequencher Connections will fetch the record and add the sequence to the session window.
You can also remove a sequence from Sequencher Connections by right-clicking on the
number of the row.
Note: The outline colors for individual and group input Connections windows are different to
help you distinguish sessions when you are working with several of them.

RUNNING SEQUENCHER CONNECTIONS

There are a number of different ways to send your data for analysis. Click on the Run All
button. It is between the two panes, to the right of the dialog. This will send all your data to all
of the Channels you have set up. Alternatively, you can send one sequence by right-clicking on
the number to the left of the sequence name in the grid and choosing Run on Each Channel
from the contextual menu. You can also send all sequences for a specific Channel by right-
clicking on the column header of the Channel and choosing Run on Each Sequence. And finally,
you can also send a single sequence for a single Channel by right-clicking in a single Channel
cell and choosing Run.

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 Figure 25-4 Session status messages

Once you have chosen Run All or any of the other Run commands, your data is submitted to
your chosen resource(s) and you can monitor the progress. The cells will change status and
color as the analysis progresses, though some states change so quickly you may not see the
change. The cells change status through Queued (white), Waiting (orange; data is in the queue
before being sent to the connected resource), Sending (turquoise; data being sent to the
connected resource), Pending (yellow; waiting for the resource to send results back), and Done
(green). There is a further Done status for previously saved results. Finally, there is a Failed
(red) status that indicates that no results were received.
You may also re-run your data by pressing the Run All button a second time. This will delete
your existing results and run all of the analyses again. If you cancel before the new results
appear, your previous results will be restored.

VIEWING RESULTS WITH SEQUENCHER CONNECTIONS

As soon as the cells change to Done or if you are working with older results and the status is
Done, your results are ready for viewing. You can view the results as all the Channels for an
individual sequence reach this state or you can wait till all the results have been received.
There are multiple different views depending on the Channel. The contents of the Web View
changes from Connections-related text to display the BLAST or Primer-BLAST results view. This
view contains the graphical overview of BLAST or Primer-BLAST results, the one-line
descriptions of the hits, and the text alignments of query versus hit. The Text view displays the
one-line descriptions and text alignments only. The XML view shows the alignments but with
XML formatting. The Sequence view displays the query sequence.

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When you add a sequence to the Connections Session by clicking on the + GenBank
Accession… button, a GenBank tab will appear. This view contains the Feature table for that
sequence.
If you have created a Primer-BLAST analysis, then you will see a Primer Picker tab when you
click on any cell containing the status of Done or Done. Finally, there is a Schematic view that
allows you to compare analyses with different parameters including Primer-BLAST so you can
view results from BLAST and Primer-BLAST at the same time within the Schematic. In order to
view any particular tab, simply click on it.
Once you have opened a tab, you may also save the results to a file by using the right-click
contextual menu.
BLAST and Primer-BLAST results are available on the NCBI website for a maximum of 36 hours.
You can check exactly when your BLAST results will expire by looking next to the RID in the
Web View. Unfortunately, NCBI does not provide similar information for Primer-BLAST.
However, once your results have expired from the NCBI website, Connections saves your
results and displays a message to that effect in the Web View, which normally displays the
NCBI website view of your results. You can still view the alignments or the primers. To view
your results, click on the Text tab. You can also view the alignments by right-clicking on the
name of the sequence and choosing Show Schematic from the context-sensitive menu.

ADDING OR REMOVING CHANNELS

In a Sequencher Connections session, a column represents a Channel (analysis) and the data to
be analyzed is represented by a row. The simplest type of session would contain a single
column and a single row. More complex sessions might contain many rows (sequences) and
columns (Channels).

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 Figure 25-5 BLAST results expired from NCBI

You can add more Channels by right-clicking in an existing Channel header row. A contextual
menu appears that has menu items for adding new BLAST Channels, Primer-BLAST Channels,
and Local-BLAST Channels. Each menu item adds the Channel before the Channel you have
selected. If you want to remove a Channel, right-click in its column header and choose the
Remove Channel menu item.
Figure 25-6 Right-click Channel menu

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Once you have added a new Channel, you can create a new series of settings using Options…
in the contextual menu (right-click in a Channel column header). You can see an example using
BLAST in the next section.
You can also cancel running Channels by right-clicking on their headers and choosing the
Cancel Running Jobs menu item.

VIEWING SAVED SESSIONS

Your sessions are saved with your project. This means that you can view your results again and
again. In order to see a saved session, go to the Window menu and choose Open Existing
Connections Session…. The Session Launcher dialog appears where you can choose a session
from the list.
Figure 25-7 Viewing saved sessions list in the Session Launcher

Clicking on a header in the Session Launcher dialog sorts the sessions by that column.
Once the session has opened, you will be able to review existing results in the Web View.
Although results do expire from the NCBI website after 36 hours, for BLAST and Primer-BLAST,
you can still see your results in the Text tab. You can also view the alignments by right-clicking
on the name of the sequence and choosing Show Schematic from the context-sensitive menu.

DELETING SAVED SESSIONS

You can delete an existing Connections session from your project. To do this, go to Window >
Delete Existing Connections Session…. A dialog appears listing all your current sessions. Select
or multi-select the sessions you want to remove and then click on the Delete Selected
Session(s) button. You cannot undo this action once the sessions have been deleted.

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BLAST

For each new Channel you add, you will see a new tab in the Channel Options dialog. There are
a number of options that can be set for BLAST. Right-click on a column header in the
Sequencher Connections dialog and choose Options… from the contextual menu. A Channel
Options dialog appears in which you can set the following: algorithm (blastn or megablast),
number of Alignments (drop-down menu), Databases (drop-down menu), number of
Descriptions (drop-down menu), the E (Expectation) Value, Filters (checkboxes), and Word size.
If you choose to change Alignments and Descriptions, you set an integer (10, 100, 250 for
example). For the E or Expectation number, you set a number above 0. You cannot set a
negative value but you can set a very small number such as 0.0001. The E value describes the
number of hits you would expect to see by chance. The lower the E value, the more significant
the match will be. If the E value is increased (larger than the default value of 10), you should
get a larger list of hits but the hits have a less significant score.
Figure 25-8 BLAST Channel options

If you want to change the database you wish to search, then you can choose a different one
from a series in the Database drop-down menu. The database you choose is also used to name
the Channel. You can apply filters to your query sequence, for example low complexity or
repeat elements, by clicking one or more of the checkboxes. If you include Masking as well as a
Filter, the mask only applies to the look-up phase of the BLAST search. It does not apply to the
extension phase of the BLAST algorithm.

PRIMER-BLAST

The Primer-BLAST resource at NCBI uses the well-known Primer3 program to design primers. It
has an added advantage in that the BLAST algorithm is then used to screen the predicted
primers against a user-selected database website to see whether there could be any potential
for unintended amplification.

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You have the option of sending the entire sequence for analysis or choosing a subset. Right-
click on the number to the left of the sequence whose range you want to specify. Choose Edit
Primer Ranges… from the contextual menu.
Figure 25-9 Right-click Sequence menu

You are presented with a dialog where you can specify From and To ranges for the forward and
reverse primers. This is so you can specify when you want primers to be located on specific
sites. For example, if you specified a Forward Primer range From: 200, To: 500, then your
forward primers would be located within these coordinates. You can specify just one primer if
you prefer (e.g. just the forward primer).
Figure 25-10 Edit Primer Ranges

Note: The position range of the primers must not overlap.

PRIMER-BLAST OPTIONS

There are a number of options you can set besides the Primer Ranges in order to refine your
primers further. Right-click on the header for the Primer-BLAST Channel and select the
Options… menu item. The Channel Options dialog opens at the Primer-BLAST tab. The options
are divided into the Primer Parameters and the Specificity Checking Parameters. You can ask
Primer-BLAST to send back more than 10 results. You can also adjust the primer melting
temperature and specify the %GC content.

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 Figure 25-11 Primer-BLAST primer parameters

To enable specificity-checking, click on the Primer Pair Specificity-Checking Parameters

button. In the dialog that opens, click on the checkbox next to Enable search for primer pairs
specific to the intended PCR template. Once you have made changes to the settings, click on
the OK button to dismiss the dialog.
Figure 25-12 Primer-BLAST primer specificity parameters

The results are displayed when the status of the job is shown as Done. Right-click on the name
of the sequence and choose Show Schematic.

SAVING PRIMERS TO SEQUENCHER PROJECT

Once the Primer-BLAST analysis has completed, you can review the proposed primers by
clicking on the Text tab. Here you will see detailed information on each primer pair. You will
notice that a new tab called Primer Picker appears whenever you click on a cell in a Primer-
BLAST analysis column. The Primer Picker contains a subset of the information contained in
the Text tab. The information consists of a checkbox, the primer pair sequences, the start point
for the primer on the original sequence, and the end point for the primer.

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 Figure 25-13 The Primer Picker tab

Click on the Primer Picker tab and select the primers you want to save to your project by
clicking on the checkbox for each pair you want. If you want to save all the primer sequences
to Sequencher, then click on the Select All button. If you do not like the selections you have
made, you can clear all the checkmarks by clicking on the Select None button.
Once you have finalized your selection, click on the Save Selected button. This button will be
greyed until you make your first primer pair selection.
Any checkboxes you select will remain checked until you close your session. The Primer-BLAST
analysis itself will remain until you a) delete the Primer-BLAST column from the session, b)
delete the sequence from the Session table, or c) delete the session itself. Any primers you
have saved to your project are unaffected by any of these deletions.
Sequencher automatically assigns the Primer Feature key to the primer sequences. It also
colors the bases of forward primers green and the reverse primers red. Once the primer
sequences are in your project, you can assemble them to other sequences.

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 Figure 25-14 Primers aligned to a sequence

If you create a contig with primers as shown in Figure 25-14, you can create a consensus
sequence that contains those primers.
Select a contig containing primers and then choose Contig>Create New Seq From Consensus….
A new dialog appears which allows you to set selected options. You can choose to include any
GenBank style feature keys by selecting the Include Features checkbox.
Figure 25-15 Create New Sequence From Consensus options

The consensus will contain the primers, but as features rather than individual sequences, as
seen in the figure below.
Figure 25-16 Consensus containing primers as features

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You will also see the primers as features in the Sequence Overview. To view the name of the
feature, place the cursor over it for a few seconds. The name will appear in a tooltip.
Figure 25-17 Primer features in sequence Overview.

If you want to keep a large number of primer sequences in your project, you can store them in
a Refrigerator. This is a special folder whose contents will be saved with your project but which
will not be assembled to any sequences unless first removed from the Refrigerator. You can
create a Refrigerator by selecting a group of primers and choosing Edit>Refrigerate. You will
be prompted to give your Refrigerator a name. Click on the OK button to dismiss this dialog.
Figure 25-18 Moving primers to a Refrigerator

To view the contents of a Refrigerator, double-click on its icon. To move any primers to your
project desktop, select the ones you want to move and click the Move Selected Items To
Project Window button.

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 Figure 25-19 Moving primers to the Project Window

LOCAL-BLAST - MAC

You need to have Local-BLAST installed on your computer in order to be able to utilize it. You
can download the program for your computer from:

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/5

You will need to download the DMG. Once the download has completed, locate the DMG and
double-click on it. The DMG will open and you will see the BLAST package installer inside. This
has a file extension of pkg. Double-click on this and follow the prompts. The installer is large so
be sure this is what you want to do before you start. Do not change the location of the
installation.
Now you will need to create a database to query. You can use your own sequences or you can
download parts of the databases that BLAST uses from ftp://ftp.ncbi.nlm.nih.gov/blast/db/.
These databases are pre-formatted and need no further attention. For more information, visit
ftp://ftp.ncbi.nlm.nih.gov/blast/documents/blastdb.html.
You will need to place the databases in the correct location. From the Finder, visit the Go menu
and choose Go to Folder…. You will see a new dialog, type in /usr/local/ncbi/blast and click on
the Go button.

5
At the time of writing this was version 2.2.40+

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 Figure 25-20 Go to the folder dialog showing blast path

You are now in the blast folder. Create a new folder here called db. This is where you place
your database files.
Figure 25-21 Creating a db folder for BLAST

LOCAL-BLAST - WINDOWS

You need to have Local-BLAST installed on your computer in order to be able to utilize it. You
can download the program for your computer from:
ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/6
You will need to download the EXE. Once the download has completed, locate the EXE and
double-click on it and follow the prompts. The installer is large so be sure this is what you want
to do before you start. Do not change the location of the installation.
Now you will need to create a database to query. You can use your own sequences or you can
download parts of the databases that BLAST uses from ftp://ftp.ncbi.nlm.nih.gov/blast/db/.
These databases are pre-formatted and need no further attention. For more information, visit
ftp://ftp.ncbi.nlm.nih.gov/blast/documents/blastdb.html.
You will need to place the databases in the correct location. The location BLAST expects
databases to be stored is pointed to by the environment variable “BLASTDB.” This environment
variable can be viewed or set using the following procedure:

6
At the time of writing this was version 2.2.28.

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• Locate the Control Panel using the Start menu or by using Search.

• In the Control Panel window, doubl- click on the System icon.

• In the popup, click on the Advanced tab.

• Click on the Environment Variables button (if BLASTDB is already set, it may be
under User variables for… or System variables).

• If BLASTDB does not yet exist, click on the New button under the User variables for
... panel (or add it as a System variable if you want it available for all users).

• Type the environment variable name BLASTDB and enter the absolute path. For
example, if the databases are in the folder “db” in C:\Program Files\NCBI\blast-
2.2.28+\, the path would be C:\Program Files\NCBI\blast-2.2.28+\db.

• Click on the OK button to dismiss any open dialogs.

Figure 25-22 Editing the Environment Variable

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From a Windows Explorer window, navigate to the folder to which BLASTDB points. This is
where you place your database files.
Figure 25-23 Creating a db folder for BLAST

WORKING WITH LOCAL-BLAST

Other than being installed locally and using databases that you have created, there are very
few differences between BLAST and Local-BLAST options. You still have the choice between
blastn and megablast. You can still mask your sequences. You can still filter for low complexity
and set the E value and word size.
Note: Unlike BLAST, Local-BLAST does not set a default database for you. You must choose a
database from those available by using the Database drop-down menu in Local-BLAST Options.
Figure 25-24 Local-BLAST parameters

CONNECTIONS SCHEMATIC

The Connection Schematic is a tool that allows you to compare the results of BLAST and
Primer-BLAST runs, using different parameters, on a single sequence. This is an immensely
powerful way of finding functional regions in your sequences. In this example, we are showing
the results of the same sequence run with BLAST against different subsets of the GenBank
collection: htgs, dbsts, human est, and mouse est. The hits are listed in a table below the
diagram.

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Right-click in the row header of the sequence you have results for and whose Channels you
would like to summarize in a schematic. The row header has a row number in it like 1, 2, 3, 4,
etc. Choose Show Schematic.
Depending on how much data is involved, this window may take a little time to build. To obtain
more information about a particular hit, hover your cursor over the line. This will get
information about that particular BLAST hit. Alternatively, you may see more detailed data in
the tables below the graphic.
You will note that each Channel in the image below is displayed in a different color. This is set
in the Channel Options. Click on the green box next to the Default Graphic Color text and
choose a color. By clicking on the appropriate radio button, you can zoom all the way in to
view the individual bases on the Schematic.
Initially, you may prefer to use the Web View to examine your results using the BLAST or
Primer-BLAST graphic. An important thing to remember is that BLAST and Primer-BLAST results
eventually expire from the NCBI website. Sequencher Connections saves the results of any
searches so that you can have a permanent record of your search. Moreover, the Schematic
not only gives you a graphic view of your results, but also allows you to compare those results
across a series of channels, dramatically enhancing the graphic overview.
Figure 25-25 The Connections Schematic showing BLAST and Primer-BLAST results

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SEQUENCHER CONNECTIONS, GROUP SEQUENCES AND MUSCLE

Group sequences are sets of sequences you suspect are related. One of the main ways of
testing relatedness is to align all the sequences together to maximize the number of matching
bases. In order to achieve this, it is often the case that many gaps will be inserted. There are
several programs that are designed for this of which ClustalW2 and MUSCLE are among the
best known. MUSCLE is a multiple-sequence alignment algorithm that is claimed to achieve
both better speed and accuracy than ClustalW2. Starting with a group of phylogenetically-
related sequences in Sequencher, it is now possible to align those sequences and create a
phylogenetic tree using the MUSCLE algorithm.
When working with grouped sequences, you have two options. You can either create a new
session for each group or you can place related groups in the same session. In the image
below, two sets of fish mitochondrial DNA data have been added to a single grouped session.
The advantage of this is that you can keep related analyses together.
Figure 25-26 Group session with two groups of data

Open a Sequencher project file containing your sequences. Then select some or all of the
sequences. From the Window menu choose Add to Connections Session... if you are creating
a grouped session for the first time. You will then have to name your session and then click on
the OK button.

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If you choose several sequences that have the same name to add to a session, Sequencher will
warn you and ask you to change the names of those sequences.
Figure 25-27 Group Session duplicate names dialog

To edit the name, click in the Name field and type a new name or add an additional character
or number if you want to preserve the majority of the name. As the name clashes are
removed, the red exclamation points will disappear. When all duplicates have been renamed,
click on the OK button. The session opens and the group is given a name (typically Group 1,
Group 2, … Group n). Change the name by typing over the selected text in the name field.
Figure 25-28 Group session with user-specified name for group

Once your session is open, you will initially see only one row in the grid bearing the name that
you gave to your sequence group. You will also note that the tab views differ from those in the
Individual BLAST Sequencher Connections dialog. You will see a Web View (which shows a
page from the Gene Codes website), an Alignment view (a text-based view of the MUSCLE
alignment), a Log view (view of the log file generated by the MUSCLE program), a Tree view
(phylogenetic tree view), and a Sequence view (the unaligned sequences).
If you want to add further groups to the session, make your selection in the Project Window,
select the Add to Connections Session… item on the Window menu, click on the Add to an

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existing Connections Session... radio button in the Session Launcher dialog, choose the
session you want to add the data to, and then click on the OK button.
Note: The outline colors for Individual and Group Connections sessions are different.
To run MUSCLE on all of the data in a session, click on the Run All button. If you have set up
several Channels and only want to use a selected Channel, right-click in the header row for that
Channel and choose the Run on Each Group menu item on the context menu. Once the status
has changed to Done, you may view the alignment in the Text view or the phylogenetic tree in
the Tree view by clicking on the appropriate tab.
Figure 25-29 Status messages in a Group session

MUSCLE OPTIONS

As with the Sequencher Connections for individual sequences, you can also configure options
for grouped sequences. Right-clicking on the MUSCLE column header invokes a contextual
menu. Choose Options....
You can change the number of iterations that MUSCLE will go through. Each iteration will try to
improve the quality of the multiple alignment. However, with very long sequences or large
numbers of sequences, it may not be practicable to perform too many iterations. At this time,
the maximum number of iterations you can choose is 5.
Another option you can set affects the speed of the alignment. When comparing two
sequences, a shortcut method whereby short regions of high similarity are found has been
implemented in MUSCLE. This speeds up the algorithm but does have an adverse affect on
accuracy. You can turn this option on or off by choosing the appropriate radio button in the
Use diagonals to speed up groupbox.

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 Figure 25-30 Options for MUSCLE algorithm

You can also change the color of the tree by clicking on the Default Graphic Color icon and
selecting a replacement for the default.

SEQUENCHER CONNECTIONS MUSCLE TREE VIEW

You can view the phylogenetic tree in the Tree view by clicking on the appropriate tab. If you
right-click within the Tree view, you will see a contextual menu that allows you to switch
between the rectangular and circular rendering of the tree.

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 Figure 25-31 Rectangular phylogram using MUSCLE alignment

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 Figure 25-32 Circular phylogram using MUSCLE alignment

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 26. APPENDIX – KEYBOARD SHORTCUTS

WINDOWS

Menu/Window Command Windows

Any view Help F1

File menu Open window or Open project (set in User Ctrl+O

Preferences)

Close window Ctrl+W

Get Info… Ctrl+I

Save Project Ctrl+S

Print Ctrl+P

Exit Ctrl+Q

Edit menu Undo Ctrl+Z

Cut Selection Ctrl+X

Copy Selection Ctrl+C

Paste Ctrl+V

Edit Comments… Ctrl+,

Select menu Select All Ctrl+A

Select None Ctrl+\

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Menu/Window Command Windows

Find bases… Ctrl+F

Find bases again Ctrl+G

Contig column Ctrl+K

Select Next Ambiguous base Ctrl+N

Select Next Contig Disagree Ctrl+D

Select Next Edited ase Ctrl+E

Select Next Low Confidence Base Ctrl+L

Select Next Met to Stop Ctrl+M

Sequence menu Mark Selection As Feature Ctrl+‘

View menu Display Format Ruler Ctrl+R

Reverse & Comp Ctrl+4

Sequenced Strand Ctrl+5

st
View submenu Display 1 frame translation Ctrl+1

nd
Display 2 frame translation Ctrl+2

rd
Display 3 frame translation Ctrl+3

Single stranded Ctrl+-

Double stranded Ctrl+=

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Menu/Window Command Windows

Window menu Chromatogram Ctrl+T

User Preferences Ctrl+U

Switch between large icons, small icons, and Alt+click in Title line
list view.

Overview Options Names at Left Ctrl+Shift+left arrow key

Sequence Editor/Contig Insertion point moves one base to the right. Right arrow key
editor

Insertion point moves one base to the left. Left arrow key

Insertion point moves 3 bases to the right. Alt+right arrow key

Insertion point moves 3 bases to the left. Alt+left arrow key

Insertion point moves to 3’ end of Ctrl+right arrow key

sequence.

Insertion point moves to 5’ end of Ctrl+left arrow key

sequence.

Summary view Display Formatting Ruler. Ctrl+Alt+R

Frame 1 Ctrl+Alt+1

Frame 2 Ctrl+Alt+2

Frame 3 Ctrl+Alt+3

User defined Ctrl+6 to Ctrl+0

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MAC

Menu/Window Command Mac

Any view Help Cmd+Shift+? or Help

Project Window Continuous selection Click first item then Shift+Click

Discontinuous selection Cmd+Click each item

Sequencher menu Quit Sequencher Cmd+Q

File menu Open window or Open project (set in Cmd+O

User Preferences

Close window Cmd+W

Get Info… Cmd+I

Save Project Cmd+S

Print Cmd+P

Edit menu Undo Cmd+Z

Cut Selection Cmd+X

Copy Selection Cmd+C

Paste Cmd+V

Edit Comments… Cmd+,

Select menu Select All Cmd+A

Select None Cmd+\

Find bases… Cmd+F

Find bases again Cmd+G

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Menu/Window Command Mac
Contig column Cmd+K

Select Next Ambiguous base Cmd+N

Select Next Contig Disagree Cmd+D

Select Next Edited Base Cmd+E

Select Next Low Confidence Base Cmd+L

Select Next Met to Stop Cmd+M

Sequence menu Mark Selection As Feature Cmd+‘

Delete Bases Fill Void From Right Delete

Delete Bases Fill Void From Left Alt+Delete

Insert Gaps & Move Bases Right Tab

Insert Gaps & Move Bases Left Alt+Tab

View menu Display Format Ruler Cmd+R

Reverse & Comp Cmd+4

Sequenced Strand Cmd+5

st
View submenu Display 1 frame translation Cmd+1

nd
Display 2 frame translation Cmd+2

rd
Display 3 frame translation Cmd+3

Single stranded Cmd+-

Double stranded Cmd+=

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Menu/Window Command Mac
Window menu Chromatogram Cmd+T

User Preferences Cmd+U

Switch between large icons, small icons Alt+click in Title line

and list view.

Overview Options Names at Left Ctrl+Alt+left arrow

Condensed Base Numbers Ctrl+Alt+C

Diagram Key Ctrl+Alt+K

Fragment Labels Ctrl+Alt+L

Fragment Names Ctrl+Alt+N

Fragment Positions Ctrl+Alt+P

Start & Stop Codons Ctrl+Alt+S

Base Numbers at Transitions Ctrl+Alt+T

Scale to Window Ctrl+Alt+W

Base numbers every X Bases Ctrl+Alt+X

Sequence Editor/Contig Insertion point moves one base to the Right arrow key
editor right

Insertion point moves one base to the Left+arrow key

left

Insertion point moves 3 bases to the Alt+right arrow key

right

Insertion point moves 3 bases to the left Alt+left arrow key

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Menu/Window Command Mac
Insertion point moves 9 bases to the Ctrl+Alt+right arrow key
right

Insertion point moves 9 bases to the left Ctrl+Alt+left arrow key

Insertion point moves to 3’ end of Cmd+right arrow key

sequence

Insertion point moves to 5’ end of Cmd+left arrow key

sequence

Continue selection to 5’ end Cmd+[

Continue selection to 3’ end Cmd+]

Summary view Display/Hide Bullets and Pluses Ctrl+Alt+B

Display/Hide Consensus Sequence Ctrl+Alt+C

Display/Hide & Dashes Ctrl+Alt+D

Display/Hide Icons Ctrl+Alt+I

Display/Hide Fragment Sequences Ctrl+Alt+F

Display/Hide Matching bases as dashes Ctrl+Alt+M

Display Formatting Ruler Cmd+R

Frame 1 Cmd+1

Frame 2 Cmd+2

Frame 3 Cmd+3

Restriction map display 1 cutter (unique cutter) Ctrl+Alt+1

options

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Menu/Window Command Mac
2 cutters Ctrl+Alt +

3 cutters Ctrl+Alt+3

4 & more cutters Ctrl+Alt+4

All cutters Ctrl+Alt+A

Cut Positions Ctrl+Alt+C

Fragment Sizes Ctrl+Alt+F

Cut Positions Ctrl+Alt+C

Fragment Sizes Ctrl+Alt+F

Multiple Lines Ctrl+Alt+M

List selected enzyme names Ctrl+Alt+N

Single Lines Ctrl+Alt+S

Text Ctrl+Alt+T

Scale to Window Ctrl+Alt+W

User defined Cmd+6 to Cmd+0

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 27. APPENDIX – ADVANCED EXPRESSIONS

A regular expression is a special way of describing a search pattern using text strings according
to certain syntax rules. Sequencher uses regular expressions to help you break down your
sequence-naming schema in order to define your Assembly Handles.
Table 27-1 Regular Expression special characters

Character Description
. Represents any character

[A-Za-z] Represents any letter

[A-Z]. Represents any capital letter

[a-z]. Represents any lowercase letter

\d Represents any digit

[0-9] Represents any digit

* A character or special character which is followed by * matches zero or more

instances of the character

+ A character or special character which is followed by + matches one or more

instances of the character

? A character or special character which is followed by ? matches zero or one

instances of the character

{N} A character or special character followed by {N}, where “N” = a whole number,
matches that number of instances of the preceding character

[] The contents of square brackets describe a list of matching items, regardless of

order.

^ Use this in square brackets to exclude the other items contained within the brackets.

\ The \ is known as the escape character and it allows the character to be interpreted
literally. It can also give a standard character a special meaning as in \d.

() Matches whatever you type within the parentheses

| The | acts as an “or”.

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You may require a more complex delimiter than those provided in the Name Delimiters drop-
down menu. You can use regular expressions to define these more complex delimiters.
Table 27-2 Examples of delimiter expressions

Delimiter Sequence Name Handle Handle Handle3 Handle4 Handle5

Expression 1 2

% Gel?%A%T7?%dog Gel? A T7? dog

\? Gel?%A%T7?%dog Gel %A%T7 %dog

\?% Gel?%A%T7?%dog Gel A%T7 dog

\?|% Gel?%A%T7?%dog Gel A T7 dog

If your sequence name is complex, you may need to write a regular expression that describes
the delimiters and the Assembly Handles. In the example below, the text between the
parentheses defines the Assembly Handles. Anything not enclosed by parentheses will be
treated as a delimiter.
(Assembly Handle 1)Delimiter(Assembly Handle 2)Delimiter(Assembly Handle 3)
You would use this kind of expression with the Expression is a delimiter box unchecked.
Regular expressions are powerful tools. There are many resources on the Internet that
describe how to use regular expressions. Here is one to help you get started:
www.zytrax.com/tech/web/regex.htm

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 28. APPENDIX - IU PAC-IUB STANDARD CODES

Code Base(s) Meaning

A A Adenosine

C C Cytosine

G G Guanine

T T Thymine

U U Uracil (RNA)

R A or G Purine

Y C or T (or U) Pyrimidine

K G or T Keto

M A or C Amino

S G or C Strong

W A or T (or U) Weak

B C, G, T (or U) Not A

D G, A, T (or U) Not C

H A, C, T (or U) Not G

V A, C, G Not T (or U)

N A, C, G, T (or U) (Any)

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 29. APPENDIX – DEFAULT FEATURE STYLES

The following table displays the default settings for the colors and styles of new features. New
features are those you create in an existing sequence or those described in an imported
sequence with a GenBank style Feature Table.
You can change the default settings in the Feature, Motif preference pane. (For more
information, refer to Chapter 23 “Customizing Sequencher and User Preferences”.

Feature Display Color Style Second

Strand

A. misc_feature

1. misc_difference √ Red

a) conflict √ Red

b) unsure √ Red

c) old_sequence √ Red

d) variation √ Red Underlined

e) modified_base √ Red

f) mutation √ Red

g) allele √ Red

2. gene

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Feature Display Color Style Second
Strand

3. misc_signal

a) promoter

1) CAAT_signal

2) TATA_signal

3) -35_signal

4) -10_signal

5) GC_signal

b) RBS

c) polyA_signal

d) enhancer

e) attenuator

f) terminator

g) rep_origin

h) oriT

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Feature Display Color Style Second
Strand

4. misc_RNA

a) prim_transcript

1) precursor_RNA

a) mRNA

b) 5'clip

c) 3'clip

d) 5'UTR

e) 3'UTR

f) exon √ Blue

g) CDS √ Blue Protein

1) sig_peptide

2) transit_peptide

3) mat_peptide

h) intron √ Cyan Invert case

i) polyA_site

j) rRNA

k) tRNA

l) scRNA

m) snRNA

n) snoRNA

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Feature Display Color Style Second
Strand

5. Immunogobulin related

a) C_region

b) D_segment

c) J_segment

d) N_region

e) S_region

f) V_region

g) V_segment

6. repeat_region

a) repeat_unit

b) LTR

c) satellite

d) transposon

7. misc_binding

a) primer_bind

b) protein_bind

c) STS

d) primer

9. misc_structure

a) stem_loop

b) D-loop

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Feature Display Color Style Second
Strand

10. gap

11. operon

12. source

Unrecognized*

*Note – The Unrecognized key is at the same level at misc_feature.

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 30. APPENDIX - FEATURE KEYS & QUALIFIERS

GENBANK FEATURE TABLES

GenBank Feature Tables provide a way of describing and locating features in a DNA sequence
using internationally agreed-upon layout and vocabulary. Although these tables look complex,
all tables are comprised of three basic elements which are described in the table below.
Table 30-1 Elements in a feature table

Element Description
Feature Key A single word or abbreviation which indicates a
functional grouping
Location The instruction for where to find the feature in the
sequence
Qualifier Additional information about the feature

Feature tables may look simple or complex depending on the number of annotations in the
table. The following figure shows a typical entry.
Figure 30-1 A simple feature table entry

Key Location/Qualifiers source 1..1509

/organism="Mus musculus"

/strain="CD1"

In the example above, the feature key is “source” and the location is represented by a range,
“1…1509”. A location can be a single base, a range of bases, a single base within a range, etc.
The qualifier, which can be free text, controlled vocabulary, citation or references numbers,
sequences, or user-defined feature labels is separated from the location by a forward slash.

For a more detailed discussion of table entries and their standard typography, see
http://www.ncbi.nlm.nih.gov/collab/FT/index.html.

The table below contains a listing of the standard feature keys which have been implemented in
Sequencher.

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 Table 30-2 Feature keys groups and hierarchies

Feature Specificity or detail Functional grouping

A. misc_feature

1. misc_difference a) conflict Difference and change

features
b) unsure

c) old_sequence

d) variation
2. gene
e) modified_base
3. misc_signal a) promoter Expression signal features
f) mutation
1) CAAT_signal
g) allele
2) TATA_signal

3) -35_signal

4) -10_signal

5) GC_signal

b) RBS

c) polyA_signal

d) enhancer

e) attenuator

f) terminator

g) rep_origin

h) oriT

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Feature Specificity or detail Functional grouping
4. misc_RNA a) prim_transcript Transcript features

1) precursor_RNA

a) mRNA

b) 5'clip

c) 3'clip

d) 5'UTR

e) 3'UTR

f) exon

g) CDS

1) sig_peptide

5. Immunogobulin a) C_region 2) transit_peptide

related
b) D_segment 3) mat_peptide

c) J_segment h) intron

d) N_region i) polyA_site
6. repeat_region a) repeat_unit Repeat features
e) S_region j) rRNA
b) LTR
f) V_region k) tRNA
c) satellite
g) V_segment l) scRNA
7. misc_binding a) primer_bind Binding features
d) transposon
m) snRNA
b) protein_bind
n) snoRNA
8. misc_recomb c) STS
a) iDNA Recombination features
9. misc_structure a) stem_loop Structure features
d) primer
b) D-loop

10. gap
11. operon
12. source

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FEATURE TABLE QUALIFIERS

Genbank Feature Qualifiers convey many different types of information. To accommodate this
breadth there are several value formats.
Table 30-3 Feature Qualifiers

Qualifier Type Description

Free text
Free text qualifiers are usually descriptive phrases
enclosed in double quotation marks.
Controlled vocabulary or
Controlled vocabulary or enumerated values qualifiers
enumerated values
are selections from a controlled vocabulary list and are
entered without quotation marks. Examples of
controlled vocabularies can be found in the Appendices
to the GenBank Feature Table documentation

(http://www.ncbi.nlm.nih.gov/collab/FT/index.html).

Citation or reference
A citation or reference number is enclosed in square
numbers
brackets to distinguish it from other numbers.
Sequences
Since 1998, it has not been acceptable to use a literal
sequence of bases as the Sequence Qualifier.

Feature labels
The feature label qualifier supports clarity in reporting by
making sure your references are unambiguous. An
example of a feature label would be alcA (see example
below).

The figure below shows a typical example of a feature table entry with qualifiers. In this
example, the feature key is CDS. The location is expressed as two numbers. The qualifiers tell
the reader that the gene is alcA, the product of the gene is alcohol dehydrogenase, and that the
evidence is experimental.

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 Figure 30-2 Qualifier examples

Key Location/Qualifiers

CDS 6006..>6253

/gene="alcA"

/experiment="experimental evidence, no additional details

recorded"

/note="ADHI; ethanol regulon"

/codon_start=1

/product="alcohol dehydrogenase I"

Information on the Feature Keys and Tables and Qualifiers has been taken from the official
GenBank documentation which can be obtained from the following URL:
http://www.ncbi.nlm.nih.gov/collab/FT/index.html.

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 31. APPENDIX - DEFAULT FEATURE QUALIFIERS

In this table you will find the default Feature Qualifier for each Feature Key as implemented in
Sequencher.

Feature Key Default Feature Qualifier

Sequencher *

A. misc_feature misc feature [note]

1. misc_difference misc difference [note]

a) conflict conflict [compare]

b) unsure unsure difference

c) old_sequence old sequence [compare]

d) variation variation [note]

e) modified_base [gene] modified base[mod_base]

Allele ** allele

Mutation ** mutation

2. gene [gene] gene

3. misc_signal [gene] misc signal

a) promoter [gene] promoter

1) CAAT_signal [gene] CAAT signal

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Feature Key Default Feature Qualifier

2) TATA_signal [gene] TATA signal

3) -35_signal [gene] -35 signal

4) -10_signal [gene] -10 signal

5) GC_signal [gene] GC signal

b) RBS [gene] RBS

c) polyA_signal [gene] polyA signal

d) enhancer [gene] enhancer

e) attenuator [gene] attenuator

f) terminator [gene] terminator

g) rep_origin [gene] replication origin

h) oriT [gene] oriT

4. misc_RNA [gene] misc RNA

a) prim_transcript [gene] primary transcript

1) precursor_RNA [gene] precursor RNA

a) mRNA [gene] mRNA

b) 5'clip [gene] 5'clip

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Feature Key Default Feature Qualifier

c) 3'clip [gene] 3'clip

d) 5'UTR [gene] 5'UTR

e) 3'UTR [gene] 3'UTR

f) exon [gene] exon

g) CDS [gene] CDS

1) sig_peptide [gene] signal peptide

2) transit_peptide [gene] transit peptide

3) mat_peptide [gene] mature peptide

h) intron [gene] intron

i) polyA_site [gene] polyA site

j) rRNA [gene] rRNA

k) tRNA [gene] tRNA

l) scRNA [gene] scRNA

m) snRNA [gene] snRNA

n) snoRNA [gene] snoRNA

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Feature Key Default Feature Qualifier

5. Immunogobulin related Immunogobulin related

a) C_region [gene] C region

b) D_segment [gene] D segment

c) J_segment [gene] J segment

d) N_region [gene] N region

e) S_region [gene] S region

f) V_region [gene] V region

g) V_segment [gene] V segment

6. repeat_region [gene] [rpt_type] repeat region [rpt_family]

a) repeat_unit [gene] [rpt_type] repeat unit

b) LTR [gene] LTR

c) satellite [gene] [rpt_type] satellite

Transposon ** transposon

7. misc_binding misc binding of [bound_moiety]

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Feature Key Default Feature Qualifier

a) primer_bind [gene] primer binding site

b) STS STS [locus_tag]

c) protein_bind [bound_moiety] protein binding site

Primer** primer

8. misc_recomb Misc recombination [gene] [map]

a) iDNA [gene] iDNA

9. misc_structure misc structure [note]

a) stem_loop [gene] stem loop

b) D-loop [gene] D-loop

10. gap gap of [estimated_length]

11. operon [operon] operon

Source [organism]

Unrecognized ***

* The Sequencher Feature Key is for personal annotation.

** Legacy keys.

*** This is used for Feature Keys which are unknown to Sequencher so that they
can be imported in to a project.

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 32. GLOSSARY

Name Description

Administrator Also known as Admin or admin user. A user account that typically has
privileges that allow the user to change system settings and install software.

Agent A region in some dialogs (and also in the Contig Editor) that contains
information in text form.

ASCII American Standard Code for Information Interchange; the most widely
accepted coding system that permits letters, numbers, and punctuation to be
represented in binary computer code.

Assemble by
A feature that uses portions of a sequence’s name to determine which other
Name sequences it will be compared against to construct a contig.

Base caller Program that analyses fluorescent trace intensities from an automated
sequencer and assigns bases together with a probability of error.

BLAST Basic Local Alignment Search Tool. Developed by NCBI. This algorithm will
identify sequences in a database that align to the query sequence. Most
searches are conducted over the internet using the BLAST servers at NCBI. The
program can also be installed on a desktop computer in which case it is called
Local-BLAST.

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Name Description

Button bar The tool region in a Sequencher window. The button bar is located just below
the title bar.

CAF A format for describing sequence assemblies

Channel The channel in a Connections Session contains a single program and associated
settings and is represented in the Session by a column.

Checkbox A control that allows you to toggle a parameter. Click on the box to turn it on
or off. When a checkbox is active, it is displayed with an “X” in the box.

Chromatogram Also known as a sequencing trace or electropherogram. This is the plot of results
from electrophoresis of a DNA sequencing reaction. The results are visualized as
traces of four separate colors where each peak represents a base.

Condition In RNA-Seq, a condition is a single sample, tissue, or treatment on a single

sample or tissue. RNA-Seq experiments usually contain at least two conditions.

Confidence A number associated with each base call and which defines the likelihood
score that a base call is incorrect. The most common scale is from 1-60, where “60”
represents a 1/106 chance of a wrong call and 20 represents a 1/102 chance.
See also Quality score.

Contig Contiguous alignment of a set of sequences to make up the sequence of

bases from a longer piece of DNA.

Cursor The indicator on a computer screen that shows the currently active location.
Cursors have various shapes, indicating different purposes and capabilities.

Data source A data source is a sequence that will be analysed in a Connections Session.

Dialog Any of several special windows that the operating system or Sequencher
displays when it needs to alert you to something you need to know, or needs
to ask you a question.

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Name Description

Delimiter Usually a character such as / or – which separates elements of a sequence

name

Elevator button A very small scroller that allows you to increase or decrease a numerical value
by using the mouse. An elevator button has an up arrow and a down arrow.

In this manual, enzyme always refers to a restriction endonuclease.

Enzyme

Exemplar Chosen or primary sequence. Literal meaning typical example or excellent

model.

External Data A tool for managing NGS, RNA-Seq, and MSA (Multiple-Sequence Alignment) run
Browser folders. With this tool, you may open the folders, delete them, view the log files,
view alignments in Tablet, and annotate the run.

External Data The location where your run folders are saved. The default location is your
Home Documents folder.

External Tools Programs that can be used by Sequencher to perform analysis. Specifically, the
External Tools are BWA, GSNAP, Maq, Velvet, MUSCLE, Tablet and which are
installed using a single installer. The RNA-Seq tools are also an example of
external tools.

Field A rectangle in which the user can (or in some cases must) enter names or
other information.

Gap An insertion or deletion between two or more aligned sequences. This may be
one or more bases long.

Handle The portion of a sequence name between two consecutive delimiters. Also
known as Assembly handle.

Large Gap An insertion or deletion consisting of 10 or more bases between two or more
aligned sequences.

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Name Description

Modal dialog A dialog that requires a response before you can continue with regular
actions in the application.

Motif In Sequencher, this is a short subsequence of 50 bases or less.

MSF Multiple Sequence Format. A format used by multiple- sequence alignment

programs.

Multiplex ID A system for tagging reads using short sequences of DNA. Used to distinguish
samples in the same sequencing reaction. Sequencher separates the samples
using these tags prior to aligning them.

NGS Also known as Next-Generation Sequencing. This term covers a number of

technologies that enable high-throughput sequencing.

NEXUS/Paup Format used by phylogenetic and cladistic software.

Non-stranded In GSNAP, this refers to protocols where reads that align 5’ to 3’ on either strand
of the genome and their reverse complements are considered.

Project A project contains sequences, information on how these sequences fit

together to form larger pieces, and parameter information, specified by the
user, that controls alignment operations.

Project window The window in which Sequencher displays the Contigs and unincorporated
Sequences of a project. Items in the Project Window can be shown as icons,
small icons, or as a sorted list.

Quality score A number associated with each base call and which defines the likelihood
that a base call is incorrect. See also Confidence score.

Radio button A control that allows you to choose among alternate possibilities. Radio
buttons always appear in sets of at least two. Click one of the buttons to
select it. A button that is selected is shown with a filled circle; buttons that
are not selected are shown with open circles.

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Name Description

Reference A sequence used as a prototype or benchmark sequence. If a sequence has

sequence been designated as Reference within Sequencher, it has special properties.

Refrigerator A special folder in which to store subsets of sequences which need to remain
in your project.

Replicate Duplicates of samples used in RNA-Seq to control variability. There are two kinds
of replicates – biological and technical. Biological replicates are separate
samples that are then processed independently. Technical replicates use the
same sample but perform the technical steps separately.

RNA-Seq This term applies to the use of NGS sequencing of transcriptomes. RNA-Seq
provides a quantifiable snapshot of the expressed RNA in the sample at a given
time.

RNA editing Conversion of Adenosine to Inosine by the ADAR gene. This can be detected by
NGS sequencing as an Adenosine to Guanine change when compared to a
Reference Sequence.

SCF Standard Chromatogram Format. A format used to store analyzed data or

fragment data for a single sample.

Schematic Visualization of BLAST, Local-BLAST, and Primer-BLAST results. The Schematic

presents a consolidated view across all analyses for an individual sequence.

Secondary peak A chromatogram peak whose height may be less than that of the primary
peak and whose presence may indicate that a heterozygote exists at that
position.

Sequence A small piece of DNA, decoded into its component bases, in order,
represented as text. Sometimes in reverse order, depending upon the
vagaries of the sequencing procedure.

The window used for editing DNA sequences. This editor looks much like a
Sequence Editor
word processor and supports the standard text editing commands: Cut, Copy,
Paste, and Undo.

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Name Description
Term used to describe a collection of channels (analyses) and data sources
Session
(sequences) in Sequencher Connections. There are two types of sessions –
individual or grouped. In Individual Sessions, the analysis operates on single
sequences although it can be applied to many sequences in parallel. In a Grouped
Session, a single analyses operates on several sequences such as in a multiple
alignment.

An on-screen control that simulates the action of a slide control. This is used as
Slider
a control for increasing/decreasing a value.

In GSNAP, this refers to protocols where only reads that align 5’ to 3’ on either
Stranded
strand of the genome are considered.

A text region at the top of a window generally describing the function of the
Title bar
window or the name of the document displayed in the window.

Identifying single bases that differ when compared to a Reference Sequence.

Variant Calling
Unlike Sanger sequencing where the traces may be examined, in NGS, reliance is
placed on the algorithm used to distinguish between a true variant and technical
problems with the sequence.

A cloning vector sequence database prepared by Friedhelm Pfeiffer, in

VecBase
collaboration with William Gilbert. A file with this name, in VecBase
(CODATA) format. Part of the Sequencher distribution.

Volcano plot Specialised form of scatter plot. Plots –log10 (p-value) against log2fold change
in expression.

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