Nextera XT DNA Library Prep

Reference Guide

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Nextera XT DNA Library Prep Reference Guide

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Table of Contents
Overview 1
Introduction 1
DNA Input Recommendations 2
Input DNA Quantification 2
Assess DNA Purity 2

PCR Amplicons 2
Additional Resources 3

Protocol 5
Introduction 5
Library Prep Workflow 7
Tagment Genomic DNA 8
Preparation 8
Procedure 8

Amplify Libraries 9
Preparation 9
Procedure 10

Clean Up Libraries 11
Preparation 11
Procedure 11

Check Library Quality 12

Normalize Libraries (Nextera XT and Nextera XT V2) 13
Preparation 14
Procedure 14

Dilute Libraries to the Starting Concentration 16

Appendix A Supporting Information 18

Nextera XT DNA Library Prep Reference Guide

How the Nextera XT Assay Works 18

Nextera XT Quality Metrics 19
Check Library Size 19
Check Library Concentration 20

Kit Contents 21
Nextera XT Library Prep Contents 22
Index Kit Contents 23

Consumables and Equipment 25

Consumables 25
Equipment 26
Thermal Cyclers 26

Acronyms 26

Technical Assistance 34

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Nextera XT DNA Library Prep Reference Guide

Overview
This guide explains how to prepare up to 384 uniquely dual-indexed paired-end libraries from DNA
using the Nextera XT DNA Library Prep workflow.
The Nextera XT workflow:
• Uses tagmentation, an enzymatic reaction, to fragment DNA and add adapter sequences in only 15
minutes.
• Uses innovative sample normalization at inputs ≥ .
• Is compatible with extracted formalin-fixed paraffin-embedded (FFPE) samples. ∆Cq ≤ 5 is
recommended for optimal performance.

Introduction
This guide explains how to prepare up to 384 dual-indexed paired-end libraries from DNA using
Nextera™ XT Library Prep workflow.
The Nextera XT DNA Library Prep protocol:
• Uses tagmentation, an enzymatic reaction, to fragment DNA and add partial adapter sequences in
only 5 minutes.
• Reduces reagent containers, pipetting, and hands-on time using. master mixed reagents.
• Requires only 1 ng input DNA.
• Supports genomes that are less than 5 Mb.

Table 1 Example Applications for Nextera Kits

Nextera XT (FC-131-1024, FC-131-1096) Nextera DNA Flex (20018704, 20018705)
Small genomes, amplicons, plasmids Human genomes, large or complex genomes
PCR amplicons (> 300 bp)* Small genomes, microbial, plasmids,
PCR amplicons (> 150 bp)
Plasmids Nonhuman mammalian genomes (eg, mouse,
rat, bovine)
Microbial genomes (eg, Prokaryotes, Archaea) Plant genomes (eg, Arabidopsis, maize, rice)
Concatenated amplicons Invertebrate genomes (eg, Drosophila)
Double-stranded cDNA –
Single-cell RNA-Seq –

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* Using > 300 bp amplicon size ensures even coverage across the length of the DNA fragment. For more
information, see PCR Amplicons on page 2.

DNA Input Recommendations

The Nextera XT protocol is optimized for 1 ng of input DNA. Quantify the starting material before
preparing libraries.
Assess DNA purity to make sure that the initial DNA sample does not contain > 1 mM EDTA and is free of
organic contaminants, such as phenol and ethanol. These substances can interfere with the Nextera
tagmentation reaction and result in assay failure.

Input DNA Quantification

The enzymatic DNA fragmentation used for this protocol is more sensitive to DNA input compared to
mechanical fragmentation. Success depends on accurate quantification of input DNA.
Use a fluorometric-based method to quantify input DNA. For example, if you use the Qubit dsDNA BR
Assay system, use 2 µl of each DNA sample with 198 µl of the Qubit working solution. Avoid methods
that measure total nucleic acid, such as NanoDrop or other UV absorbance methods.

Assess DNA Purity

UV absorbance is a common method used for assessing the purity of a DNA sample. The ratio of
absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is
optimized for DNA with 260/280 absorbance ratio values of 1.8–2.0, which indicates a pure DNA
sample.
For a secondary indication of sample purity, use the ratio of absorbance at 260 nm to absorbance at
230 nm. Target a 260/230 ratio of 2.0–2.2. Values outside this range indicate the presence of
contaminants. For a complete list of contaminants, including sources, avoidance, and effects on the
library preparation, see the Nextera XT Troubleshooting Technical Note.
Dilute the starting material in 10 mM Tris-HCI, pH 7.5–8.5. Incomplete tagmentation caused by
contaminants can cause library preparation failure, poor clustering, or low quality sequencing results.

PCR Amplicons
When starting with PCR amplicons, the PCR amplicon must be > 300 bp. The standard clean-up
protocol depletes libraries < 500 bp. Therefore, Illumina recommends that amplicons < 500 bp undergo
a 1.8 x Illumina Purification Beads volume normal ratio to supernatant during Clean Up Libraries. Shorter
amplicons can otherwise be lost during the library cleanup step.

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Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing
coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon
target region, design primers to extend beyond the target region by 50 bp per end.

Additional Resources
The Nextera XT DNA Library Prep support pages on the Illumina website provide software, training
resources, product compatibility information, and the following documentation. Always check support
pages for the latest versions.
• Compatible products and requirements for recording sample information, sequencing libraries, and
analyzing data.
• Questions and answers about using the kit.
• Training videos about the kit and courses for related products and subjects.
• The latest versions of the kit documentation.

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Table 2 Sample Input Recommendations

Resource Description
Custom Protocol Selector A tool for generating end-to-end instructions tailored to your library
prep method, run parameters, and analysis method, with options to
refine the level of detail.
Nextera XT DNA Library Prep Provides a checklist of steps for the experienced users.
Kit Checklist (document #
1000000006566)
Nextera XT Library Prep Kit Provides an interactive checklist of user-supplied consumables and
Consumables and equipment.
Equipment List (document #
1000000006567)
Index Adapters Pooling Provides pooling guidelines and dual-index strategies for using the 10
Guide (document base pair IDT for Illumina DNA/RNA UD Indexes or 8 base pair
#1000000041074) Nextera XT and Nextera XT v2 Indexes with the Nextera XT DNA
Library Prep kit.
Illumina Adapter Sequences Provides the nucleotide sequences that comprise Illumina
(document # oligonucleotides used in Illumina sequencing technologies.
1000000002694)
Infinium HD FFPE QC Assay Provides the protocol to assess DNA input quality for FFPE samples.
Protocol (document #
15020981)
Illumina Free Adapter Provides the protocol to block excess free adapter, minimize index
Blocking Reagent (document hopping, and enhance data quality.
# 1000000047585)
IDT for Illumina DNA/RNA UD Provides information about IDT for Illumina DNA/RNA Unique Dual
Indexes support page (UD) indexes.

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Protocol
Introduction
This chapter describes the Nextera XT DNA protocol.

• Review the planned complete sequencing workflow, from sample through analysis, to ensure
compatibility of products and experiment parameters.
• Before proceeding, confirm kit contents and make sure that you have the required components,
equipment, and consumables. This protocol requires library prep reagents and index adapters.
Index adapters are sold separately. See Supporting Information on page 18.
• Follow the protocols in the order shown, using the specified volumes and incubation parameters.

Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.
Avoiding Cross-Contamination
• When adding or transferring samples or reagent master mixes, change tips between each sample.
• When adding index adapters with a multichannel pipette, change tips between each row or each
column. If using a single channel pipette, change tips between each sample.
• [Tubes] Open only one index adapter tube at a time to prevent misplacing caps. Remove unused
index adapter tubes from the working area.
Sealing the Plate
• Always seal the 96-well plate with the adhesive seal using a rubber roller to cover the plate before
the following steps in the protocol:
– Shaking steps
– Thermal cycling steps
– Centrifuge steps
• Microseal 'A' adhesive film is used for thermal cycling steps to prevent evaporation.
• Microseal 'B' adhesive seals are effective at -40°C to 110°C and suitable for skirted or semiskirted
PCR plates. Use Microseal 'B' seals for thermal cycling or short-term storage.
• Microseal ‘F’ foil seals are effective at temperatures down to -70°C and are suitable for storing the
96-well plates containing the final libraries long term.
IPB 100 ml Bottle Resuspension

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Manually mix the bead reagent container by manual inversion. Inversion mixing constitutes turning a
container upside down and then returning it to its upright position.
1. Mix the bead reagent by inversion, at a rate of at least a single inversion per second.
2. Rotate bottle 90 degrees every 30s, and continually inverting for a total of 2 minutes.
3. After this time, visually inspect the inside of the container for any solid material still adhering to the
walls.
4. If container inner walls are free of any residual material, bead reagent can be considered fully
resuspended.
5. If reagent is still found on the inner walls, repeat the mixing step for additional 1 minute.
6. Repeat visual inspection of the container walls to ensure complete mixing. If necessary, repeat the
mixing step (5).
Preparing IDT for Illumina DNA/RNA Unique Dual (UD) Indexes Plate
Prepare IDT for Illumina DNA/RNA UD Indexes as follows.
® ®
• Nextera XT is compatible with IDT for Illumina DNA/RNA Unique Dual (UD), IDT for Illumina
Nextera DNA Unique Dual (UD), and Nextera DNA Combinatorial Dual (CD) Indexes.
• Pipette slowly to minimize foaming.
• Each index plate is for single use only.
® ®
• IDT for Illumina DNA/RNA UD Indexes use 10 base pair index codes that differ from Nextera
XT,and Nextera XT v2 indexes, which use eight base pair index codes. Confirm that your
sequencing system is configured for 10 base pair index codes.
• Centrifuge at 1000 × g for 1 minute to settle liquid away from the seal.
• [< 96 samples] Pierce the foil seal on the index adapter plate using a new pipette tip for each well
for only the number of samples being processed.
• [96 samples] Align a new Eppendorf 96-well PCR plate above the index adapter plate and press
down to puncture the foil seal on all 96 wells. Press down slowly to avoid tipping the volume over.
• Discard the empty Eppendorf plate used to puncture the foil seal.

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Library Prep Workflow

The following diagram illustrates the Nextera XT DNA Library Prep workflow. Safe stopping points are
marked between steps.
Time estimates are based on processing 8 samples.

Figure 1 Nextera XT DNA Library Prep Workflow

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Tagment Genomic DNA

This step uses the Nextera transposome to tagment gDNA, which is a process that fragments and tags
DNA with adapter sequences.

Consumables
• Amplicon Tagment Mix (ATM)
• Tagment DNA Buffer (TD)
• Neutralize Tagment Buffer (NT)
• 96-well PCR plate
• Microseal 'B' adhesive seals

Preparation
1. Prepare the following consumables:

Item Storage Instructions

ATM -25°C to -15°C Thaw on ice. Invert the thawed tubes 3–5 times, and then
centrifuge briefly.
TD -25°C to -15°C Thaw on ice. Invert the thawed tubes 3–5 times, and then
centrifuge briefly.
NT 15°C to 30°C Check for precipitates. If present, vortex until all particulates are
resuspended.

2. Save the following TAG program on the thermal cycler:

• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 55°C for 5 minutes
• Hold at 10°C

Procedure
1. Add the following volumes in the order listed to each well of a new 96-well PCR plate.
• TD (10 µl)
• 1 ng DNA (5 µl)
2. Pipette to mix.
3. Add 5 µl ATM to each well.

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4. Pipette 10 times to mix, and then seal the plate.

5. Centrifuge at 280 × g at 20°C for 1 minute.
6. Place on the preprogrammed thermal cycler and run the TAG program. When the program reaches
10°C, immediately proceed to step 7 because the transposome is still active.
7. Add 5 µl NT to each well.
8. Pipette 10 times to mix, and then seal the plate.
9. Centrifuge at 280 × g at 20°C for 1 minute.
10. Incubate at room temperature for 5 minutes.

Amplify Libraries
This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds the
Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation.
To confirm indexes selected for low plexity pooling have the appropriate color balance, see the Index
Adapters Pooling Guide (document #1000000041074).
Index adapter tubes or plates are ordered separately from the library prep components. For a list of
compatible index adapters for use with this protocol, see Kit Contents on page 21.

Consumables
• Nextera PCR Master Mix (NPM)
• Index adapters (tubes or plates)
• Microseal 'A' adhesive film
About Reagents
• Index adapter plates
– A well may contain > 10 µl index adapters.
– Do not add samples to the index adapter plate.
– Each well of the index plate is single use only.
• Index adapter tubes
– Open only one index adapter tube at a time to prevent misplacing caps. Alternatively, use fresh
caps after opening each tube.

Preparation
1. Prepare the following consumables:

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Item Storage Instructions

Index adapters -25°C to -15°C Thaw at room temperature.
[Tubes] Vortex to mix, and then centrifuge
briefly.
[Plates] Spin briefly before use.
NPM -25°C to -15°C Thaw on ice for 20 minutes.

2. Save the following NXT PCR program on a thermal cycler:

• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 72°C for 3 minutes
• 95°C for 30 seconds
• 12 cycles of:
• 95°C for 10 seconds
• 55°C for 30 seconds
• 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 10°C

Procedure
1. Add the following index adapter volumes per sample according to your index adapter kit type.

Index Adapter Kit Type Volume of Index Adapter per Sample

index adapter tubes 5 µl i7 adapter
5 µl i5 adapter
index adapter plate 10 µl prepared i7 and i5 index adapters

2. Add 15 µl NPM to each well.

3. Pipette 10 times to mix, and then seal the plate.
4. Centrifuge at 280 × g at 20°C for 1 minute.
5. Place on the preprogrammed thermal cycler and run the NXT PCR program.

SAFE STOPPING POINT

If you are stopping, store at 2°C to 8°C for up to 2 days. Alternatively, leave on the thermal cycler for up
to 24 hours.

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Clean Up Libraries
This step uses single-sided bead purification to purify amplified libraries.

Consumables
• Illumina Purification Beads (IPB)
• Freshly prepared 80% ethanol (EtOH)
• Resuspension Buffer (RSB)
• 96-well 0.8 ml polypropylene deepwell storage plate (MIDI plate) (2)
• 96-well PCR plate
• Microseal 'B' adhesive seal
• Microseal 'F' foil seals
• Nuclease-free water
About Reagents
• Illumina Purification Beads
– Must be at room temperature before use.
– Resuspend before each use.
– Resuspend frequently to make sure that beads are evenly distributed.
– Aspirate and dispense slowly due to the viscosity of the solution.

Preparation
1. Prepare the following consumables:

Item Storage Instructions

IPB 15°C to 30°C Resuspend IPB beads.
RSB -25°C to -15°C Thaw and bring to room temperature. Vortex to mix.
RSB can be stored at 2°C to 8°C after the initial thaw.

2. Prepare fresh 80% EtOH from absolute ethanol.

Procedure
1. Centrifuge at 280 × g at 20°C for 1 minute to collect contents at the bottom of the well.
2. Transfer 50 µl supernatant from each well of the PCR plate to corresponding wells of a new MIDI
plate.

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The ratio of supernatant to volume of IPB is 3:2. If you transfer less than 50 µl supernatant,
adjust the volume of IPB accordingly.

3. If you are using standard DNA input, add 30 µl IPB to each well containing supernatant.
4. If you are using small PCR amplicon sample input, add the IPB volume according to your input size in
the table.

Input Size (bp) IPB Recommendation IPB Volume (µl)

300–500 1.8x IPB 90
> 500 0.6x IPB 30
(0.5x IPB for ≥ 2 x 250 cycles) (25 µl for ≥ 2 x 250 cycles)

5. Seal the plate, and then use a plate shaker at 1800 rpm for 2 minutes.
6. Incubate at room temperature for 5 minutes.
7. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
8. Without disturbing the beads, remove and discard all supernatant.
9. Wash two times as follows.
a. With the plate on the magnetic stand, add 200 µl fresh 80% EtOH without mixing.
b. Incubate for 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant.
10. Use a 20 µl pipette to remove and discard residual EtOH.
11. Air-dry on the magnetic stand for 15 minutes.
12. Remove from the magnetic stand.
13. Add 52.5 µl RSB to the beads.
14. Seal the plate, and then use a plate shaker at 1800 rpm for 2 minutes.
15. Incubate at room temperature for 2 minutes.
16. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
17. Transfer 50 µl supernatant to a new 96-well PCR plate.

SAFE STOPPING POINT

If you are stopping, seal the plate with Microseal 'B' adhesive seal or Microseal 'F' foil seal and store at
-25°C to -15°C for up to 7 days.

Check Library Quality

1. Run 1 µl undiluted library on an Agilent Technology 2100 Bioanalyzer using a High Sensitivity DNA
kit.

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Typical libraries show a broad size distribution of ~250–1000 bp, as shown in the top panel. Various
libraries can be sequenced with average fragment sizes as small as 250 bp or as large as 1500 bp.

Figure 2 Example Bioanalyzer Trace

Normalize Libraries (Nextera XT and

Nextera XT V2)
This process normalizes the quantity of each library made with Nextera XT Index v2 or Nextera XT
Index Kits to ensure more equal library representation in the pooled library.
Do not follow the normalization protocol and instead manually normalize libraries:
• If you are using IDT for Illumina Nextera UD Indexes.
• If the final library yield is < 10 nM.
• If your sequencing system uses onboard denaturation.

Consumables
• Library Normalization Additives 1 (LNA1)
• Library Normalization Beads 1 (LNB1)
• Library Normalization Wash 1 (LNW1)
• Library Normalization Storage Buffer 1 (LNS1)
• 0.1 N NaOH (fewer than 7 days old) (3 ml per 96 samples)
• 96-well 0.8 ml polypropylene deep-well storage plate (MIDI plate)
• 96-well PCR plate
• 15 ml conical tube
• Microseal 'B' adhesive seals

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• About Reagents
• Vortex LNA1 vigorously to make sure that all precipitates have dissolved. Inspect in front of a light.
• Vortex LNB1 vigorously, with intermittent inversion (at least 1 minute). Repeat until all beads are
resuspended and no beads are present at the bottom of the tube when it is inverted.
• Always use a wide-bore pipette tip for LNA1.
• Mix only the required amounts of LNA1 and LNB1 for the current experiment. Store the remaining
LNA1 and LNB1 separately at the recommended temperatures.
• Aspirate and dispense beads slowly due to the viscosity of the solution.
This set of reagents contains potentially hazardous chemicals. Personal injury can occur
through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
including eye protection, gloves, and laboratory coat appropriate for risk of exposure.
Handle used reagents as chemical waste and discard in accordance with applicable regional,
national, and local laws and regulations. For additional environmental, health, and safety
information, see the SDS at support.illumina.com/sds.html.

Preparation
1. Prepare the following consumables:

Item Storage Instructions

LNA1 -25°C to -15°C Prepare under a fume hood.
Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
LNB1 2°C to 8°C Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
LNW1 2°C to 8°C Bring to room temperature. Use a 20°C to 25°C water bath as
needed.
LNS1 Room Bring to room temperature.
temperature

Procedure
1. Transfer 20 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI
plate.
2. Combine the following volumes in a 15 ml conical tube to prepare the LN master mix. Multiply each
volume by the number of samples being processed.
• LNA1 (46 µl)
• LNA2 (8 µl)

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Reagent overage is included in the volume to ensure accurate pipetting.

3. Pipette 10 times to mix.
4. Pour the LN master mix into a trough.
5. Use a 200 µl multichannel pipette to transfer 45 µl LN master mix to each well.
6. Seal the plate, and then use a plate shaker at 1800 rpm for 30 minutes.
7. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
8. Without disturbing the beads, remove and discard all supernatant.
9. Wash two times as follows.
a. Add 45 µl LNW1 to each well.
b. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
c. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
d. Without disturbing the beads, remove and discard all supernatant.
10. Add 30 µl 0.1 N NaOH to each well.
11. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
12. Add 30 µl LNS1 to each well of a new 96-well PCR plate labeled SGP.
13. After the 5 minute elution completes, make sure that all samples in the MIDI plate are resuspended.
If they are not, resuspend as follows.
e. Pipette 10 times to mix or lightly tap the sample plate on the bench.
f. Seal the plate, and then use a plate shaker at 1800 rpm for 5 minutes.
14. Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
15. Transfer 30 µl supernatant from each well of the MIDI plate to the corresponding well of the SGP
plate.
16. Seal the sample plate, and then centrifuge at 1000 × g for 1 minute.

At this point, the libraries are single-stranded DNA, which resolves poorly on an agarose gel or
Bioanalyzer chip. For quality control, use the double-stranded DNA saved from step 17 of the
cleanup procedure.

SAFE STOPPING POINT

If you are stopping, store at -25°C to -15°C for up to 7 days.

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Dilute Libraries to the Starting

Concentration
This step dilutes libraries to the starting concentration for your sequencing system and is the first step
in a serial dilution. After diluting to the starting concentration, libraries are ready to be denatured and
diluted to the final loading concentration.
For sequencing, Illumina recommends the read lengths indicated on the Nextera XT DNA Library Prep
Product Compatibility support site page. If you would like additional overlapped reads, raw coverage, or
adjusted the IPB recommendations for ≥ 2 x 250 cycles, you can sequence up to 2 x 250 or 2 x 300, but
it is not required.
IDT for Illumina DNA/RNA UD Indexes uses 10 base pair index codes that differ from the Nextera XT, and
Nextera XT v2,, which use eight base pair index codes. This change in base pair index codes can require
adjustments to your sequencing run set up.
1. Calculate the molarity value of the library or pooled libraries using the following formula.
• For libraries qualified on a Bioanalyzer, use the average size obtained for the library.
• For all other qualification methods, use 600 bp as the average library size.

2. Using the molarity value, calculate the volumes of RSB and library needed to dilute libraries to the
starting concentration for your system.
3. Dilute libraries using UNB:
• Libraries quantified as a multiplexed library pool—Dilute the pool to the starting
concentration for your system.
• Libraries quantified individually—Dilute each library to the starting concentration for your
system. Add 10 µl of each diluted library to a tube to create a multiplexed library pool.
4. Follow the denature and dilute instructions for your system to dilute to the final loading
concentration.
• For the iSeq 100 System, see the system guide for dilution instructions (libraries are
automatically denatured).
• For the NovaSeq 6000 System, see the system guide for pool and denature instructions.
• For the HiSeq 4000 and HiSeq 3000 Systems, see the cBot 2 or cBot system guide for reagent
preparation instructions.
• For all other systems, see the denature and dilute libraries guide.

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Optimize concentrations for your workflow and quantification method over subsequent sequencing
runs or by flow cell titration.

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Appendix A Supporting Information

Supporting Information
The protocol described in this guide assumes that you have reviewed the contents of this section,
confirmed protocol contents, and obtained all required consumables and equipment.
Introduction
The protocol described in this guide assumes that you have reviewed the contents of this section,
confirmed protocol contents, and obtained all required consumables and equipment.

How the Nextera XT Assay Works

The Nextera XT DNA Library Prep uses an engineered transposome to tagment genomic DNA, which is
a process that fragments DNA and then tags the DNA with adapter sequences in one step. Limited-
cycle PCR uses the adapters to amplify the insert DNA. The PCR step also adds index adapter
sequences on both ends of the DNA, which enables dual-indexed sequencing of pooled libraries on
Illumina sequencing platforms.

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A. Nextera XT transposome with adapters combined with template DNA

B. Tagmentation to fragment and add adapters
C. Limited-cycle PCR to add index adapter sequences

Nextera XT Quality Metrics

Two factors can cause cluster density fluctuations in libraries prepared with the Nextera XT DNA Library
Prep:
• An average sample size that is too large or too small after tagmentation.
• A final sample concentration that is too low due to a low yield when starting the bead-based
normalization step.
To troubleshoot fluctuations in cluster density, consider checking library size and library concentration.
For more information, see Nextera XT Library Prep: Tips and Troubleshooting (Pub. No. 770-2015-015).

Check Library Size

Larger molecules cluster less efficiently than smaller molecules. If the fragment size after tagmentation
is larger than expected, low cluster numbers are possible. The inverse is also true. The average
expected library size after tagmentation is between 400 bp and 1.2 kb.

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Check the library size with a high sensitivity Bioanalyzer trace after the PCR cleanup step. Look for a
long low plateau. Alternatively, PCR-amplify the library with qPCR primers and run the product on an
agarose gel. The sequence for these primers is available in the Sequencing Library qPCR Quantification
Guide (document # 11322363).
• Short libraries indicate too little input DNA—Requantify the input DNA with a fluorometric method.
Start with 10%–25% more input DNA. If the library peak is below 400 bp and you want to continue
with this library, dilute the library further.
• Long libraries indicate too much input DNA or the presence of inhibitors—Start with less input
DNA, make sure that the input DNA is free from inhibitors, and repeat the quantification step.
For more information on library dilution, see the denature and dilute libraries guide for your sequencing
system.

Check Library Concentration

Bead-based normalization is most efficient when the library yield after amplification is 10–15 nM, or
higher. Measure library concentration using high sensitivity dsDNA Qubit after library cleanup, and
measure library size with a Bioanalyzer to calculate molarity.
If you are starting with high-quality DNA and see low yield after library cleanup, there are possible
issues with IPB cleanup or the amplification step. If results show either condition, confirm proper
storage of the PCR master mix at -25°C to -15°C in a no-frost freezer. Confirm minimal freeze-thaw
cycles.

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Nextera XT DNA Library Prep Reference Guide

Kit Contents
Completing the Nextera XT protocol requires library prep reagents and index adapters.

Component Kit Options Catalog #

Library prep reagents Nextera XT DNA Library Prep FC-131-1024
(24 Samples)
Nextera XT DNA Library Prep FC-131-1096
(96 Samples)
Index adapters IDT for Illumina Nextera UD Indexes Set A 20027213
(96 Indexes, 96 Samples)
IDT for Illumina Nextera UD Indexes Set B 20027215
(96 Indexes, 96 Samples)
IDT for Illumina Nextera UD Indexes Set C 20027215
(96 Indexes, 96 Samples)
IDT for Illumina Nextera UD Indexes Set D 20027216
(96 Indexes, 96 Samples)
IDT for Illumina Nextera UD Indexes Set A, B, 20027217
C, D
(384 Indexes, 384 Samples)
Nextera XT Index Kit (24 Indexes, 96 FC-131-1001
Samples)
Nextera XT Index Kit v2 Set A FC-131-2001
(96 Indexes, 384 Samples)
Nextera XT Index Kit v2 Set B FC-131-2002
(96 Indexes, 384 Samples)
Nextera XT Index Kit v2 Set C FC-131-2003
(96 Indexes, 384 Samples)
Nextera XT Index Kit v2 Set D FC-131-2004
(96 Indexes, 384 Samples)
[Optional] Additional Bag of 48 Index Adapter replacement caps, 15026585
Accessories for Nextera orange
XT Index Kit Bag of 32 Index Adapter replacement caps, 15026586
white
TruSeq Index Plate Fixture Kit FC-130-1005

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Nextera XT DNA Library Prep Reference Guide

Sequencing primers provided in TruSeq v3 reagent kits are not compatible with Nextera XT libraries.
Sequencing Nextera XT libraries on a HiSeq 2500 using TruSeq v3 reagents requires the sequencing
primers provided in the Illumina TruSeq Dual Index Sequencing Primer Box. This box is provided in a
paired-end (PE-121-1003) and single-read (FC-121-1003) version. One box is needed for each run.

Nextera XT Library Prep Contents

Nextera XT Sample Prep Kit– Box 1 of 2
These reagents are shipped at -25°C to -15°C. Promptly store reagents at the indicated tube
temperature to ensure proper performance.

Tube Quantity Storage

Acronym Reagent Name
24 Samples 96 Samples Temperature

1 1 ATM Amplicon Tagment Mix -25°C to -

15°C
1 2 TD Tagment DNA Buffer -25°C to -
15°C
1 1 HT1 Hybridization Buffer -25°C to -
15°C
1 1 NPM Nextera PCR Master Mix -25°C to -
15°C
1 4 RSB Resuspension Buffer -25°C to -
15°C
1 1 LNA1 Library Normalization -25°C to -
Additives 1 15°C
1 2 LNW1 Library Normalization Wash 2°C to 8°C
1

Nextera XT Sample Prep Kit– Box 2 of 2

These reagents are shipped at -25°C to -15°C. Promptly store reagents at the indicated tube
temperature to ensure proper performance.

Tube Quantity Storage

Acronym Reagent Name
24 Samples 96 Samples Temperature

1 1 NT Neutralize Tagment Buffer Room

temperature

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Nextera XT DNA Library Prep Reference Guide

Tube Quantity Storage

Acronym Reagent Name
24 Samples 96 Samples Temperature

1 1 LNB1 Library Normalization Beads 2°C to 8°C

1
1 1 LNS1 Library Normalization Room
Storage Buffer 1 temperature

Index Kit Contents

For index adapter sequences, see Illumina Adapter Sequences (document # 1000000002694).

IDT for Illumina Nextera DNA UD Indexes (96 Indexes, 96 Samples) Plate
Format, Store at -25°C to -15°C

Quantity Description
1 96 Dual Adapter Index Plate

Nextera XT Index Kit v2 (96 indexes, 384 Samples)

Index Adapters, Store at -25°C to -15°C

Set Quantity Description

8 tubes Index Primers, S502, S503, S505–S508,
S510, and S511
A
12 tubes Index Primers, N701–N707, N710–N712,
N714, and N715
8 tubes Index Adapters: S502, S503, S505–
S508, S510, and S511
B
12 tubes Index Adapters: N716, N718–N724, and
N726–N729
8 tubes Index Adapters: S513, S515–S518, and
S520–S522
C
12 tubes Index Adapters: N701–N707, N710–
N712, N714, and N715

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Nextera XT DNA Library Prep Reference Guide

Set Quantity Description

8 tubes Index Adapters: S513, S515–S518, and
S520–S522
D
12 tubes Index Adapters: N716, N718–N724, and
N726–N729

Index Adapter Replacement Caps, Store at 15°C to 30°C

Quantity Description
1 bag i7 Index Tube Caps, Orange
1 bag i5 Index Tube Caps, White

Nextera XT Index Kit

(24 Indexes, 96 Samples)

Index Adapters, Store at -25°C to -15°C

Quantity Description
4 tubes Index Adapters: S502–S504 and S517
6 tubes Index Adapters: N701–N706

Index Adapter Replacement Caps, Store at 15°C to 30°C

Quantity Description
1 bag i7 Index Tube Caps, Orange
1 bag i5 Index Tube Caps, White

TruSeq Index Plate Fixture Kit, Store at Room Temperature (Optional)

Each TruSeq Index Plate Fixture Kit contains two fixtures to help arrange index primers before
dispensing to a 96-well plate during library amplification. The fixture pairs with both the 24-sample kit
and 96-sample kit.

Quantity Description
2 TruSeq Index Plate Fixture

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Nextera XT DNA Library Prep Reference Guide

Consumables and Equipment

Make sure that you have the required consumables and equipment before starting the protocol.
The protocol has been optimized and validated using the items listed. Comparable performance is not
guaranteed when using alternate consumables and equipment.

Consumables
Consumable Supplier
10 µl pipette tips General lab supplier
10 µl multichannel pipettes General lab supplier
10 µl single channel pipettes General lab supplier
1000 µl pipette tips General lab supplier
1000 µl multichannel pipettes General lab supplier
1000 µl single channel pipettes General lab supplier
200 µl pipette tips General lab supplier
200 µl multichannel pipettes General lab supplier
200 µl single channel pipettes General lab supplier
96-well storage plates, round well, Fisher Scientific, catalog # AB-0859
0.8 ml (MIDI plate)
Illumina Purification Beads Illumina, 1 x 100 ml, catalog # 20060057
Illumina, 4 x 100 ml, catalog # 20060058
Distilled water General lab supplier
Ethanol 200 proof (absolute) for molecular Sigma-Aldrich, product # E7023
biology (500 ml)
Microseal 'A' film Bio-Rad, catalog # MSA-5001
Microseal 'B' adhesive seals Bio-Rad, catalog # MSB-1001
Microseal 'F' foil seals Bio-Rad, catalog # MSF-1001
NaOH 1 N, pH > 12.5, molecular biology grade General lab supplier
RNase/DNase-free multichannel reagent VWR, catalog # 89094-658
reservoirs, disposable
Ultrapure water General lab supplier
Hard-Shell 96-well PCR plates Bio-Rad, catalog # HSP-9601

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Nextera XT DNA Library Prep Reference Guide

Equipment
Equipment Supplier
High-Speed microplate shaker VWR, catalog # 13500-890 (110 V/120 V)
VWR, catalog # 14216-214 (230 V)
Magnetic stand-96 Thermo Fisher Scientific, catalog # AM10027
Microplate centrifuge General lab supplier
Vortexer General lab supplier

Thermal Cyclers
Use the following recommended settings for selected thermal cycler models. Before performing library
prep, validate any thermal cyclers not listed.

Thermal Cycler Temp Mode Lid Temp Vessel Type

Bio-Rad DNA Engine Tetrad 2 Calculated Heated, Polypropylene
Constant at plates and tubes
100°C
MJ Research DNA Engine Tetrad Calculated Heated Plate
Eppendorf Mastercycler Pro S Gradient S, Heated Plate
Simulated Tube

Acronyms
Acronym Definition
ATM Amplicon Tagment Mix
HT1 Hybridization Buffer
IPB Illumina Purification Bead
LNA1 Library Normalization Additives 1
LNB1 Library Normalization Beads 1
LNS1 Library Normalization Storage Buffer 1
LNW1 Library Normalization Wash 1
NT Neutralize Tagment Buffer

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Nextera XT DNA Library Prep Reference Guide

Acronym Definition
NPM Nextera PCR Master Mix
RSB Resuspension Buffer
SGP Storage Plate
TD Tagment DNA Buffer
UD Unique Dual Index

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Nextera XT DNA Library Prep Reference Guide

Revision History
Document Date Description of Change

Document # August 2021 Add IPB bead resuspension section to tips and
15031942 v06 techniques
Replaced AMPure XP with IPB Bead
Replaced Vortex verbiage with resuspend
Changed storage temperature and instructions in
Clean Up Libraries preparation.
IPB bead added to consumables and acronyms
sections

Document # May 2019 Added information on IDT® for Illumina®–Nextera™

15031942 v05 UD Indexes sets A, B, C, and D, including kit
contents, preparation procedures, and additional
resources.
Removed plate layout information.
Removed the Pool Libraries section and moved the
Check Library Quality section before the Normalize
Libraries section.
Revised Additional Resources to provide more
clarity on the resources available.
Revised language throughout document to provide
consistency across other Nextera library preparation
reference guides.
Added protocol for diluting libraries to the starting
concentration.
Removed obsolesced Nextera XT Index Kit (96
Indexes, 384 Samples) (# FC-131-1002) from Kit
Contents.

Document # January Added information on reviewing sequencing

15031942 v04 2019 workflows to ensure compatibility with library prep
methods.

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Nextera XT DNA Library Prep Reference Guide

Document Date Description of Change

Document # February Updated the normalize libraries procedure to

15031942 v03 2018 indicate that shaking samples after the five-minute
elution is necessary only if samples are not
resuspended.
Reorganized kit contents information, including
renaming some sections to match kit labeling and
identify storage temperature.
Corrected the diagram that shows how the Nextera
XT assay works to clarify each transposome dimer
has two of the same adapter color.

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Nextera XT DNA Library Prep Reference Guide

Document Date Description of Change

Document # April Added the following information:

15031942 v02 2017 • Supported genome size of < 5 Mb.
• The ratio of absorbance that indicates
contaminants.
• Recommendations for PCR amplicons.
• AMPure XP bead recommendations for runs ≥ 2 ×
250 cycles.
• Reagent and library volumes in the PCR plate after
the tagmentation and amplification steps.
• Beckman Coulter Genomics item # A63880 for
Agencourt AMPure XP, 5 ml.
• Illumina catalog # PE-121-1003 and # FC-121-
1003 for the TruSeq Dual Index Sequencing
Primer Box.
Added the following technical notes to the list of
additional resources:
• Best Practices for Standard and Bead-Based
Normalization in Nextera XT DNA Library
Preparation Kits (Pub. No. 470-2016-007)
• Nextera XT Library Prep: Tips and
Troubleshooting (Pub. No. 770-2015-015)
Consolidated steps in the pool libraries procedure.
Identified the NaOH consumable as molecular
biology grade.
Specified the use of molecular-grade water or 10
mM Tris-HCl, pH 7.5–8.5 to dilute starting material
for DNA quality assessment.
Specified proceeding immediately when
tagmentation is complete so that neutralization
occurs while the transposome is active.
Specified a thaw time of 20 minutes for NPM
(Nextera PCR Master Mix).
Updated the normalize libraries procedure to apply
to various sample numbers, not only 96.
Updated TCY plate to Hard-Shell 96-well PCR plate,
skirted.
Updated magnetic stand supplier to Thermo Fisher
Scientific.
Corrected the catalog numbers for Nextera kits
provided in the introduction.
Document # 15031942 v06 Corrected the illustration showing how the Nextera 30
assay works.
For Research Use Only. Not for use in diagnostic procedures.
Nextera XT DNA Library Prep Reference Guide

Document Date Description of Change

Document # January Updated design of workflow diagram.

15031942 v01 2016 Renamed and combined some procedures as
needed to improve continuity.
Simplified consumables information at the beginning
of each section.
Revised step-by-step instructions to be more
succinct.
Removed reference to obsolete Experienced User
Cards and added reference to the Custom Protocol
Selector.
Clarified AMPure XP bead recommendation for
nonamplicon applications. See Clean Up Libraries.
Added information about normalizing low yield
libraries. See Normalize Libraries.
Corrected index adapter labels on the assay
diagram.

15031942 Rev. E January Corrected kit contents for Nextera XT DNA Library
2015 Preparation Index Kit v2 Set A (FC-131-2001) to
include index N715.

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Nextera XT DNA Library Prep Reference Guide

Document Date Description of Change

15031942 Rev. D September Added info for new index kits that enable
2014 preparation of up to 384 indexed paired-end
libraries.
Updated DNA Input Recommendations for diluting
starting material and the potential results of
incomplete tagmentation.
Added new Nextera XT Quality Metrics with new
information on how to troubleshoot fluctuations in
cluster density.
Removed Dual Indexing Principle and Low Plexity
Pooling Guidelines sections. This information can be
found in the Nextera Low-Plex Pooling Guidelines
Tech Note on the Nextera XT DNA Library Prep
support page.
References to read lengths on the MiSeq were
updated for v3 chemistry.
Added instructions for alternate tip if processing
fewer than 24 samples while transferring LNB1
beads in Library Normalization.
Added NaOH 1N pH > 12.5 to the Consumables and
Equipment list as a user-supplied consumable.
Removed Tween 20 from Consumables and
Equipment list. Consumable not used in protocol.

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Nextera XT DNA Library Prep Reference Guide

Document Date Description of Change

15031942 Rev. C October Modifications were added in PCR Clean-Up for

2012 2x300 runs on the MiSeq.
New section for clustering samples on the HiSeq,
HiScanSQ, and GAIIx. See Clustering Samples for
HiSeq, HiScanSQ, and GAIIx.
The Dual Indexing Principle section listed incorrect
catalog numbers for the Nextera XT Index kits. The
correct catalog numbers are now listed.
Emphasized making sure the NT (Neutralize
Tagment Buffer) and LNS1 (Library Normalization
Storage Buffer 1) reagents are at room temperature
before use in the protocol.
Removed reference to Tris-Cl 10 mM, pH8.5 with
0.1% Tween 20 from the User-Supplied
Consumables table because it is not used in this
library preparation.

15031942 Rev. B July 2012 Emphasized making sure the NT (Neutralize

Tagment Buffer) and LNS1 (Library Normalization
Storage Buffer 1) reagents are at room temperature
before use in the protocol.
Removed reference to Tris-Cl 10 mM, pH8.5 with
0.1% Tween 20 from the User-Supplied
Consumables table because it is not used in this
library preparation.

15031942 Rev. A May 2012 Initial release.

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Technical Assistance
For technical assistance, contact Illumina Technical Support.
Website: www.illumina.com
Email: [email protected]

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Nextera XT DNA Library Prep Reference Guide

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Product documentation—Available for download from support.illumina.com.

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