METHODS GUIDE

Methods
for RNA
sequencing
Illumina solutions for
profiling RNA, from
targeted panels to the
whole transcriptome

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Table of contents

01 Introduction
Nucleic acid extraction .................................................................................... 5
Library preparation .......................................................................................... 5
Sequencing....................................................................................................... 6
Data analysis..................................................................................................... 7
Insights.............................................................................................................. 7

02 Method 1: mRNA-Seq
Relevant applications . ..................................................................................... 8
Step-by-step overview .................................................................................... 9

03 Method 2: Targeted RNA-Seq

Relevant applications . ..................................................................................... 12
Step-by-step overview .................................................................................... 13

04 Method 3: Total RNA-Seq

Relevant applications . ..................................................................................... 16
Step-by-step overview .................................................................................... 17

05 Support that never stops

Trusted technology, trusted partner.................................................................20
Learn more.........................................................................................................20

06 Ordering information
Library preparation............................................................................................21
Sequencing system...........................................................................................21
Data analysis......................................................................................................22

07 References
References.........................................................................................................23

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01 Introduction

As researchers gain deeper insights into the genome, they

learn how factors beyond the genetic sequence influence
development, disease states, and more. To truly understand
what is occurring, scientists are turning towards a multiomics
approach, looking at the transcriptome, epigenome, and
proteome, in addition to the genome, to find comprehensive
answers. In this guide, we will focus on next-generation
sequencing (NGS) workflows for studying the transcriptome. Key benefits:

NGS-based RNA sequencing (RNA-Seq) offers a highly RNA-Seq offers several advantages over
sensitive method for analyzing gene expression across other RNA analysis methods, such as qPCR
the transcriptome, illuminating changes and characterizing and gene expression arrays, including:
various RNA forms with no prior knowledge of the
transcriptome required. This technique uncovers transcript • Covering a wider dynamic range than
isoforms, gene fusions, and single nucleotide variants (SNVs) gene expression arrays, resulting in
in a single experiment.1-3 Bulk RNA-Seq is a well-established greater sensitivity and accuracy5
method that measures average RNA expression in cell • Capturing both known and novel
populations. Ideal for newcomers to NGS, bulk RNA-Seq is features, enabling analysis of the
increasingly used in translational and clinical cancer research, transcriptome without a reference2
benefiting from its simplicity and the depth of information
• Offering a rich view of the transcriptome
RNA-Seq provides.4
beyond expression profiling, detecting
alternative splice sites, gene fusions,
Overview of the RNA-Seq workflow
and allele-specific expression

Illumina RNA-Seq workflows integrate RNA extraction,

library preparation, sequencing, and data analysis to support
transcriptome studies (Figure 1).

Figure 1: RNA sequencing workflow

Library preparation Sequencing Data analysis Insights

Adapters are added Libraries are hybridized Variants are called, Variant interpretation,
to isolated to flow cells expression levels visualization,
cDNA fragments and sequenced accessed and reporting

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STEP 1

RNA extraction
Many commercial RNA extraction and purification kits are available. Choose a kit or method that produces the highest-
quality nucleic acids possible for your specific targets and from your specific sample types.

STEP 2

Library preparation
Following nucleic acid isolation, scientists can prepare nucleic acid libraries—a collection of similarly sized fragments
that have known oligonucleotide adapter sequences attached to the 5′ and 3′ ends of the strands that are loaded onto
an instrument for sequencing. The Illumina RNA library prep portfolio offers a range of solutions to support various
sequencing methods and sample types, including formalin-fixed paraffin-embedded (FFPE) tissue (Table 1). Illumina
RNA library prep solutions offer flexibility, scalability, and performance with a rapid, automation-friendly workflow
option to prepare sequencing-ready libraries in a single day. The RNA library preparation products mentioned in this
guide are representative of the portfolio and are not meant to be a comprehensive list of all available options. View the
full RNA library prep portfolio.

Table 1: A comparison of select Illumina RNA preparation kitsª

Illumina RNA Prep Illumina Stranded Total RNA

Parameter Illumina Stranded mRNA Prep with Enrichment Prep with Ribo-Zero Plus

Coding transcriptome with Coding transcriptome with

Area of interest Noncoding and coding RNA
high-quality samples FFPE samples

Measure genes and transcripts while

Quantify gene expression, identify Analyze expression in a focused set of
detecting known and novel features;
known and novel isoforms, detect genes of interest, obtain quantitative
Description targeted hybridization removes
gene fusions, and measure allele- expression information, detect small
abundant rRNA to focus on high-
specific expression variants and gene fusions
value portions of the transcriptome

Tagmentation-based RNA library

Mechanism of PolyA selection upfront of rRNA depletion ahead of
prep followed by hybrid–capture
action ligation-based RNA library prep ligation-based RNA library prep
enrichment

10 ng high-quality RNA, 1 ng high-quality RNA,

Input 25–1000 ng high-quality RNA
20 ng FFPE RNA 10 ng FFPE RNA

Hands-on time < 3 hr < 2 hr < 3 hr

Total assay time 6.5 hr < 9 hr ~ 7 hr

Automation
Yes Yes Yes
capability

a. More RNA library preparation kits are available at illumina.com/techniques/sequencing/ngs-library-prep/rna.html.

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STEP 3

Sequencing
After libraries are prepared, they are ready for sequencing. Regardless of your research question, flexible Illumina
sequencing systems can help you find answers using simple push-button workflows (Table 2). For studies focused on
a small amount of information, ie, prokaryotic species or small targeted RNA oncology panels, researchers can use a
benchtop sequencing system, such as the MiSeqTM i100 Series or NextSeqTM 1000 and NextSeq 2000 Systems. For large-
scale studies, researchers can use high-throughput instruments like the NovaSeqTM 6000 System and NovaSeq X Series,
and multiplex up to 384 samples with unique dual indexes.

Table 2: Select examples of sequencing systems

NextSeq 1000 and

System MiSeq i100 Series NovaSeq 6000 System NovaSeq X Series
NextSeq 2000 Systems

Expansive application Extraordinary throughput

Simplified operations and Immense discovery power
System overview breadth and proven and transformative
fast, flexible sequencing for deeper insights
performance economics

Output range 1.5–30 Gb 10–540 Gba 65 Gb–6 Tb 165 Gb–16 Tb

Single reads per

5–100M 100M–1.8B 800M–10B 1.6–26B
flow cell

Maximum read
2 x 300 bp 2 x 300 bp 2 × 250 bp 2 × 150 bp
length

a. Maximum specifications based on a P4 flow cell run; P4 flow cells are available for the NextSeq 2000 System only.

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STEP 4

Data analysis
RNA-Seq data can be easily and securely transferred, stored, and analyzed in the Illumina cloud-computing platforms:
BaseSpace TM Sequence Hub and Illumina Connected Analytics. For users without bioinformatics expertise, BaseSpace
Sequence Hub is recommended for its intuitive interface, easy run setup and monitoring, and simplified push-button
secondary analysis. For more advanced users, Illumina Connected Analytics supports customization with highly
configurable, scalable analysis. Both platforms offer in-cloud access to DRAGENTM secondary analysis pipelines for
accurate and efficient analysis of RNA-Seq data.

STEP 5

Insights
Outputs from secondary analysis pipelines can be ingested into reporting and exploration software, such as:

• Correlation Engine—analyze private omics data with highly curated public data to help put data into biological context
• lllumina Connected Insights—streamline variant interpretation and reporting for somatic oncology research
applications
• PartekTM FlowTM software—perform statistical analysis and explore data with interactive, customizable, and
publication-ready visualizations

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02 Method 1: mRNA-Seq
Messenger RNA sequencing (mRNA-Seq) sensitively and accurately quantifies gene expression, identifies known and
novel isoforms in the coding transcriptome, and measures allele-specific expression. Protein-coding genes with polyA
tails are selected ahead of library preparation in this method. Using mRNA-Seq to study the coding transcriptome,
researchers can focus on a smaller, more manageable portion of the transcriptome that will provide information relevant
to their area of interest.

Relevant applications

Gene expression profiling for disease research

To understand normal cell development and disease mechanisms, researchers frequently investigate differential
expression during development, in specific tissues, or in response to varying conditions. mRNA-Seq shows exceptional
performance in profiling genes with low expression levels. It is being used to assess gene expression profiles for the
study of complex diseases and laying the groundwork for advances in precision medicine by identifying potentially
therapeutic biomarkers.6

Data from mRNA-Seq experiments can offer insight into the gene networks and pathways involved in complex disease
and cell biology mechanisms.7,8 For example, transcriptomic analysis is helping researchers compare brain regions with
different pathology to identify meaningful gene expression changes in Alzheimer’s disease (AD).7,9 Differential expression
profiling is also revealing the pathogenesis of heart failure and identifying gene signatures to detect heart disease.10-14

Biomarker profiling for drug development

mRNA-Seq is increasingly being utilized to discover and profile RNA-based drug response biomarkers with the aim of
improving the efficiency and success rate of the drug development process. While several technologies have been used
for this application, the capabilities of mRNA-Seq promise to be of particular benefit.15-17

Differential expression profiles and gene fusions detectible through RNA analysis have been shown to associate with a
range of response characteristics, including efficacy, the incidence of adverse effects, pharmacodynamics, and other
attributes.18-21 Such biomarkers have therefore become an invaluable tool in multiple components of the development
process, such as informing the interpretation of clinical trial data, allowing more efficient stratification of trial cohorts,
and identifying neoantigen candidates for immunotherapy.22,23 RNA-based biomarkers may also provide a foundation for
the development of companion diagnostics, including for compounds that have previously failed clinical trials.

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Step-by-step overview

mRNA-Seq has five basic steps: RNA extraction, library preparation, sequencing, analysis, and insights (Figure 2).

Figure 2: mRNA-Seq workflow.

Library
RNA extraction Sequencing Data analysis Insights
preparation

QIAGEN Illumina Stranded Illumina DRAGEN RNA app Partek Flow

RNeasy Kit mRNA Prep sequencing software
system
Correlation Engine
Illumina Connected
Insights

STEP 1

RNA extraction
For mRNA-Seq, it is crucial to use high-quality mRNA as input. There are several commercial options for mRNA isolation,
depending on the sample type; the QIAGEN RNeasy for RNA kit is recommended for use with Illumina mRNA-Seq
workflows. Effectively evaluating mRNA quality is a critical step in successful RNA sequencing and can be achieved by
measuring mean mRNA fragment size.

STEP 2 Table 3: Illumina Stranded mRNA Prep specifications

Library preparation
Feature Specification

Illumina Stranded mRNA Prep delivers accurate, RNA input amount 25–1000 ng high-quality RNA
unbiased detection of the coding transcriptome with
Total assay time 6.5 hr
precise measurement of strand information (Table 3).
Hands-on time < 3 hr

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STEP 3

Sequencing
Several sequencing systems can be used for mRNA-Seq. The one chosen depends on several factors, including the
application, study size, throughput requirements, and more (Table 4).

Table 4: Experimental parameters for performing mRNA-Seq on different sequencing systems

Single reads per flow No. of samples Recommended

System Flow cell
cell per flow cellª read length

50M 50M 2
MiSeq i100 Plus
2 x 75 bp
System
100M 100M 4

P1 100M 4

NextSeq 1000 and P2 400M 16

NextSeq 2000 2 x 75 bp
Systems P3ᵇ 1.2B 48

P4ᵇ 1.8B 72

SP 1.6B 64

S1 3.2B 128
NovaSeq 6000
2 x 75 bp
System
S2 8.2B 328

S4 8–10B 384

10B 10B 384

NovaSeq X Series 2 x 75 bp
25B 26B 384

a. Based on 25M reads per sample. Sufficient gene expression and most use cases of fusion calling.
b. Flow cells are only available on the NextSeq 2000 System.

STEP 4

Data analysis
For secondary analysis of mRNA-Seq data, Illumina recommends using the DRAGEN RNA pipeline, which performs
RNA quantification, gene fusion detection, and small variant calling in one integrated workflow (Table 5). This pipeline is
available on-premises with a DRAGEN Server, on the cloud-based BaseSpace Sequence Hub and Illumina Connected
Analytics platforms, and onboard select sequencing systems, including the NextSeq 1000 and NextSeq 2000 Systems
and NovaSeq X Series.

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STEP 5

Insights
After secondary data analysis, results can be transferred to Correlation Engine to understand the biological effects
of gene expression changes (Table 5). Correlation Engine contains knowledge-based gene sets and results from
thousands of public studies that inform biological interpretation. Connect differential gene expression data from RNA-
Seq experiments with disease associations or visualize correlated genes. Gain further insights with Partek Flow software
using robust statistical algorithms and information-rich visualizations. Additionally, Illumina Connected Insights can be
used to streamline interpretation and reporting for research applications.

Table 5: Illumina mRNA-Seq analysis software

Pipeline Application Input Access point

DRAGEN RNA Offers multiple operating modes, FASTQ • DRAGEN Server

including reference-only alignment • BaseSpace Sequence Hub
and annotation-assisted alignment with Optional: Custom reference Gene • Illumina Connected Analytics
gene fusion detection. The gene fusion Annotation File (GTF, GFF, or GFF3) • Onboard NextSeq 1000/2000
module uses the DRAGEN RNA spliced Systems, NovaSeq X Series
aligner to perform split-read analysis on
supplementary (chimeric) alignments to
detect potential breakpoints.

RNA-Seq Processes sequencing data from csv, txt, xlsx files • Correlation Engine
interpretation mRNA to estimate transcript abundance
and identify differentially expressed
transcripts across samples

RNA-Seq visualization Provides differential analysis, clustering, tsv, csv, txt, gz, FASTQ, BAM • Partek Flow software
and statistical analysis and data exploration plots

Illumina Connected Supports streamlined interpretation and VCF • Illumina Connected Insights with
Insights reporting from DRAGEN software for automated ingestion of VCF files
oncology research applications

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03 Method 2: Targeted RNA-Seq

Targeted RNA-Seq is a highly accurate method for selecting and sequencing specific transcripts of interest that offers
both quantitative and qualitative information. Targeted RNA-Seq can be achieved via either enrichment or amplicon-
based approaches, both of which enable gene expression analysis in a focused set of genes of interest. Here, we focus
on enrichment assays, which provide the ability to detect both known and novel gene fusion partners in many sample
types, including FFPE tissue.

Relevant applications

Variant detection for cancer research

Targeted RNA-Seq is a critical tool for direct measurement of the functional consequences of mutations. Despite the
average cancer containing about 46 mutations, only five to eight are necessary for initiation.24 Genomic profiling alone
is insufficient to differentiate these driver mutations from passenger mutations, or those that do not influence cancer
initiation or progression. Measurement of gene expression patterns and mutation consequences using targeted RNA-Seq
enables large-scale, unbiased differentiation of factors crucial for cancer progression, resulting in more thorough and
accurate cancer modeling.

Detection and characterization of respiratory pathogens

Targeted RNA-Seq provides an effective method for rapid and accurate identification of respiratory pathogens.
Combining Illumina RNA Prep with Enrichment with target-specific probe panels enables targeted sequencing of
different subsets of respiratory pathogens. For example, the Respiratory Virus Oligo Panel v2 targets SARS-CoV-2 and
other common respiratory viruses in a single assay.25 Alternatively, Illumina offers the Respiratory Pathogen ID/AMR
Panel, which targets ~280 respiratory pathogens, including viruses, bacteria, and fungi, and associated antimicrobial
resistance (AMR) markers.26

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Step-by-step overview

Targeted RNA-Seq has five basic steps: RNA extraction, library preparation and enrichment, sequencing, data analysis,
and insights (Figure 3).

Figure 3: Targeted RNA-Seq workflow.

Library
RNA extraction Sequencing Data analysis Insights
preparation

QIAGEN Illumina RNA Prep Illumina DRAGEN RNA app, Partek Flow
RNeasy Kit with Enrichment sequencing DRAGEN Microbial software
system Enrichment Plus app
Illumina Connected
Insights

STEP 1

RNA extraction
For targeted RNA-Seq, high-quality intact RNA or RNA isolated from FFPE samples can be used as input. There are
several commercial options for RNA isolation, depending on the sample type; the QIAGEN RNeasy for RNA kit is
recommended for use with intact samples and QIAGEN RNeasy FFPE for use with FFPE tissue. Effectively evaluating RNA
quality is a critical step in successful RNA-Seq and can be achieved by measuring mean RNA fragment size before library
prep.

STEP 2

Library preparation
Illumina RNA Prep with Enrichment provides a fast, integrated workflow for producing enriched and indexed sequencing
libraries (Table 6). The kit enables rapid, targeted interrogation of an expansive number of target genes with exceptional
capture efficiency and coverage uniformity using various prebuilt probes or custom probes for maximum flexibility.27

Table 6: Illumina RNA Prep with Enrichment specifications

Feature Specification

RNA input amount 10 ng high-quality RNA, 20 ng FFPE RNA

Total assay time < 9 hr

Hands-on time < 2 hr

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STEP 3

Sequencing
There are many panels that can be used to select the targets of interest. In this example workflow, we focus on using the
Illumina Exome Panel to analyze the coding transcriptome. The sequencing system used depends on several factors,
including the application, study size, throughput requirements, and more (Table 7).

Table 7: Experimental parameters for performing targeted RNA-Seq on different sequencing systems

Single reads per flow No. of samples Recommended

System Flow cell
cell per flow cellª read length

25M 25M 8

MiSeq i100 Plus System 50M 50M 16 2 x 100 bp

100M 100M 33

P1 100M 33
NextSeq 1000 and
2 x 100 bp
NextSeq 2000 Systems
P2 400M 133

a. Based on 3M clusters per sample using the TruSight RNA Pan-Cancer Panel.

STEP 4

Data analysis
Illumina recommends using the DRAGEN RNA pipeline
or the Enrichment panel-specific analysis workflow, ie,
DRAGEN Microbial Enrichment Plus (Table 8). These
pipelines are available on-premises with a DRAGEN
Server, on cloud-based platforms, including BaseSpace
Sequence Hub and Illumina Connected Analytics, and
onboard select sequencing systems.

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STEP 5

Insights
After secondary analysis, further explore data with Partek Flow software using statistical analysis and interactive
visualizations. For somatic oncology applications using targeted RNA-Seq, Illumina Connected Insights enables QC,
annotation, interpretation, curation of SNVs, splice variants, and fusion variants from RNA (expression analysis coming
in future) with subsequent report generation. RNA may be assessed together with DNA or sequentially, and with either
option, results may be merged into a single research report if desired.

Table 8: Illumina mRNA-Seq analysis software

Pipeline Application Input Access point

DRAGEN RNA Offers multiple operating modes, FASTQ • DRAGEN server

including reference-only alignment • BaseSpace Sequence Hub
and annotation-assisted alignment Optional: Custom reference Gene • Illumina Connected Analytics
with gene fusion detection. The gene Annotation File (GTF, GFF, or GFF3) • Onboard NextSeq 1000/2000
fusion module uses the DRAGEN RNA Systems, NovaSeq X Series
spliced aligner to perform split-read
analysis on supplementary (chimeric)
alignments to detect potential
breakpoints.

DRAGEN Microbial Provides secondary analysis of FASTQ, BCL • BaseSpace Sequence Hub
Enrichment Plus common Illumina infectious disease • Illumina Connected Analytics
and microbiology enrichment panels • Onboard MiSeq i100 Series
(ie, VSP, RVEK, RPIP)

RNA-Seq Supports differential analysis, tsv, csv, txt, gz, FASTQ, BAM • Partek Flow
visualization and clustering, and data exploration plots
statistical analysis

Illumina Connected Supports streamlined interpretation VCF • Illumina Connected Insights with
Insights and reporting from DRAGEN software automated ingestion of VCF files
for oncology research applications

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04 Method 3: Total RNA-Seq

In addition to proteins, genes encode a vast array of nonprotein-coding elements that play an instrumental role in
orchestrating how a cell is organized from a transcriptional regulation perspective. A major strength of total RNA‑Seq
lies in its ability to identify novel features of the transcriptome. NGS-based total RNA-Seq can sequence the whole
transcriptome, including both coding and noncoding transcripts, without the limitation of probe design, delivering a high
resolution, base-by-base view of coding and multiple forms of noncoding RNA activity. This provides a comprehensive
picture of gene expression across the full transcriptome at a specific moment in time. With total RNA‑Seq, the whole
transcriptome—including both known and unknown regions—is captured.1-4

Relevant applications

Discovery of pathways associated with disease

Investigations into the transcriptomic differences between cancer samples and non-cancerous tissue have been shown
to be useful in differentiating cancer subtypes, assessing the impact of mutations and identifying biomarkers and other
variables. Combining total RNA-Seq with DNA sequencing and methylation analyses successfully classified meningioma
tumors into distinctive molecular groups that correlated with predicted clinical outcomes, suggesting they are capable of
informing medical therapies.28

Assessment of therapy response

Total RNA-Seq has the potential to identify genes and pathways associated with biological response or lack of response
to novel drug therapies in model systems or retrospective studies of tissue samples. As part of a multiomic profiling
study on tumor samples from melanoma patients undergoing anti-PD-1 immunotherapy, total RNA-Seq identified an
interferon gamma (INFγ) expression pattern that, along with other biomarkers, robustly predicted successful response to
immunotherapy.26

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Step-by-step overview

Total RNA-Seq has five basic steps: RNA extraction, ribosomal RNA (rRNA) depletion (optional) and library preparation,
sequencing, data analysis, and insights (Figure 4).

Figure 4: Total RNA-Seq workflow.

Library
RNA extraction Sequencing Data analysis Insights
preparation

QIAGEN Illumina Stranded Illumina DRAGEN RNA app, Partek Flow

RNeasy Kit Total RNA Prep with sequencing Microbiome software
Ribo-Zero Plus system Metatranscriptomics
Correlation Engine
app
Illumina Connected
Insights

STEP 1

RNA extraction
For whole-transcriptome methods, you can use high-quality RNA or degraded samples as input. There are several
options for RNA isolation kits depending on your sample type. Illumina recommends the QIAGEN RNeasy for extraction
of RNA from cells and QIAGEN RNeasy FFPE for extracting RNA from FFPE tissue. Effectively evaluating RNA quality is a
critical step in successful total RNA-Seq and can be achieved by measuring mean RNA fragment size before library prep.

STEP 2

Library preparation Table 9: Illumina Stranded Total RNA Prep

with Ribo-Zero Plus specifications
Illumina Stranded Total RNA Prep with Ribo-Zero Feature Specification
Plus enables preparation of whole-transcriptome
sequencing-ready libraries from low RNA inputs and RNA input amount 1 ng high-quality RNA, 10 ng FFPE RNA

low-quality samples to support a wide range of RNA-Seq Total assay time ~7 hr

applications. The kit includes Ribo-Zero Plus for single-
tube depletion of rRNA and globin RNA from multiple Hands-on time < 3 hr

species to facilitate rich transcriptome analyses

(Table 9).30

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STEP 3

Sequencing
The sequencing system used depends on several factors, including the application, study size, throughput requirements,
and more (Table 10).

Table 10: Experimental parameters for performing total RNA-Seq on different sequencing systems

Single reads per flow No. of samples Recommended

System Flow cell
cell per flow cellª read length

P1 100M 2

NextSeq 1000 and P2 400M 8

NextSeq 2000 2 x 100 bp
Systems P3ᵇ 1.2B 24

P4ᵇ 1.8B 36

SP 1.6B 32

S1 3.2B 64
NovaSeq 6000
2 x 100 bp
System
S2 8.2B 164

S4 10B 200

10B 10B 200

NovaSeq X Series 2 x 100 bp
25B 26B 384c

a. Based on 50M reads per sample.

b. P3 and P4 flow cells are only available on the NextSeq 2000 System.
c. Based on Illumina indexes. Additional indexes can be added.

STEP 4

Data analysis
Illumina recommends using the DRAGEN RNA pipeline for secondary analysis of whole-transcriptome libraries. The
DRAGEN RNA pipeline is available on-premises with a DRAGEN Server, on the cloud-based BaseSpace Sequence Hub
and Illumina Connected Analytics platforms, and onboard select sequencing systems (Table 11).

For studies that include analysis of metatranscriptomes from complex microbiome samples, such as human
gastrointestinal (GI) tract, the Microbiome Metatranscriptome application on BaseSpace Sequence Hub provides
taxonomic and functional profiling.

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STEP 5

Insights
For downstream tertiary analysis, output from the DRAGEN RNA pipeline can be automatically ingested into Illumina
Connected Insights for variant interpretation and reporting. Results can also be transferred to Correlation Engine for
gaining insights into the biological effects of gene expression changes, and to Partek Flow software for further statistical
analysis and information-rich visualizations.

Table 11: Illumina total RNA-Seq analysis software

Pipeline Application Input Access point

DRAGEN RNA Offers multiple operating modes, FASTQ • DRAGEN Server

including reference-only alignment and • BaseSpace Sequence Hub
annotation-assisted alignment with gene Optional: Custom reference Gene • Illumina Connected Analytics
fusion detection Annotation File (GTF, GFF, or GFF3) • Onboard NextSeq 1000/2000
Systems, NovaSeq X Series
The gene fusion module uses the
DRAGEN RNA spliced aligner to perform
split-read analysis on supplementary
(chimeric) alignments to detect potential
breakpoints

Microbiome Performs taxonomic and pathway FASTQ • BaseSpace Sequence Hub

Metatranscriptomics enrichment analysis on microbiome
derived RNA libraries

RNA-Seq Processes sequencing data from csv, txt, xlsx files • Correlation Engine
interpretation mRNA to estimate transcript abundance
and identify differentially expressed
transcripts across samples

RNA-Seq visualization Supports differential analysis, clustering, tsv, csv, txt, gz, FASTQ, BAM • Partek Flow software
and statistical analysis and data exploration plots

Illumina Connected Supports streamlined interpretation and VCF • Illumina Connected Insights with
Insights reporting from DRAGEN software for automated ingestion of VCF files
oncology research applications

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05 Support that never stops

Illumina strives to be the best partner possible. With a global presence, you can rely on our support to facilitate your
success. Technical support is available via phone five days a week or access online support 24/7, worldwide and in
multiple languages, with rapid response time near most major metropolitan areas. Illumina provides excellent product
consistency, supply, and quality enabled by a mature global manufacturing infrastructure.

Trusted technology, trusted partner

As a preferred NGS platform provider, Illumina has shipped over 20,000 sequencing systems globally. Illumina NGS
technology is cited in over 421,000 peer-reviewed publications—5× more than all other NGS technologies combined.31
Building on decades of expertise, Illumina has a relentless commitment to innovation and building future NGS capabilities
and applications.

Learn more

Illumina RNA sequencing

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06 Ordering information
Library preparation
Product Catalog no.

Illumina Stranded mRNA Prep, Ligation (16 samples) 20040532

Illumina Stranded mRNA Prep, Ligation (96 samples) 20040534

Illumina RNA Prep with Enrichment, (L) Tagmentation (16 samples) 20040536

Illumina RNA Prep with Enrichment, (L) Tagmentation (96 samples) 20040537

Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (16 samples) 20040525

Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (96 samples) 20040529

Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus Microbiome (96 samples) 20072063

Illumina DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples)ª 20091654

Illumina DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples)ª 20091656

Illumina DNA/RNA UD Indexes Set C, Tagmentation (96 Indexes, 96 Samples)ª 20091658

Illumina DNA/RNA UD Indexes Set D, Tagmentation (96 Indexes, 96 Samples)ª 20091660

PhiX Control Kit, v3 FC-110-3001

a. 384 indexes total available when combining sets A, B, C, and D.

Sequencing systems
System Catalog no.

MiSeq i100 Plus System 20115695

NextSeq 1000 System 20038898

NextSeq 2000 System 20038897

NovaSeq 6000 System 20012850

NovaSeq X System 20084803

NovaSeq X Plus System 20084804

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Data analysis
Product Catalog no.

BaseSpace Sequence Hub Professional Annual Subscriptionª 20042109

BaseSpace Sequence Hub Enterprise Annual Subscriptionª 15066411

Illumina Connected Analytics Professional Annual Subscription 20044876

Illumina Connected Analytics Enterprise Annual Subscription 20038994

Illumina Connected Insights–Oncology Genome Equivalent Sample – VCF 20090138

Illumina Connected Insights Starter Implementation Package 20071787

Illumina Connected Insights Expanded Implementation Package 20071787 (as scoped)

Partek Flow software Contact an Illumina sales representative

Correlation Engine Contact an Illumina sales representative

DRAGEN Server 20051343

a. BaseSpace Sequence Hub subscriptions include complimentary iCredits for running analysis apps and data storage. Additional iCredits are available for purchase.

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07 References

1. Ozsolak F, Milos PM. RNA sequencing: advances, challenges 13. Meng Z, Chen C, Cao H, Wang J, Shen E. Whole transcriptome
and opportunities. Nat Rev Genet. 2011;12(2):87-98. doi:10.1038/ sequencing reveals biologically significant RNA markers
nrg2934 and related regulating biological pathways in cardiomyocyte
2. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool hypertrophy induced by high glucose. J Cell Biochem.
for transcriptomics. Nat Rev Genet. 2009;10:57-63. doi:10.1038/ 2019;120(1):1018-1027. doi:10.1002/ jcb.27546
nrg2484 14. Andersen JD, Jacobsen SB, Trudsø LC, Kampmann ML, Banner
3. Wilhelm BT, Landry JR. RNA-Seq—quantitative measurement J, Morling N. Whole genome and transcriptome sequencing of
of expression through massively parallel RNA-Sequencing. post-mortem cardiac tissues from sudden cardiac death victims
Methods. 2009;48:249-57. doi:10.1016/j.ymeth.2009.03.016 identifies a gene regulatory variant in NEXN. Int J Legal Med.
4. Wang Y, Mashock M, Tong Z, et al. Changing Technologies of 2019;133(6):1699-1709. doi:10.1007/s00414-019-02127-9
RNA Sequencing and Their Applications in Clinical Oncology. 15. Zhao S, Fung-Leung W-P, Bittner A, Ngo K, Liu X. Comparison of
Front Oncol. 2020;10:447. Published 2020 Apr 9. doi:10.3389/ RNA-seq and microarray in transcriptome profiling of activated
fonc.2020.00447 T cells. PLoS ONE. 2014;9(1):e78644. doi:10.1371/journal.
5. Su Z, Labaj PP, Li S, et al. A comprehensive assessment of pone.0078644
RNA-Seq accuracy, reproducibility and information content 16. Atak ZK, Gianfelici V, Hulselmans G, et al. Comprehensive
by the Sequencing Quality Control Consortium. Nat Biotech. analysis of transcriptome variation uncovers known and novel
2014;32:903-914. doi:10.1038/nbt.2957 driver events in T-cell acute lymphoblastic leukemia. PLoS
6. Xu J, Gong B, Wu L, Thakkar S, Hong H, Tong W. Comprehensive Genet. 2013;9(12):e1003997. doi:10.1371/journal.pgen.1003997
Assessments of RNA-seq by the SEQC Consortium: FDA-Led 17. Kumar-Sinha C, Kalyana-Sundaram S, Chinnaiyan AM.
Efforts Advance Precision Medicine. Pharmaceutics. 2016;8(1):8. Landscape of gene fusions in epithelial cancers: seq and ye
Published 2016 Mar 15. doi:10.3390/pharmaceutics8010008 shall find. Genome Med. 2015;7:129. Published 2015 Dec 18.
7. Crist AM, Hinkle KM, Wang X, et al. Transcriptomic analysis doi:10.1186/s13073-015-0252-1
to identify genes associated with selective hippocampal 18. Docking TR, Parker JDK, Jadersten M, et al. A clinical
vulnerability in Alzheimer's disease. Nat Commun. transcriptome approach to patient stratification and
2021;12(1):2311. doi:10.1038/s41467-021-22399-3 therapy selection in acute myeloid leukemia. Nat Commun.
8. Allen M, Wang X, Burgess JD, et al. Conserved brain 2021;12(1):2474 doi:10.1038/s41467-021-22625-y
myelination networks are altered in Alzheimer's and 19. Figgett WA, Monaghan K, Ng Milica, et al. Machine learning
other neurodegenerative diseases. Alzheimers Dement. applied to whole-blood RNA-sequencing data uncovers distinct
2018;14(3):352-366. doi:10.1016/j.jalz.2017.09.012 subsets of patients with systemic lupus erythematosus. Clin
9. Guennewig B, Lim J, Marshall L, et al. Defining early changes Transl Immunology. 2019;8(12):e01093
in Alzheimer's disease from RNA sequencing of brain regions 20. Lu L, Zhang H, Pang J, Hou G, Lu M, Gao X. ERG rearrangement
differentially affected by pathology. Sci Rep. 2021;11(1):4865. as a novel marker for predicting the extra-prostatic extension of
doi:10.1038/s41598-021-83872-z clinically localised prostate cancer. Oncol Lett. 2016;11(4):2532-
10. Zhang X, van Rooij JGJ, Wakabayashi Y, et al. Genome-wide 2538. doi:10.3892/ol.2016.4282
transcriptome study using deep RNA sequencing for myocardial 21. Perez-Gracia JL, Sanmamed MF, Bosch A, et al. Strategies
infarction and coronary artery calcification. BMC Med to design clinical studies to identify predictive biomarkers in
Genomics. 2021;14(1):45. doi:10.1186/s12920-020- 00838-2 cancer research. Cancer Treat Rev. 2017;53:79-97. doi:10.1016/j.
11. Luo X, Yin J, Dwyer D, et al. Chamber-enriched gene expression ctrv.2016.12.005
profiles in failing human hearts with reduced ejection fraction. 22. Fang B, Mehran RJ, Heymach JV, Swisher SG. Predictive
Sci Rep. 2021;11(1):11839. doi:10.1038/s41598-021-91214-2 biomarkers in precision medicine and drug development against
12. Jain PN, Robertson M, Lasa JJ, et al. Altered metabolic and lung cancer. Chin J Cancer. 2015;34(7):295-309. doi:10.1186/
inflammatory transcriptomics after cardiac surgery in neonates s40880-015-0028-4
with congenital heart disease. Sci Rep. 2021;11(1):4965. 23. Zhao X, Modur V, Carayannopoulos LN, Laterza OF. Biomarkers
doi:10.1038/s41598-021-83882-x in Pharmaceutical Research. Clin Chem. 2015;61(11):1343-1353.
doi:10.1373/clinchem.2014.231712

For Research Use Only. Not for use in diagnostic procedures. M-GL-00034 v1.0 | 23
 RNA SEQUENCING

24. Pon JR, Marra MA. Driver and passenger mutations in

cancer. Annu Rev Pathol. 2015;10:25-50. doi:10.1146/annurev-
pathol-012414-040312
25. Illumina. Surveillance of infectious disease through wastewater
sequencing Application Note. illumina.com/content/dam/
illumina/gcs/ assembled-assets/marketing-literature/covidseq-
wastewater-epidemiology-app-note-m-gl-00429.pdf.
Published 2022. Accessed April 24, 2024.
26. Illumina. Rapid detection of respiratory pathogens using the
MiniSeq System Application Note. illumina.com/content/
dam/illumina/ gcs/assembled-assets/marketing-literature/
respiratory-pathogen-panel-miniseq-app-note-m-gl-01508.
pdf. Published 2023. Accessed April 24, 2024.
27. Illumina. Illumina RNA Prep with Enrichment Data Sheet.
illumina.com/content/dam/illumina/gcs/assembled-assets/
marketing-literature/ illumina-rna-prep-enrichment-data-
sheet-m-gl-02145.pdf. Published 2023. Accessed April 24,
2024.
28. Nassiri F, Liu J, Patil V, et al. A clinically applicable
integrative molecular classification of meningiomas. Nature.
2021;597(7874):119-125. doi:10.1038/s41586-021-03850-3
29. Newell F, Pires da Silva I, Johansson PA, et al. Multiomic
profiling of checkpoint inhibitor-treated melanoma: Identifying
predictors of response and resistance, and markers of
biological discordance. Cancer Cell. 2022;40(1):88-102.e7.
doi:10.1016/j.ccell.2021.11.012.
30. Illumina. Illumina Stranded Total RNA Prep, Ligation with Ribo-
Zero Plus Data Sheet. illumina.com/content/dam/illumina/gcs/
assembledassets/ marketing-literature/illumina-stranded-
total-rna-prep-data-sheet-m-gl-02148.pdf. Published 2020.
Accessed August 20, 2024.
31. Data calculations on file. Illumina, Inc. 2022.

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