Application Note: RNA Sequencing

NextSeq Series RNA-Seq Solution®

An accessible, cost-effective solution that fully characterizes RNA transcript changes for a deeper
understanding of biology.
A Flexible RNA-Seq SolutionHighlights• Clearest, Most Complete Transcriptome View for
Any Species
Accurate transcriptome-wide abundance measurement anddiscovery of gene fusions, cSNPs, and noveltranscript isomers

The NextSeq Series RNA-Seq solution enables rapid profiling, anddeep investigation of the whole transcriptome (Figure 1). Researchers
can choose from a complete suite of library preparation kits, enabling
RNA-Seq studies to be tailored for whole-transcriptome or focused
RNA analysis of any species, across a wide range of sample types.

• Accessible End-to-End Solution
Comprehensive suite of library preparation solutions andsimple-to-use data analysis pipeline• Speed, Power, and Flexibility
Complete runs in less than 18 hours with ability to switch
between 2 output modes to support full range ofproject needs• End-to-End Illumina Scientific Support
Illumina scientists and engineers are there every step of theway, providing installation, training, applications, and data
analysis supportIntroduction
The NextSeq Series RNA Sequencing (RNA-Seq) Solution delivers theclearest, most complete view of the transcriptome, making it moreaccessible than ever before. It leverages industry-leading Illumina
next-generation sequencing (NGS) technology, the broadest rangeof library preparation solutions, and a new data analysis suite thatdelivers expert-preferred tools in a simple, intuitive user interface.
The flexible and scalable NextSeq Series of sequencing systems canbe tailored to meet researchers’ changing needs as projects evolve.
Ensuring the optimal balance of run time, read budget, and sample
throughput, the NextSeq Series RNA-Seq Solution supports a rangeof studies, from basic gene expression profiling to complex
whole-transcriptome analysis.

PrepSequence

AnalyzeShare

5.5 hours hands-ona

10 minutes hands-on

5 minutes hands-on,
on-site or in cloud

Secure,unlimited storage

8HOURS

15-18 <4HOURS

HOURS PER SAMPLEINSTANTLY

Figure 1: NextSeq Series Sample-to-Answer RNA-Seq Sequencing
Workflow*—The NextSeq Series features a simple workflow that delivers highly
accurate sequencing data.

*Times vary by experiment and assay type. Details shown are for an mRNA expression
profiling experiment assuming 2 × 75 bp on instrument. Analysis results include differential
expression and identification of alternative transcripts.

For Research Use Only. Not for use in diagnostic procedures.

Figure 2: NextSeq System—The NextSeq Series of sequencing systems
leverages the latest advances in SBS chemistry and the industry’s
simplest workflow.

The NextSeq Series provides the power and versatility needed toaddress the full range of transcriptome analysis needs (Figure 2). Dualsequencing output modes enable researchers to select the optimalbalance between sample number and reads per sample. For example,gene expression profiling (the measurement of gene-level abundanceacross known features) can be performed efficiently at high-throughput
capacity with up to 40 samples† in a single run. Offering the power
to discover novel features, whole-transcriptome analysis can query
coding and noncoding RNA at up to 8 samples per run; researchers
can also analyze coding RNA at up to 16 samples per run (Table 1).
Data analysis can be performed in BaseSpace®—the Illumina cloudand onsite genomics computing environment—using intuitive apps
developed for analyzing Illumina RNA-Seq data. A part of the Illumina
industry-leading NGS ecosystem, the NextSeq Series affords
researchers with access to the world’s largest collection of commercial
and open source data analysis software tools for performing additional
downstream analysis.
The NextSeq Series offers cross-application flexibility, enabling
researchers to transition easily between sequencing projects (Figure 3).
The system is compatible with the industry’s widest range of library
preparation kits from Illumina and third parties, enabling an easytransition between Illumina RNA-Seq, whole-genome sequencing(WGS), exome sequencing, and other applications. For example,
researchers can pair RNA-Seq with exome sequencing on theNextSeq Series to assess whether coding variants impact transcript† Expression profiling assumes 10 M reads per sample

Application Note: RNA Sequencing
expression or perform ChIP-Seq to analyze DNA-protein interactions
and better characterize functional regulation. With the NextSeq 550
System, researchers can perform NGS and array scanning on thesame platform for further exploration or confirmation of copy numbervariants detected through sequencing.

Whole Genome Sequencing

RNA Sequencing

Advantages of RNA-Seq Over Gene Expression Arrays
RNA-Seq is increasingly the platform of choice for thought
leaders, providing a detailed snapshot of the transcriptome ata given point in time. It offers numerous advantages over gene
expression arrays, including:•Requires no a priori knowledge of the transcriptome•

Provides qualitative and quantitative transcriptome analysis:•Sequence and variant information•

Greater accuracy of fold-change measurement•

Better specificity•

May be applied to any species, even if a reference sequence
is not available•

A better value, delivering advantages at lower price / sample
than many gene expression arrays

Exome Sequencing

Figure 3: NextSeq Series Sequencing Applications—The flexible NextSeq
Series enables researchers to transition seamlessly between applications toadvance their research.

Broad Suite of RNA Library Preparation Options
The NextSeq Series RNA-Seq Solution offers an extensive portfolio oflibrary preparation solutions addressing the full range of transcriptome
study needs. Researchers can choose the kit that best fits theirexperimental design goals, while addressing common challenges,
including poor RNA quality or limited available sample input amount.
The TruSeq® Stranded RNA suite of library preparation kits deliver
precise measurement of strand orientation and the highest uniformityof coverage across transcripts, ensuring the most efficient use ofread output. TruSeq Stranded mRNA provides a cost-efficient option
for coding RNA-focused analysis, while TruSeq Stranded Total RNA
enables whole-transcriptome analysis, capturing coding and multiple
forms of noncoding RNA to obtain a complete picture of biology.
TruSeq Stranded Total RNA configurations also offer highly robust
performance in low quality, formalin-fixed, paraffin-embedded(FFPE) samples.
Additional solutions from Epicentre® (an Illumina company) provide
complementary fits for specialized application needs. ScriptSeqTM kits
provide fast total assay time and whole-transcriptome solutions foryeast and bacterial studies.

Industry-Leading Read Quality
With the highest read output of any desktop sequencer, the NextSeq
Series generates higher read counts per sample, enabling researchers
to empower their studies with increased dynamic range, moreaccurate fold-change estimates, and more sensitive detection ofgenes, transcripts, and differential expression. New NextSeq v2
reagent kits are optimized to improve base calling and data quality
even further.
The NextSeq Series dual output modes enable researchers to optimize
study designs based on sample number and output requirements.
If higher sample throughput is needed, studies can be scaled up to
hundreds of samples per run using the Illumina HiSeq® 2500 System(Table 1).

Value of Paired-End Sequencing
With the versatile NextSeq Series, researchers can perform
single-read or paired-end sequencing. Single-read sequencing isa highly economical option for gene expression profiling. However,paired-end RNA-Seq offers critical advantages. Read depth
information generated from both ends of an insert allows transcript
isomers to be differentiated effectively, providing more accurate

Table 1: Illumina RNA-Seq Solutions

Samples Per Run
StudyMeasurement

Typical Reads Per Sample

NextSeq Series

HiSeq 2500 System

Mid Output

High Output

Rapid Run

High Output

Gene Expression
Profiling

Gene-level abundance across
known features

10 M(1 × 75 bp)

1340

60

400mRNA-Seq

Coding RNA abundance anddiscovery

25 M(2 × 75 bp)41624160

Total RNA-Seq

Coding and noncoding RNA
abundance and discovery

50 M(2 × 75 bp)

281280For Research Use Only. Not for use in diagnostic procedures.

Application Note: RNA Sequencing
detection and quantification of transcript-level abundance. Paired-end
sequencing also enables potential duplicate reads to be retained,
increasing the percentage of aligned reads. Paired-end information
substantially enhances the sensitivity to detect gene fusions andinsertion/deletion variants.

Simplified Bioinformatics in BaseSpaceTranscriptome data generated on the NextSeq Series is instantly andsecurely transferred, stored, and analyzed in BaseSpace (Cloud orOnsite), delivering transcript profiles in a single day (Figure 4). New
Illumina RNA-Seq Software Apps in BaseSpace provide
expert-preferred data analysis tools (TopHat/Cufflinks) packaged in an
intuitive, click-and-go user interface designed for informatics novices.
These apps deliver preconfigured workflows that support a range ofcommon transcriptome data analysis needs. TopHat 2 provides highconfidence alignment for abundance measurement and the detectionof splice junctions, gene fusions, and cSNPs. CuffDiff enables sensitivetranscript discovery and differential expression analysis. TopHat
Fusion delivers robust, high-confidence detection of gene fusions,while the Illumina Isaac1 pipeline delivers reliable variant calling.
Simple-to-follow prompts guide users through the entire process,starting from selecting the files generated by the sequencer, to filteringand visualizing analyzed data and results. RNA-Seq Apps Software
generates output files that can be directly input into a broad range ofavailable downstream analysis tools.

Figure 4: Storage and Analysis of NextSeq Series Data in the BaseSpace
Cloud—NextSeq Series data can be securely and seamlessly uploaded to theBaseSpace cloud for fast, cost-effective analysis and storage.

The BaseSpace environment includes a growing community ofBaseSpace app software tools for visualization, analysis, and sharing.
Because Illumina NGS technology is the most established and broadly
adopted sequencing solution, users can leverage the industry-leading
Illumina NGS ecosystem, the world’s largest collection of commercial
and open-source data analysis software tools.

To learn more about RNA-Seq Software Apps, go to
www.illumina.com/transcriptome.

Summary
The NextSeq Series RNA-Seq Solution offers a true solution, providing
unprecedented access to the clearest, most complete view of thetranscriptome. Dual output modes ensure cost-efficiency acrosstranscriptome project types, from gene-level expression profiling towhole-transcriptome discovery. Combining the power, speed, andflexibility of the NextSeq Series with the industry’s most comprehensive
library preparation portfolio and user-friendly RNA-Seq Software Apps,
the NextSeq Series RNA-Seq Solution enables researchers to gain adeeper understanding of the transcriptome.

Learn More
Go to www.illumina.com/rna to learn more about the next revolution inRNA sequencing.

Join the Illumina Community
With a NextSeq System in their laboratory, researchers join aworldwide community of over 60,000 scientists using Illumina
technology for their research studies. Illumina schedules
community events throughout the year, bringing researchers
together to share ideas. User group meetings, scientificsymposiums, and blog forums provide venues to discuss new
research methods and breakthrough studies.
An integral part of the Illumina community is our dedicatedservice and support team, consisting of more than 300 people
worldwide, 75% of whom have advanced degrees. Illumina
technical support begins when the NextSeq System is delivered,with Illumina scientists and engineers assisting with systeminstallation and setup, and the training of laboratory personnel.
They are there 24/7 globally to answer questions every step of
the way, giving researchers the peace of mind to focus on theirnext research study.
As researchers’ needs change, new systems are brought into
the laboratory, or new methods are undertaken, the Illumina
support and training teams are there to provide assistance. Inaddition to on-site support, training courses (via webinar or at anIllumina facility) are available to bring laboratory personnel quickly
up to speed.

Table 2: NextSeq Series Performance Parameters

Flow Cell Configuration
High-Output Flow Cell
Up to 400 M single reads
Up to 800 M paired-end reads
Mid-Output Flow Cell
Up to 130 M single reads
Up to 260 M paired-end reads

Read Length (bp)Output (Gb)

Run Time

2 × 150

100–12029 hours

2 × 7550–60

18 hours

1 × 7525–3011 hours

2 × 150

32–39

26 hours

2 × 75

16–1915 hours

Data Quality

Required Input> 75% higher than Q30
at 2 × 150 bp

100 ng–1 μg withTruSeq RNA Library Prep Kits

Total times include cluster generation, sequencing, and base calling on a NextSeq System. Install specifications are based on Illumina PhiX control library at supported cluster densities (between129 and 165 K/mm² clusters passing filter). Actual performance parameters may vary based on sample type, sample quality, and clusters passing filter. The percentage of bases > Q30 is averagedover the entire run.

For Research Use Only. Not for use in diagnostic procedures.

Application Note: RNA Sequencing
Table 4: NextSeq Series Specifications

Ordering Information

Instrument Configuration

System Name

Catalog No.

RFID tracking for consumables

NextSeq 500 System

SY-415-1001

Instrument Control Computer (Internal)a

NextSeq 550 System

SY-415-1002

Output Kit Name

Catalog No.

NextSeq 500 Mid-Output Kit (150 cycles)

FC-102-1001

NextSeq 500 Mid-Output Kit (300 cycles)

FC-404-1003

NextSeq 500 High-Output Kit (75 cycles)

FC-404-1005

Operating Environment

NextSeq 500 High-Output Kit (150 cycles)

FC-404-1002

Temperature: 19°C to 25°C (22°C ± 3°C)

NextSeq 500 High-Output Kit (300 cycles)

FC-404-1004

Humidity: Noncondensing 20%–80% relative humidity

NextSeq 500 Mid-Output v2 Kit (150 cycles)

FC-404-2001

Altitude: Less than 2,000 m (6,500 ft)

NextSeq 500 High-Output v2 Kit (150 cycles)

FC-404-2002

Air Quality: Pollution degree rating of II

NextSeq 500 Mid-Output v2 Kit (300 cycles)

FC-404-2003

Ventilation: Up to 2,048 BTU/hr @ 600 W

NextSeq 500 High-Output v2 Kit (300 cycles)

FC-404-2004

For Indoor Use Only

NextSeq 500 High-Output v2 Kit (75 cycles)

FC-404-2005

Light Emitting Diode (LED)

Library Preparation Kit Name

Catalog No.

520 nm, 650 nm; Laser diode: 780 nm, Class IIIb

TruSeq Stranded mRNA LT Set A

RS-122-2101

Dimensions

TruSeq Stranded mRNA LT Set B

RS-122-2102

W×D×H: 53.3 cm × 63.5 cm × 58.4 cm (21.0 in × 25.0 in × 23.0 in)

TruSeq Stranded mRNA HT

RS-122-2103

Weight: 83 kg (183 lbs)

TruSeq Stranded Total RNA LT(with Ribo-Zero Human/Mouse/Rat) Set ARS-122-2201

TruSeq Stranded Total RNA LT(with Ribo-Zero Human/Mouse/Rat) Set B

RS-122-2202

TruSeq Stranded Total RNA HT(with Ribo-Zero Human/Mouse/Rat)

RS-122-2203

TruSeq Stranded Total RNA LT(with Ribo-Zero Gold) Set A

RS-122-2301

TruSeq Stranded Total RNA LT(with Ribo-Zero Gold) Set B

RS-122-2302

TruSeq Stranded Total RNA HT(with Ribo-Zero Gold)

RS-122-2303

TruSeq Stranded Total RNA LT(with Ribo-Zero Plant) Set A

RS-122-2401

TruSeq Stranded Total RNA LT(with Ribo-Zero Plant) Set B

RS-122-2402

TruSeq Stranded Total RNA HT(with Ribo-Zero Plant)

RS-122-2403

TruSeq Stranded Total RNA LT(with Ribo-Zero Globin) Set A

RS-122-2501

TruSeq Stranded Total RNA LT(with Ribo-Zero Globin) Set B

RS-122-2502

TruSeq Stranded Total RNA HT(with Ribo-Zero Globin)

RS-122-2503

TruSeq RNA Sample Prep Kit v2 Set A(48 reactions)RS-122-2001

TruSeq RNA Sample Prep Kit v2 Set B(48 reactions)RS-122-2002

TruSeq Small RNA Library Prep Kit Set A

RS-200-0012

TruSeq Small RNA Library Prep Kit Set B

RS-200-0024

TruSeq Small RNA Library Prep Kit Set C

RS-200-0036

TruSeq Small RNA Library Prep Kit Set D

RS-200-0048

Base Unit: Dual Intel Xeon ES-2448L 1.8 GHz CPU
Memory: 96 GB RAM
Hard Drive: 750 GB
Operating System: Windows 7 embedded standard

Crated Weight: 151.5 kg (334 lbs)

Power Requirements
100–120 VAC 15 A
220–240 VAC 10 A

Radio Frequency Identifier (RFID)
Frequency: 13.56 MHz
Power: Supply current 120 mA, RF output power 200 mW

Product Safety and Compliance
NRTL certified IEC 61010-1
CE marked
FCC/IC approveda. Computer specifications are subject to change.

Reference1.

Raczy C, Petrovski R, Saunders CT, et al. Isaac: Ultra-fast whole genome
secondary analysis on Illumina sequencing platforms. Bioinformatics.
2013;29:2041-2043.

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, BaseSpace, Epicentre, HiSeq, NextSeq, ScriptSeq, TruSeq, andthe pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries.
Pub. No. 770-2013-051 Current as of 02 June 2015