Data Sheet: Illumina® Sequencing

TruSeq™ RNA and DNA Library Preparation Kits v2
Master-mixed reagents, optimized adapter design, and a flexible workflow provide a simple, costeffective method for preparing RNA and DNA samples for scalable next-generation sequencing.

Highlights• Simple Workflow for RNA and DNA:
Master-mixed reagents and minimal hands-on steps.
• Scalable and Cost-Effective Solution:
Optimized formulations and plate-based processing
enables large-scale studies at a lower cost.
• Enhanced Multiplex Performance:
Twenty-four adaptor-embedded indexes enable highthroughput processing and greater application flexibility.
• High-Throughput Gene Expression Studies:
Gel-free, automation-friendly RNA library preparation
for rapid expression profiling.

Introduction
Illumina next-generation sequencing (NGS) technologies continue toevolve, offering increasingly higher output in less time. Keeping pacewith these developments requires improvements in library preparation.
To maximize the benefits of NGS and enable delivery of the highest
data accuracy, Illumina offers the TruSeq RNA and DNA Library Preparation Kits (Figure 1).
The TruSeq RNA and DNA Library Preparation Kits provide a simple,
cost-effective solution for generating libraries from total RNA or genomic
DNA that are compatible with Illumina’s unparalleled sequencing output.
Master-mixed reagents eliminate the majority of pipetting steps andreduce the amount of clean-up, as compared to previous methods,
minimizing hands-on time. New automation-friendly workflow formats
enable parallel processing of up to 96 samples. This results in economical, high-throughput RNA or DNA sequencing studies achieved with theeasiest-to-use library preparation workflow offered by any NGS platform.

The gel-free protocol for TruSeq DNA library preparation is available
for target enrichment using the TruSeq Exome Enrichment or TruSeq
Custom Enrichment kits.
TruSeq library preparation makes RNA sequencing for high-throughput
experiments more affordable, enabling gene expression profilingstudies to be performed with NGS at a lower cost than arrays. It also
provides a cost-effective DNA sequencing solution for large-scale
whole-genome resequencing, targeted resequencing, de novo sequencing, metagenomics, and methlyation studies.

Enhanced Multiplex Performance
TruSeq kits take advantage of improved multiplexing capabilities toincrease throughput and consistency, without compromising results.
Both the RNA and DNA preparation kits include adapters containing
unique index sequences that are ligated to sample fragments at thebeginning of the library construction process. This allows the samples
to be pooled and then individually identified during downstream
analysis. The result is a more efficient, streamlined workflow that leads
directly into a superior multiplexing solution. There are no additional
PCR steps required for index incorporation, enabling a robust, easyto-follow procedure. With 24 unique indexes available, up to 384
samples can be processed in parallel on a single HiSeq 2000 run.

TruSeq RNA Library Preparation
With TruSeq reagents, researchers can quickly and easily prepare
samples for next-generation sequencing (Figure 2). Improvements in
the RNA to cDNA conversion steps have significantly enhanced theoverall workflow and performance of the assay (Figure 3).

Figure 1: TruSeq Library Preparation Kits

Simple and Cost-Effective Solution
Whether processing samples for RNA-Seq, genomic sequencing, orexome enrichment, the TruSeq kits provide significantly improved library
preparation over previously used methods. New protocols reduce thenumber of purification, sample transfer, and pipetting steps. The new
universal, methylated adaptor design incorporates an index sequence at
the initial ligation step for improved workflow efficiency and more robust
multiplex sequencing. For maximum flexibility, the same TruSeq kit canbe used to prepare samples for single-read, paired-end, and multiplexed sequencing on all Illumina sequencing instruments.
TruSeq DNA and RNA Library Prep kits include gel-free protocols
that eliminate the time-intensive gel purification step found in othermethods, making the process more consistent and fully automatable.

TruSeq Library Preparation Kits are available for both genomic DNA andRNA samples.

Data Sheet: Illumina® Sequencing
Figure 2: Optimized TruSeq RNA Library Preparation

Figure 3: TruSeq RNA Library Preparation Reagents
Provide Significant Savings in Time and Effort

Starting with total RNA, mRNA is polyA-selected and fragmented. It thenundergoes first- and second-strand synthesis to produce products readyfor library construction (Figure 4).

Starting with total RNA, the messenger RNA is first purified using
polyA selection (Figure 2A), then chemically fragmented and converted
into single-stranded cDNA using random hexamer priming. Next, thesecond strand is generated to create double-stranded cDNA (Figure2B) that is ready for the TruSeq library construction workflow (Figure 4).
Efficiencies gained in the polyA selection process, including reduced
sample transfers, removal of precipitation steps, and combining ofelution and fragmentation into a single step, enable parallel processingof up to 48 samples in approximately one hour. This represents a 75%
reduction in hands-on time for this portion of library construction. Improving performance, the optimized random hexamer priming strategy
provides the most even coverage across transcripts, while allowinguser-defined adjustments for longer or shorter insert lengths.
Eliminating all column purification and gel selection steps from theworkflow removes the most time-intensive portions, while improving theassay robustness. It also allows for decreased input levels of RNA—aslow as 100 ng— and maintains single copy per gene sensitivity.

TruSeq DNA Library Preparation
The TruSeq DNA Library Preparation Kits are used to prepare DNAlibraries with insert sizes from 300–500 bp for single, paired-end, andmultiplexed sequencing. The protocol supports shearing by either
sonication or nebulization with a low input requirement of 1 ug of DNA.

Current
Methods

TruSeqMethods

Savings

No. of Steps

49

1831

Time (hours)

16

1225%

Compared to current methods for preparing mRNA samples for sequencing,use of the TruSeq reagents significantly reduces the number of steps andhands-on time.

Table 1: Savings When Processing 96 Samples> 50% of pipetting steps eliminated> 50% of reagent tubes eliminated> 75% of clean-up steps eliminatedSequence-Ready Libraries> 50% of sample transfer steps eliminatedLibrary construction begins with either double-stranded cDNA synthesized from RNA or fragmented gDNA (Figure 4A). Blunt-end DNA
fragments are generated using a combination of fill-in reactions andexonuclease activity (Figure 4B). An ‘A’- base is then added to theblunt ends of each strand, preparing them for ligation to the sequencing adapters (Figures 4C). Each adapter contains a ‘T’-base overhang
on 3’-end providing a complementary overhang for ligating the adapter

Compared to previous kits, processing multiple samples with thenew TruSeq Library Preparation Kits provides significant reductions
in library construction costs, the number of steps, hands-on time,and PCR dependency.

Data Sheet: Illumina® Sequencing
Figure 4: Adapter Ligation Results in Sequence-ReadyConstructs without PCR

to the A-tailed fragmented DNA. These newly redesigned adapters
contain the full complement of sequencing primer hybridization sites
for single, paired-end, and multiplexed reads. This eliminates the needfor additional PCR steps to add the index tag and multiplex primer
sites (Figure 4D). Following the denaturation and amplification steps(Figure 4E), libraries can be pooled with up to 12 samples per lane
(96 sample per flow cell) for cluster generation on either cBot or theCluster Station.
Master-mixed reagents and an optimized protocol improve the library
construction workflow, significantly decreasing hands-on time andreducing the number of clean-up steps when processing samples forlarge-scale studies (Table 1). The simple and scalable workflow allows
for high-throughput and automation-friendly solutions, as well as
simultaneous manual processing for up to 96 samples. In addition,enhanced troubleshooting features are incorporated into each stepof the workflow, with quality control sequences supported by Illumina
RTA software.

Enhanced Quality Controls
Specific Quality Control (QC) sequences, consisting of doublestranded DNA fragments, are present in each enzymatic reactionof the TruSeq library preparation protocol: end repair, A-tailing, andligation. During analysis, the QC sequences are recognized by the RTA
software (versions 1.8 and later) and isolated from the sample data.
The presence of these controls indicates that its corresponding step
was successful. If a step was unsuccessful, the control sequences will
be substantially reduced. QC controls assist in comparison betweenexperiments and greatly facilitate troubleshooting.

Designed For Automation
The TruSeq Library Preparation Kits are compatible with high-throughput, automated processing workflows. Library preparation can be
performed in standard 96-well microplates with master-mixed reagent
pipetting volumes optimized for liquid-handling robots. Barcodes onreagents and plates allow end-to-end sample tracking and ensurethat the correct reagents are used for the correct protocol, mitigating
potential tracking errors.

Part of an Integrated Sequencing Solution
Samples processed with the TruSeq Library Preparation Kits can beamplified on either the cBot Automated Cluster Generation Systemor the Cluster Station and used with any of Illumina’s next-generation
sequencing instruments, including HiSeq™ 2000, HiSeq 1000,HiScan™SQ, Genome AnalyzerIIx (Figure 5).

Summary
Library construction begins with either fragmented genomic DNA or doublestranded cDNA produced from total RNA (Figure 4A). Blunt-end fragments
are created (Figure 4B) and an A-base is then added (Figure 4C) to prepare
for indexed adapter ligation (Figure 4D). Final product is created (Figure 4E),
which is ready for amplification on either the cBot or the Cluster Station.

Illumina’s new TruSeq Library Preparation Kits enable simplicity, convenience, and affordability for library preparation. Enhanced multiplexing
with 24 unique indexes allows efficient high-throughput processing.
The pre-configured reagents, streamlined workflow, and automationfriendly protocol save researchers time and effort in their next-generation sequencing pursuits, ultimately leading to faster discovery andpublication.
Learn more about Illumina next-generation sequencing solutions at
www.illumina.com/sequencing.

Data Sheet: Illumina® Sequencing
Figure 5. Illumina’s Complete Sequencing Solution

Ordering Information
ProductCatalog No.

For RNA Preparation
TruSeq RNA Library Preparation Kit v2, Set A(12 indexes, 48 samples)

RS-122-2001

TruSeq RNA Library Preparation Kit v2, Set B(12 indexes, 48 samples)

RS-122-2002

For DNA Preparation
TruSeq DNA Library Preparation Kit v2, Set A(12 indexes, 48 samples)

FC-121-2001

TruSeq DNA Library Preparation Kit v2, Set B(12 indexes, 48 samples)

FC-121-2002

For Cluster Generation on cBot and Sequencing on theHiSeq 2000/1000 and HiScanSQ
TruSeq Paired-End Cluster Kit v3—cBot—HS(1 flow cell)

PE-401-3001

TruSeq Single-Read Cluster Kit v3—cBot—HS(1 flow cell)

GD-401-3001

For Cluster Generation on cBot and Sequencing on the
Genome AnalyzerIIx
TruSeq Paired-End Cluster Kit v2—cBot—GA(1 flow cell)

PE-300-2001

TruSeq Single-Read Cluster Kit v2—cBot—GA(1 flow cell)

GD-300-2001

For Cluster Generation on the Cluster Station and Sequencing
on the Genome AnalyzerIIx

The TruSeq Library Preparation Kits readily fit in with Illumina’s advanced
next-generation sequencing solutions.

TruSeq Paired-End Cluster Kit v5—CS—GA(1 flow cell)

PE-203-5001

TruSeq Single-Read Cluster Kit v5—CS—GA(1 flow cell)

GD-203-5001

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their respective owners.
Pub. No. 770-2009-039 Current as of 17 November 2014