Data Sheet: Targeted Resequencing

TruSeq® Custom Amplicon v1.5
A new and improved amplicon sequencing solution for interrogating custom regions of interest.
Highlights• Simple assay customization
Easily create projects that include up to 1536 amplicons
in a single reaction, across a range of amplicon sizes andreference genomes• Optimized reagents
Achieve high performance from as little as 50 ng of DNA,even in challenging GC-rich regions• Rapid sample processing
Prepare up to 96 reactions simultaneously using simple
plate-based format, and standard lab equipment• Easy, integrated analysis
Access automated, design-specific variant calling, and data
analysis with preconfigured MiniSeq® and MiSeq® software

Design and order custom oligo probes
using DesignStudio
Generate template library using
the TruSeq Custom Amplicon Assay Kit
Attach sample indices to libraries using
the TruSeq Custom Index Kit

Normalize and pool libraries

Perform automated cluster generationand sequencing on the MiSeq System

View data using the Illumina Amplicon Viewer

Introduction
The TruSeq Custom Amplicon Library Preparation Kit allows
researchers to sequence hundreds of genomic regions covering as
little as 2 kb up to 650 kb of cumulative sequence. This highly targeted
approach enables a wide range of applications for discovering,
validating, and screening genetic variants in a rapid and efficientmanner.
Requiring as little as 50 ng of starting gDNA, the TruSeq Custom
Amplicon assay is amenable to a wide range of samples, even thoseavailable in limited quantity. With the ability to generate up to 1,536
amplicons per reaction and integrated indexes to support up to 96
samples per MiSeq run, the TruSeq Custom Amplicon assay provides
an unprecedented level of sample multiplexing.* DesignStudio™
supports TruSeq Custom Amplicon designs with a range of ampliconsizes optimized for different sample types (eg formalin-fixed paraffinembedded) and reference genomes (eg human, mouse, rat, andbovine). Entire projects can now be accomplished in only days,
leveraging the long paired-end–read capability, speed, and high data
quality of the MiSeq System.
The TruSeq Custom Amplicon Library Preparation Kit offers a fully
integrated DNA-to-data solution, including convenient online probe
design and ordering, a streamlined workflow, and automated dataanalysis. The Illumina Amplicon Viewer software allows researchers toeasily review project data, including variants detected, across multiple
MiSeq runs.

* Also compatible with the MiniSeq, NextSeq® Series, and HiSeq® Series
Systems.
For Research Use Only. Not for use in diagnostic procedures.

Figure 1: TruSeq Custom Amplicon Workflow—The TruSeq Custom Amplicon
assay is a fully integrated end-to-end amplicon sequencing solution, including
online probe design and ordering, assay, sequencing, automated data
analysis, and offline software for reviewing results.

TruSeq Custom Amplicon Workflow Overview
The entire TruSeq Custom Amplicon process takes only 2 days togo from DNA to data. Researchers can initiate a project by enteringtarget regions of the genome into DesignStudio software (Figure 1).
After a custom design has been ordered, oligonucleotide probes
are synthesized and pooled into a Custom Amplicon Tube (CAT)containing all the probes necessary to generate up to 1536 attempted
amplicons per reaction. Sample-specific indexes are then added toeach library by PCR using common primers from the TruSeq Amplicon
Index Kit. An integrated bead-based normalization procedure enables
simple volumetric pooling of libraries before sequencing on theMiSeq System and avoids laborious quantification methods. Pooledlibraries are then loaded into the MiSeq System for automated cluster
generation, sequencing, and data analysis, including variant calling.
Finally, the Illumina Amplicon Viewer can be used to view project data
across many sequenced samples.

Data Sheet: Sequencing

Simplified Probe DesignDesign TruSeq Custom Amplicon probes targeting user-defined
regions using DesignStudio, a free, easy-to-use online software toolthat provides optimized coverage and estimates total project pricing(Figure 2). After logging on to a personalized account and naming
the project, researchers can select coordinates for targeting genomic
regions of interest. Probe design is automatically performed by using
an algorithm that considers a range of factors, including GC content,specificity, probe interaction, and coverage. Candidate amplicons
are visualized and assessed using estimated success scores. Probes
can be filtered with user-defined tags, and then added to, or removed
from, the design. After visualization and QC, the CAT design can thenbe ordered along with suggested accessory kits.
Typical design success, depending upon custom-selected content,
is 90% or better for desired bases (Table 1). In some cases, higher
or lower success rates can be achieved, depending upon thesequence context (eg, regions of homology or %GC content). To
use DesignStudio, customers will need an account on the Illumina
ecommerce system. Request an account at icom.illumina.com/Account/Register.

Optimized Assay Chemistry
The TruSeq Custom Amplicon v1.5 assay chemistry begins withhybridizing 2 custom-designed probes upstream and downstream of
the region of interest (Figure 3). Each probe contains unique, targetspecific sequence as well as a universal adapter sequence that is used
in a subsequent amplification reaction. The addition of an improved
hybridization buffer, OHS2, ensures that the probes are optimallybound across regions of varying GC-richness and improves assay
performance in potentially difficult- to-address regions (Figure 4). A
proprietary extension-ligation reaction extends across the region ofinterest, followed by ligation to unite the 2 probes and yield a libraryof new template molecules with common ends. During this step, anenhanced extension-ligation mix, ELM4, increases library yield anduniformity (Table 2).
While the increase in uniformity may appear modest, many amplicons
that are poorly represented when using ELM3 achieve significantly
higher read counts with ELM4, enabling higher confidence for variant
calling in these regions. Extension-ligation templates are PCRamplified and 2 unique, sample-specific indexes are incorporated.
Final reaction products are converted to a single-stranded, adapterligated, normalized library using a bead-based protocol. The
sequence-ready library can be stored in the optimized LNS2 storage
buffer for up to 3 weeks, or loaded directly onto the MiSeq System
without additional processing.†† Also compatible with the MiniSeq, NextSeq Series, and HiSeq Series Systems.

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

Figure 2: DesignStudio for Custom Probe Design—The Manage TargetsScreen shows a Target Region view in DesignStudio software. Easily visualize
targeted genomic regions and attempted amplicons to assess design coverageand score. Summary metrics for the entire project are on the left pane, along withproject information and user-defined labels for convenient data sorting during thedesign phase.

Table 1: DesignStudio Parameters for Custom AmpliconParameters

ProjectA

Project
B

ProjectCProjectD

Total Sequence Input (bp)34,76769,423

205,441

4,572

Total Number of Amplicons

293

264

1,434114

Designability Score (% BaseCoverage)

98.3

97.0

93.2

91.3

Data Sheet: Sequencing
Use DesignStudio to create custom oligo capture probes
flanking each region of interest
CustomProbe 1

Region of interest

CustomProbe 2

CAT (custom amplicon tube)

CAT probes hybridize to flanking
regions of interest in unfragmented gDNA
CustomProbe 1

CustomProbe 2

Extension/Ligation between Custom Probes
across regions of interest

PCR adds indices and sequencing primers
P7

Index 1

P5

Index 2

Uniquely tagged amplicon library readyfor cluster generation and sequencing
P7

Index 1

Index 2

P5Figure 3: TruSeq Custom Amplicon Workflow—The TruSeq Custom Amplicon assay is a simple and streamlined method for capturing and amplifying targeted regionsof interest.

3000

OHS1OHS2Read Counts

2500
2000
1500
1000
500
0Region 1 Region 2 Region 3 Region 4Region 5 Region 6(~40% GC) (~40% GC) (~80% GC) (~80% GC) (~80% GC) (~80% GC)

Figure 4: DeOHS2 Increases Read counts for GC-Rich Targets—The optimized OHS2 hybridization buffer with TruSeq Custom Amplicon v1.5 significantly
improves assay performance in challenging GC-rich regions.

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

Data Sheet: Sequencing
Table 2: DesignStudio Parameters for Custom Amplicon
Coverage Uniformitya
Design# Amplicons

ELM3

ELM4

Read Countb
IncreaseAmplicon

ELM3

ELM4

11483

93%

94%

1%

110315

2

217

89%

91%

2%

2663

31483

86%

87%

1%

3828

41420

86%

87%

1%

48

22

115

83%

85%

2%

87%

89%

2%

5Averagea. Coverage uniformity (defined as total bases present at > 0.2× mean coverage), across designs and in poorly represented regions, increases with ELM4 extensionligation mix.
b. Read count for representative under-performing amplicons increases significantly with the addition of ELM4 extension-ligation mix.

B100

10090908080

Percent On-Target Reads

Percent Amplicons CoveredA7060
504030

7060
50
4030

2020

101000Sample 1Sample 2

0.1× mean

Sample 3

0.2× mean

Sample 4Sample 5

Sample 1

Sample 2Sample 3

Sample 4

Sample 5

0.5× mean

Figure 5: High Coverage Uniformity and Specificity—A. Uniformity measured as reads mapping to target regions out of total reads per run, is shownfor 5 samples of a representative target panel. For all samples, > 85% of bases are covered at 0.1× and 0.2× of the mean coverage (blue and purplebars, respectively), and > 65% of bases are covered at 0.5× of the mean coverage (green bars). B. Specificity defined as percentage of filtered reads
corresponding to user-defined targets. > 90% of reads were on target.

The TruSeq Custom Amplicon Library Preparation Kit supports highlevels of multiplexing, while providing excellent specificity and uniformity.
An example TruSeq Custom Amplicon experiment was performed byfollowing the workflow described in Figure 1. Representative uniformity
data, with percent of bases at least 0.2× the mean sequencing depth,
are shown for 5 samples prepared with the TruSeq Custom Amplicon
assay and sequenced using the MiSeq System (Figure 5A). Specificity
data, percent of sequenced bases passing filter aligning to desired
target regions, for samples are shown in Figure 5B. Coverage uniformity
and specificity were excellent, with > 85% of bases covered at > 0.2×of the mean coverage and > 90% of reads on target. These dataillustrate how the TruSeq Custom Amplicon assay can be used torapidly generate desired amplicons in a highly multiplexed reaction forsequencing with the MiSeq System.‡ The TruSeq Custom Amplicon
assay is highly concordant with genotype information using theInfinium® assay (99.4%, 27,500/27,654 calls, data not shown). As
shown in Figure 6, read counts per amplicon are highly reproducible
across different users, with R2 = 0.92.

‡ Also compatible with the MiniSeq, NextSeq Series, and HiSeq Series Systems.
For Research Use Only. Not for use in diagnostic procedures.

Read Counts per Amplicon (User 2)

Excellent Data Quality

350

R2 = 0.92300250200150100500050100150200250300

350

Read Counts per Amplicon (User 1)
Figure 6: High Assay Reproducibility—High reproducibility of Project A readcounts across 2 different users (R2 = 0.92).

Data Sheet: Sequencing

Figure 7: Illumina Amplicon Viewer—Streamlined, intuitive analysis toolbars in Amplicon Viewer. Top pane: visualize coverage levels of each amplicon or
across entire genome regions (shown) and view quality scores, as well as variant calls in the middle and lower panes, respectively. Left scroll bars allow users
to navigate through samples and targets, and track variants.

After the sequencing run on the MiSeq System, data are automatically aligned using the MiSeq Reporter and can be visualized using theIllumina Amplicon Viewer. As shown in Figure 7, large genomic regions
containing multiple amplicons can be viewed, or zoomed in to view
individual amplicons. Quality scores are tracked, and variant calls areeasily and intuitively visualized. Target and sample statistics are managed in simple drop-down menus. From design to analysis, the TruSeq
Custom Amplicon user experience is customized and streamlined, andkeeps project data highly accessible.

SummaryTruSeq Custom Amplicon v1.5 is a powerful targeted resequencing
solution that provides highly multiplexed amplicon sequencing
performance with an optimized chemistry workflow and integrated
analysis. Paired with the MiSeq System,§ The TruSeq Custom
Amplicon v1.5 assay produces remarkably high-quality datafor up to 96 samples in a single run. The robust combination of
the TruSeq Custom Amplicon assay and the MiSeq System has
driven studies comprising tens of thousands of samples, including
hundreds of thousands of amplicons. Using TruSeq Custom
Amplicon v1.5 with the MiSeq System enables the highest quality,
most reliable, and simplest approach to deep interrogation ofgenomic regions of interest.

§ Also compatible with the MiniSeq, NextSeq Series, and HiSeq Series Systems.
For Research Use Only. Not for use in diagnostic procedures.

TruSeq Custom Amplicon Specifications
Specification

Value

Minimum input DNA
Attempted amplicons per reaction
Content range
Specificity50 ngaa

2–650 kba

Coverage uniformity (> 0.2x mean)

16–1,536> 70%
aSpecies available

Samples types

Amplicon sizes
a. Target values will vary due to custom designs.

> 80%
Human, mouse,rat, and bovine
Fresh, frozen,and FFPE
150, 175, 250,
and 425 bp

Data Sheet: Sequencing

Ordering Information
Kit

Catalog No.

TruSeq Custom Amplicon v1.5 Kit (96 Samples)

FC-130-1001

TruSeq Custom Amplicon Index Kit(96 indexes, 384 Samples)

FC-130-1003

TruSeq Index Plate Fixture Kit

FC-130-1005

TruSeq Index Plate Fixture and Collar Kit (2 Each)

FC-130-1007

Learn More
Go to www.illumina.com/products/truseq_custom_amplicon.ilmn tolearn more about the new and improved TruSeq Custom Amplicon v1.5
Assay.

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

Data Sheet: Sequencing

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

Data Sheet: Sequencing

Illumina, Inc. • 1.800.809.4566 toll-free • 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, DesignStudio, HiSeq, MiniSeq, MiSeq, NextSeq TruSeq, and the pumpkin orange color are trademarks orregistered trademarks of Illumina, Inc. and/or its affiliates in the U.S. and/or other countries.
Pub. No. 770-2011-020 Current as of 23 November 2015