Prep are Lib rary | Seq uence | Analyze Data

Nextera™ DNA Exome
Simple workflow streamlines library preparation and exome enrichment to accelerate humanexome sequencing.
Highlightsllll

Flexibility for Different Study Designs
Optimized chemistry accommodates low input amounts

Table 1: Exome Content with Nextera DNA Exome and TruSeq
DNA Exome
Coverage Specification

Nextera DNA Exome or TruSeq DNA
Exome

Efficient Library Prep and Exome Enrichment
Rapid, automation-friendly workflow completes in < two days,
with only three hours of hands-on timeTarget Size

45 MbNo. of Target Exons

214,405

Accurate Variant Calls
High coverage and on-target sequencing result in accurate
data

Percent of Exome Covered (by Database)Integrated Workflow Solution
Comprehensive workflow streamlines exome sequencing
from library prep through data analysis

Introduction
Exome sequencing has gained recognition in the scientificcommunity as a powerful method for discovering potential causative
variants for genetically driven diseases.1–3 Nextera DNA Exome,
previously sold as the TruSeq™ Rapid Exome Kit, delivers a fastexome sequencing workflow, combining library preparation andexome enrichment into a single, streamlined process with no need for
DNA shearing equipment.

Target Content

coding exons

RefSeqa

99.45%

CCDSb

98.83%ENSEMBLc

99.68%

GENCODE v19d

99.68%

a. RefSeq - NCBI Reference Sequence Database.
www.ncbi.nlm.nih.gov/refseq/. Accessed February 11, 2015.
b. CCDS - Consensus CDS (CCDS) Database.
www.ncbi.nlm.nih.gov/projects/CCDS/CcdsBrowse.cgi.
Accessed February 11, 2015.
c. ENSEMBL - Ensembl Genome Browser. www.ensembl.org/index.html.
Accessed February 11, 2015.
d. GENCODE - GENCODE Project: Encyclopedia of genes and gene variants.
www.gencodegenes.org/. Accessed February 11, 2015.

Flexibility for Different Study Designs
Nextera DNA Exome is optimized for 50 ng of input DNA. Enhanced
transposome chemistry results in reduced bias, providing consistentlibrary preparation and exome enrichment across a range of DNA
quantities (Figure 1). The transposome enzyme is highly tolerant ofvarying DNA input amounts, accommodating minor inconsistencies
in DNA quantification. The kit supports library pooling before
enrichment, enabling labs to run up to 12 samples simultaneously,
depending on sample processing volume. Nextera DNA Exomefeatures an automation-friendly workflow to simplify and streamline
lab processes even further.

Focused Exonic Content
Nextera DNA Exome is optimized to provide uniform and specificcoverage of 45 Mb of exonic content. The probe set is designed toenrich 214,405 exons (Table 2). This focused design, paired withuniform and specific enrichment, enables comprehensive exome
sequencing and reliable identification of true, coding variants.

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

Figure 1: Consistent Tagmentation for Varying Input Amounts—Enhanced
transposome chemistry is highly tolerant of varying DNA input amounts, reducing
bias and providing consistent results. Libraries were assessed on a Bioanalyzer
instrument.

Prep are Lib rary | Seq uence | Analyze Data

Efficient Library Preparation and Exome
Enrichment
Nextera DNA Exome provides library preparation and exome
enrichment in less than two days, with just three hours of hands-ontime (Figure 3). Library preparation begins with tagmentation, in which
genomic DNA is simultaneously fragmented and tagged withadapters (Figure 3A). Tagmented DNA is amplified and sequencing
indexes are added by PCR (Figure 3B). Up to 12 libraries are pooled
together, and the library pool is concentrated and libraries are
denatured into single-stranded DNA (Figure 3C). Biotin-labeledprobes specific to the targeted regions are used for two rounds ofhybridization (Figure 3D and 3E). The pool is enriched for the desired
regions using streptavidin beads that bind to the biotinylated probes.
Biotinylated DNA fragments bound to the streptavidin beads aremagnetically pulled down from the solution. The enriched DNAfragments are eluted from the beads and further amplified by PCR(Figure 3F). Amplified libraries are cleaned up and ready forsequencing (Figure 3G).

Accurate Variant Calls
Libraries prepared with Nextera DNA Exome deliver high coverage,
with 85% of reads covered at 20× depth (Figure 2). Such highcoverage results in accurate variant calls. Over 99.58% of variant
calls made using Nextera DNA Exome match standard reference
data in the National Institute of Standards and Technology (NIST)
database (Figure 4).5, 6
Nextera DNA Exome delivers on average 75% of on-target
sequencing reads (Figure 6). This high on-target percentage requires
fewer sequencing cycles to reach desired coverage levels, but stillachieves uniform coverage for high-confidence results. It alsoenables sequencing of more exomes per run, allowing labs tomaximize their budgets (Table 2).

Figure 2: Coverage Efficiency at Varying Depths—Nextera DNA Exome delivers
exceptional coverage across varying sequencing depth, with > 80% of targets
covered up to 20× depth.

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

Figure 3: Efficient and Fast Workflow—Nextera DNA Exome completes library
preparation and exome enrichment in less than two days and includes a safe
stopping point for greater flexibility.

Figure 4: High Correlation with NIST Database—Single nucleotide variant (SNV)
calls made with Nextera DNA Exome demonstrate high concordance withstandard reference data. The Centre de’Etude du Polymorphism Humain (CEPH)
DNA sample NA12878 was sequenced to 100× coverage depth. Precision isdefined as the probability that a called variant is accurate. Recall is defined as theprobability of calling a validated variant.

Prep are Lib rary | Seq uence | Analyze Data

Figure 5: Exome Sequencing Workflow—Nextera DNA Exome is part of an integrated exome sequencing workflow that includes library preparation, sequencing, and data
analysis.

Integrated Sequencing Workflow

Figure 6: On-Target Enrichment—Nextera DNA Exome delivers on average 75%
of on-target sequencing reads at 4 Gb per exome for efficient, cost-effectivesequencing.

Table 2: Throughput Comparison with Nextera DNA Exomea
Sequencing System
MiSeq Series

No. of Exomes per Run No. of Exomes per Run
at 50×at 100×1N/AMid-Output Flow Cell

32High-Output Flow Cell

126HiSeq 2500 System
Rapid-Run Mode (DualFlow Cell)

2412HiSeq 2500 System
High-Output Mode (DualFlow Cell)

156

78

HiSeq 3000 System

96

48

HiSeq 4000 System (DualFlow Cell)

192

96

NextSeq Series

HiSeq Series

a. Estimated number of exomes sequenced per run is calculated with a mean
coverage of 50× and 100×, respectively. Illumina recommends a 2 × 75 bp
read length on all sequencers when using Nextera DNA Exome.

*Data calculations on file. Illumina, Inc., 2015.

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

Nextera DNA Exome is part of a cohesive, supported solution thatguides researchers from library preparation through data analysis(Figure 5). The kit combines library preparation and exome
enrichment, eliminating the need to purchase indexes, sample
purification beads, or other ancillary materials. All components of
Nextera DNA Exome are designed, optimized, and analyticallyvalidated together, eliminating the need to evaluate multiple,
disparate components. Expert Illumina scientists provide a single
source of technical and field support for every stage of the workflow.
By joining the Illumina community, researchers can harness theexpertise of the Illumina support team and collaborate with the large
network of scientists using Illumina technology.
Nextera DNA Exome is compatible with the MiSeq™, NextSeq™,HiSeq™, and NovaSeq™ Series of sequencing systems. Illumina
sequencing systems use sequencing by synthesis (SBS) chemistry,
used to generate more than 90% of the world's sequencing data.*
Sequencing data are transferred automatically from Illumina systems
to BaseSpace ® Sequence Hub, the Illumina genomics computingenvironment. BaseSpace Sequence Hub removes much of thecomplexity from the typical analysis workflow, simplifying data
analysis and biological interpretation. BaseSpace Sequence Huboffers an established ecosystem of integrated data analysis tools
designed for biologists. With BaseSpace Apps, expert-preferredanalysis tools are packaged in an intuitive, user-friendly interface, sothat any researcher can access trusted analysis pipelines without
previous bioinformatics experience (Figure 7). Researchers canchoose to analyze exome data using the BWA Enrichment App,
which uses the industry-standard BWA/GATK method, or the Isaac™
Enrichment App, which uses the fast and accurate Illumina pipeline.6
For biologists investigating the genetic basis of disease, theVariantStudio App enables identification and functional interpretationof disease-associated single nucleotide variants (SNVs) andinsertions and deletions (indels). Researchers can rapidly filter andisolate consequential variants to enrich sequencing data with

Prep are Lib rary | Seq uence | Analyze Data

biological context. Significant findings are exported in concise
reports. The VariantStudio App enables researchers to explore
biological significance in a few simple steps.

and easy-to-use analysis tools, Nextera DNA Exome enables
researchers to implement exome sequencing efficiently and costeffectively.

Learn MoreTo learn more about exome sequencing, visit
www.illumina.com/techniques/sequencing/dnasequencing/targeted-resequencing/exome-sequencing.html.

Ordering Information

Figure 7: Simplified Data Analysis with BaseSpace Apps—Nextera DNA Exome
sequencing data can be easily and securely uploaded to BaseSpace Sequence
Hub and analyzed with the BWA Enrichment App. Results are provided in easy-toread formats.

Exome Sequencing Performance Comparison
Illumina offers two integrated workflow solutions for exome
sequencing. Workflows are also available that combine Illumina
library prep with TruSeq DNA Exome or Nextera DNA Exome, followedby exome enrichment using xGen ® Universal Blockers, xGen
Lockdown Reagents, and xGen Exome Research Panel v1.0,
available from IDT (Table 3).

ProductCatalog No.

Nextera Exome Kit (24 samples)

20020616

Nextera Exome Kit (96 samples)

20020617

References
1.

Litchfield K, Summersgill B, Yost S, et al. Whole-exome sequencing reveals
the mutational spectrum of testicular germ cell tumours. Nat Commun.
2015;6:5973.

2.

Srivastava S, Cohen JS, Vernon H, et al. Clinical whole exome sequencing inchild neurology practice. Ann Neurol . 2014;76:473–483.

3.

Worthey EA, Mayer AN, Syverson GD, et al. Making a definitive diagnosis:
successful clinical application of whole exome sequencing in a child withintractable inflammatory bowel disease. Genet Med. 2011;13:255–262.

4.

Standard Reference Data (www.nist.gov/srd). Accessed 11 February 2015.

5.

Genome in a Bottle Consortium | Advances in Biological and Medical
Measurement Science (sites.stanford.edu/abms/giab). Accessed 20 February

Table 3: Exome Workflow Performance Comparison
Metric

TruSeqxGena

NexteraxGena

TruSeqExomeNextera
ExomeDNA Input

100 ng50 ng

100 ng50 ng

Sample Types

DNADNADNA andFFPE

DNA

Hands-OnTime

5 hours

2 hours

6 hours

3 hours

Total AssayTime

2.5 days

2 days

2.5 days

2 daysHybridization
Time

4 hours

4 hours

16 hours

2 hours

On-Target %

> 91%

> 92%

> 80%

> 75%

2015.
6.

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

% Coverage at
> 95%
> 85%
> 90%
> 85%
20×ba. Specifications for Illumina-IDT exome enrichment workflows are based on
preliminary data posted on BaseSpace Sequence Hub.
b. Percent coverage at 20× was determined for TruSeq-xGen and NexteraxGen kits with 3.5 Gb of sequencing. Percent coverage at 20× wasdetermined for TruSeq DNA Exome and Nextera DNA Exome with 8 Gb ofsequencing.

Summary
Nextera DNA Exome offers a simple, streamlined method for identifying
and understanding coding variants with exceptional data accuracy.
The fast library preparation and exome enrichment workflow delivers
sequencing-ready libraries less than two days and provides flexibility inproject planning according to sample volume. As part of acomprehensive workflow consisting of leading sequencing technology
Illumina, Inc. • 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.
© 2017 Illumina, Inc. All rights reserved. Illumina, BaseSpace, HiSeq, Isaac, MiSeq, Nextera, NextSeq, NovaSeq, TruSeq, and the pumpkin orange color are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. xGen is a trademark of Integrated DNA Technologies, Inc. in the U.S. and/or
other countries Pub. No. 770-2015-018-B