Design Content | Prepare Library | Sequence | Analyze DataTumor exome sequencing with Illumina DNA
Prep with Enrichment and the DRAGEN™
Platform
Combining Illumina DNA Prep with Enrichment for exome sequencing with the analyticalpower of DRAGEN enables efficient and accurate variant analysis in FFPE tumor samples.
Introduction
During tumor progression, cells acquire mutations in their DNA thatdrive hallmarks of cancer, such as proliferation, survival, and immune
escape. Understanding these mutations can help researchers
understand the biology of the disease, pave the way for newbiomarkers, and elucidate potential therapeutic targets. Because
the exome represents less than 2% of the genome, cancer exome
sequencing using next-generation sequencing (NGS) technology
is a cost-effective method to investigate associated mutations.1–3
Unfortunately, most exome sequencing solutions have 2-3 day
workflows, require large amounts of DNA, and are not compatible withformalin-fixed, paraffin embedded (FFPE) tissue samples.
To help researchers take advantage of the information within theexome, Illumina offers Illumina DNA Prep with Enrichment (formerlyNextera™ Flex for Enrichment) and the DRAGEN (Dynamic ReadAnalysis for GENomics) Bio-IT Platform. Illumina DNA Prep withEnrichment delivers high-quality exome sequencing libraries bycombining bead-based library preparation chemistry with a simplified,single hybridization for enrichment. The resulting workflow has
the fewest number of steps and the fastest turnaround time in theIllumina enrichment portfolio.4 The DRAGEN platform provides
ultra-rapid secondary analysis of NGS data from genomes, exomes,and transcriptomes. Without compromising accuracy, the DRAGEN
platform delivers quickness, flexibility, and cost efficiency, enablinglabs of all sizes and disciplines to do more with their NGS data.5
The DRAGEN Enrichment App is the recommended method foranalyzing enrichment data, including exomes. It delivers a full suite ofenrichment-specific metrics and reporting with greater accuracy and inless time than previous analysis solutions.5

This application note demonstrates a streamlined, comprehensive
exome sequencing workflow that integrates Illumina DNA Prep withEnrichment, proven Illumina sequencing, and highly accurate analysis
with the DRAGEN platform for identification of disease-associatedvariants in tumor samples (Figure 1).

MethodsSamples
Exome sequencing and variant analysis were performed using nine
unique FFPE tumor–normal sample pairs (Table 1) and Quantitative
Multiplex Reference Standard formalin-compromised DNA (fcDNA)(Horizon Discovery, Catalog no. HD799).

Library preparation
After extraction with the AllPrep DNA/RNA FFPE Kit (QIAGEN, Catalogno. 80234), sequencing libraries were prepared from either 50 ng or
10 ng of DNA input using Illumina DNA Prep with Enrichment andthe Illumina Exome Panel. Single-plex enrichment reactions wereperformed with IDT for Illumina DNA Unique Dual Indexes using anovernight hybridization at 58oC.

Sequencing
All libraries were sequenced on a NovaSeq™ 6000 System withthe appropriate flow cell and run parameters (Table 2). Samples
can also be run on the NextSeq™ 1000 and NextSeq 2000
Sequencing Systems.
For a full list of kits and reagents for this workflow, see the Appendix.

FFPEspecimens

Sample prep

Library prep
and enrichment

Sequence

Analysis andvariant calling

Supports multiple
tissue types

QIAGEN AllPrep
DNA/RNA Kit

Illumina DNA Prepwith Enrichment

NovaSeq 6000 orNextSeq 1000/2000
SystemDRAGEN Enrichment AppDRAGEN Somatic AppFigure 1: Comprehensive exome sequencing workflow—By combining Illumina DNA Prep with Enrichment with proven Illumina sequencing and ultra-rapid analysis
with the DRAGEN platform, Illumina offers an integrated, streamlined workflow using exome sequencing to interrogate FFPE tumor samples.

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

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Design Content | Prepare Library | Sequence | Analyze Data

Table 1: Tumor–normal sample pair information

Table 2: Exome sequencing run parametersVendor

SourceStatusExternal ID∆Cq

Horizon Discovery

N/A

Reference standard

HD799

N/A

Bio-Options

UterusBenign adjacent

NP-18449-1

0.26

NextSeq 1000 or NextSeq 2000 System

Bio-Options

UterusUterine cancer

NP-18449-4

0.38

NovaSeq 6000 System

Bio-Options

Colon

Benign adjacent

NP-18676-3

1.77

Bio-Options

Colon

Colon cancer

NP-18676-2

0.40

OutDo Biotech

Skin

Benign adjacent15-21711-3

1.68

OutDo Biotech

Skin

Melanoma

15-21711-5

1.82

Bio-Options

Colon

Benign adjacent

NP-17874-3

1.35

Bio-Options

Colon

Colon cancer

NP-17874-2

2.01

OutDo Biotech

Skin

Benign adjacent15-10691-12

1.62

OutDo Biotech

Skin

Melanoma

15-10691-7

2.07

Bio-Options

StomachBenign adjacent

NP-16695-4

2.22

Bio-Options

StomachGastric cancer

NP-16695-1

1.79

Bio-Options

UterusBenign adjacentNP-12844-3

2.27

Bio-Options

UterusUterine cancer

NP-12844-2

2.77

Bio-Options

Pancreas

Benign adjacent

NP-18638-6

3.62

Bio-Options

Pancreas

Pancreatic cancer

NP-18638-5

3.74

Bio-Options

UterusBenign adjacent

NP-13587-4

4.34

Bio-Options

UterusUterine cancer

NP-13587-3

4.24

Data analysis
After sequencing was complete, data was securely streamed directlyfrom the instrument into the cloud ecosystem for push-button analysis
using DRAGEN applications available through BaseSpace™ Sequence
Hub (Table 3). The DRAGEN Enrichment App was used to alignsequences from the individual samples to the reference genome,
producing BAM alignment files and quality control and analysis metrics
for exomes. The alignment BAM files were input into the DRAGEN
Somatic App, which performed variant calling and germline variant
filtering, generating a single VCF file with somatic variants from theinput tumor–normal pairs (Figure 2).
Table 3: DRAGEN analysis pipelines
Analysis tool

Application
DRAGEN
Enrichment App

Sequence mapping and alignment toreference, includes enrichment metrics

DRAGEN
Somatic App

Somatic variant detection in tumor samples,
includes tumor-only and tumor–normal
modes

Sequencing system

Tumor sample

FASTQ

Tumor alignment

Secondarymetrics and BAM

P2

3

SP/S1/S2/S4

6/13/33/80

Results
To demonstrate the performance of DRAGEN for exome sequencing
analysis, formalin-compromised and FFPE tumor–normal samples
were evaluated.

Variant calling with formalin-compromised referenceResults with the formalin-compromised reference sample HD799
show that 100% of engineered small variants present (Table 4) weredetected with 50 ng of input (Figure 3A), and all but one variant (95.8%
analytical sensitivity) were detected with 10 ng of input (Figure 3B).
Table 4: HD799 small variants and variant allele frequency (VAF)
Gene

Variant

Expected VAF

KRAS

G12D

6.0%

PIK3CA

E545K

9.0%

cKIT

D816V

10.0%

BRAF

V600E

10.5%

NRAS

Q61K

12.5%

KRAS

G13D

15.0%

PIK3CA

H1047R

17.5%

EGFR

G719S

24.5%Variant calling with FFPE tumor samples
Nine FFPE tumor samples were evaluated with different input
amounts. Results show significant correlation for variant allele
frequencies between technical replicates for samples NP-18449-4 andNP-18676-2 with DNA inputs of 50 ng and 10 ng (Figure 4).

High-performing sequencing metrics
To further demonstrate the analytical performance of DRAGEN apps,
various sequencing and enrichment metrics were evaluated. Highperforming metrics were achieved for all samples, including medianfragment length (Figure 5A), percent aligned reads (Figure 5B), padded
unique read enrichment (Figure 5C), mean target coverage depth(Figure 5D), percent target coverage at 50× (Figure 5E), and coverage
uniformity (Figure 5F).

DRAGEN Somatic App

Germline alignment
Aligning
and QC

No. of samples
per runThe number of samples per run is based on a single flow cell in which 250 million reads aregenerated per sample.

DRAGEN Enrichment App
Normal sample

Flow cell

Germline variants
Variantcalling

Tumor variants

Germline
filtering

Somaticvariants

VCF/gVCF

Figure 2: Tumor–normal variant analysis with DRAGEN apps—FASTQ files automatically generated after a sequencing run are processed by the DRAGEN
Enrichment App to generate BAM alignments and secondary analysis metrics. The BAM files are input into the DRAGEN Somatic App for variant calling and germline
filtering, which results in a single VCF file containing somatic variants.

2 | 1170-2020-002-A

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

Design Content | Prepare Library | Sequence | Analyze DataA.

A. Median fragment length
35%

Observed VAF (%)

30%
25%
20%
y = 1.1x-0.03
15%
10%5%

rep 1
rep 2
rep 3

0%0%

5%10%20%15%25%30%

B. Percent aligned reads

35%

Expected VAF (%)

B.
35%

Observed VAF (%)

30%
25%20%
y = 1.11x-0.01
15%
10%

C. Padded unique read enrichment

5%

rep 1
rep 2
rep 3

0%
0%

5%

10%

20%

15%

25%

30%

35%

Expected VAF (%)

Figure 3: Detection of engineered variants in HD799 at different inputs—Allengineered small variants in control sample HD799 were successfully detectedwith (A) 50 ng and (B) 10 ng of DNA input, except for the NRAS Q61K variant in
replicate 2 (lack of blue triangle at 12.5% VAF).
B.

NP18449 50 ng

NP18676 50 ng

1.0

1.00.80.8

0.6

y = 1 x-0, r2 = 0.97
0.4

0.2

Observed VAF

Observed VAF

A.

0.0
0.0

0.2

0.4

0.6

0.8

0.6

y = 1 x-0, r2 = 0.91
0.4

0.2

Replicate 1
Replicate 2
Replicate 3

Replicate 1
Replicate 2
Replicate 3

0.0
0.0

1.0

0.2

0.8

0.8

0.6

y = 1 x-0, r2 = 0.97
0.4

Replicate 1
Replicate 2
Replicate 3

0.0
0.00.20.4

0.6

Reference VAF

0.6

0.8

1.00.81.0

E. Percent target coverage at 50×

NP18676 10 ng
1.0Observed VAF

Observed VAF

D.

NP18449 10 ng
1.00.2

0.4

Reference VAF

Reference VAF

C.

D. Mean target coverage depth

0.6

y = 1 x-0, r2 = 0.89
0.4

0.2

Replicate 1
Replicate 2
Replicate 3

0.0
0.0

0.2

0.4

0.6

0.8

1.0

Reference VAF

F. Uniformity of coverage

Figure 4 Variant detection in FFPE tumor samples at different inputs—
Significant correlation was observed for variant allele frequencies between
technical replicates for FFPE tumor samples at DNA inputs of 50 ng and 10 ng.

Figure 5: High-performing sequencing metrics—Samples are graphed left
to right in order of decreasing quality, as indicated by ∆Cq (blue line). Highperforming metrics were achieved for all samples at inputs of 10 ng (red) and50 ng (blue).

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

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Design Content | Prepare Library | Sequence | Analyze Data

Summary

References

This application note describes an exome sequencing workflowfor FFPE tumor samples using Illumina DNA Prep with Enrichment
for library preparation, the NovaSeq 6000 System for sequencing,and DRAGEN apps for data analysis. Results demonstrate the highanalytical sensitivity and precision of DRAGEN for variant detection,
particularly in FFPE tumor samples of varying quality and input
amount.

1.

2.
3.

4.

Learn more

5.

To learn more about Illumina DNA Prep with Enrichment, visit www.
illumina.com/illumina-dna-prep-enrichment

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.
Srivastava S, Cohen JS, Vernon H, et al. Clinical whole exome sequencing
in child neurology practice. Ann Neurol. 2014;76:473–483.
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.
llumina (2020). Illumina DNA Prep with Enrichment Data Sheet. AccessedFebruary 21, 2020.
Illumina (2019). Illumina DRAGEN Bio-IT Platform Data Sheet. Accessed
February 21, 2020.

To learn more about Illumina sequencing systems, visit www.illumina.
com/systems/sequencing-platforms.html
To learn more about DRAGEN, visit www.illumina.com/products/bytype/informatics-products/dragen-bio-it-platform.html

Appendix
Recommended library preparation, enrichment, and sequencing reagents for exome sequencing
Sequencing System

Sequencing reagents

Catalog no.

NextSeq 2000 System

NextSeq 1000/2000 P2 Reagents (200 cycles)

20040557

NovaSeq 6000 SP Reagent Kit (200 cycles)

20040326

NovaSeq 6000 S1 Reagent Kit (200 cycles)

20012864

NovaSeq 6000 S2 Reagent Kit (200 cycles)

20012861

NovaSeq 6000 System

NovaSeq 6000 S4 Reagent Kit (200 cycles)

20027466

ProductDescription

Catalog no.

Illumina DNA Prep with Enrichment, (S) Tagmentation, 96 samples

96 samples, 8 12-plex enrichment reactions

20025524

Illumina DNA Prep with Enrichment, (S) Tagmentation, 16 samples

16 samples, 16 1-plex enrichment reactions

20025523

Illumina DNA Prep, (S) Tagmentation, 96 samples

96 samples

20025520

Illumina DNA Prep, (S) Tagmentation, 16 samples

16 samples

20025519

Illumina Exome Panel

8 enrichment reactions

20020183

IDT for Illumina Nextera DNA Unique Dual Indexes Set A,Tagmentation (96 indexes, 96 samples)

96 indexes, 96 samples

20027213

IDT for Illumina Nextera DNA Unique Dual Indexes Set B,Tagmentation (96 indexes, 96 samples)

96 indexes, 96 samples

20027214

IDT for Illumina Nextera DNA Unique Dual Indexes Set C,Tagmentation (96 indexes, 96 samples)

96 indexes, 96 samples

20042666

IDT for Illumina Nextera DNA Unique Dual Indexes Set D,Tagmentation (96 indexes, 96 samples)

96 indexes, 96 samples

20042667

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