Application Note: Cancer Genomics

Interrogating Cancer-Predisposing Germline
Variants on the MiniSeq™ System
Identify predisposition genomic variants linked to cancer using the most accessible sequencing
platform from Illumina.

Highlights• Streamlined Sample-to-Data Germline Testing Solutions
Simple workflows with minimal hands-on time
• Comprehensive Variant Detection with Expert-Selected
and Custom Panels
Complete assays to detect predisposition genes, variants,
and hotspots in a broad spectrum of cancers• Easy Data Analysis
Walk-away, intuitive data analysis performed locally or in thecloud• End-to-End Illumina Support
Expert Illumina specialists available globally to provide
installation, training, and support

Introduction
Next-generation sequencing (NGS) technology has led to recent
breakthroughs in cancer research, including associations between
genomic variants and cancer predisposition.1-4 With the ability
to sequence multiple genes and samples simultaneously, highthroughput NGS offers distinct advantages over traditional capillaryelectrophoresis (CE)/Sanger sequencing methods and PCR-based
genotyping.
Sanger sequencing is the current gold standard method and offers aquick and simple workflow. Yet it has low scalability due to increasing
sample input requirements, low discovery power, and low analyticalsensitivity.5, 6 PCR-based genotyping also offers a quick and simple

Germline Screening

Design Content

workflow and high analytical sensitivity. However, it can only interrogate
a limited set of mutations, has virtually no discovery power, and is not
scalable when processing multiple samples or the input requirement
is high.7 NGS offers higher analytical sensitivity (down to 1% allele
frequency limit of detection), higher discovery power with the ability toprofile hundreds of genes simultaneously, and increased resolution.8,
9Labs worldwide are taking advantage of NGS to examine multiple
cancer-predisposing alterations with a lower cost, faster turnaroundtime, and lower tissue requirements compared to Sanger/CEsequencing and PCR-based genotyping.
Using targeted sequencing methods, cancer researchers can focus
on a select set of genes, gene regions, or hotspots with known orsuspected predispositions toward cancer (Figure 1). Focused panels
for targeted cancer sequencing featuring expert-selected content

Prepare Library

Sequence

8-24 hours≤ 24 hours

TruSight® Cancer Prep Kits
• TruSeq® Custom Amplicon Prep Kits

MiniSeq System< 1 day
For Custom Panels Only

Figure 1: Targeted Sequencing Enables Variant Detection—Targeted
sequencing enables researchers to detect predisposition variants linked to abroad spectrum of cancers.

•Analyze Data &Annotate Variants< 2 hours
Local Run Manager• BaseSpace® Apps
• VariantStudio•

Figure 2: MiniSeq System Germline Screening Workflow—The integrated workflow enables streamlined library preparation, sequencing, and data analysis, allowing
for cost-effective studies for a broad range of samples.

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

Application Note: Cancer Genomics
significantly narrow the scope of a sequencing project, reducing costand data analysis burdens. Because it assesses a limited set of genes,
targeted cancer sequencing allows for deeper coverage of theseregions of interest.

Target Enrichment Chemistry

The MiniSeq System delivers a clear, complete, cost-effective toolset
for targeted cancer sequencing. It harnesses industry-leading Illumina
NGS technology, used in > 90% of all NGS around the world, withover 26,000 peer-reviewed publications in all.*
The MiniSeq System is supported by a suite of Illumina library
preparation solutions and simple, streamlined sample-to-data
workflows (Figure 2). These were developed by Illumina scientists andoptimized for the MiniSeq System following industry guidelines andexpert recommendations. The BaseSpace Environment enables labs
to analyze, archive, and share sequencing data securely, and delivers
expert-selected tools in an intuitive user interface that simplifies
informatics analysis. The Illumina service and support team is available
globally throughout the entire workflow, from library preparation to data
analysis, to offer training, assistance, and answer questions 24 hours aday, 5 days a week.
The MiniSeq System is the most affordable Illumina sequencing
system to acquire, and it is cost-efficient to run, even for low numbersof samples. It makes the quality and reliability of Illumina NGS
accessible to labs of all sizes. With the MiniSeq System, the move totargeted cancer sequencing is easier than ever.

Simple, Streamlined Workflows
MiniSeq System workflows simplify germline variant detection andenable researchers to maximize productivity (Figure 2). Researchers
can use a fixed panel of 94 genes and 284 single nucleotide
polymorphisms (SNPs) known to be associated with a predisposition
toward cancer with the TruSight Cancer Sequencing Panel. Also,
researchers can perform customized germline profiling with userdefined panels of up to 1536 amplicons generated with the TruSeq
Custom Amplicon Low Input Library Prep Kit.

Library Preparation
Illumina methods for library preparation include capture-basedtarget enrichment and amplicon generation (Figure 3). With target
enrichment, specific regions of interest are captured by hybridization
to biotinylated probes, then isolated by magnetic pulldown. Thishighly multiplexed approach enables a wide range of applications forthe discovery, validation, or profiling of genetic variants, including the
TruSight Cancer panel.
The amplicon generation method employed by TruSeq Custom
Amplicon Low Input uses a hybridization-extension-ligation approach.
This creates a single strand template from a double-stranded DNA
population that is later amplified via PCR.
TruSight Cancer Library Prep
The TruSight Cancer Sequencing Panel is compatible withTruSight
Rapid Capture kits, providing a single, integrated library preparationand enrichment workflow. This rapid prep requires only 50 ng of input
DNA, takes 1.5 days, and eliminates the need for mechanical shearing.

*Denature double-stranded DNA library. Hybridize
biotinylated probes to target regions. Enrich withmagnetic beads conjugated to streptavidin.

Amplicon Generation Chemistry

CustomProbe 1P7Index 1

CustomProbe 2

Index 2

P5Probes hybridize to flanking regions of interestin unfragmented gDNA. Extenstion-ligation between
probes across target region. Sequencing primers andindexes are added with PCR.

Figure 3: Library Preparation Methods — Illumina methods for sequencing
library preparation include targeted enrichment and amplicon generation.

The TruSight Rapid Capture Sample Prep Guide is an easy-to-follow
protocol for preparing DNA sequencing libraries. It leads users througheach step of library preparation, listing necessary reagents andindicating safe stopping points.
TruSeq Custom Amplicon Low Input Library Prep
The TruSeq Custom Amplicon Low Input assay empowers researchers
to create a custom amplicon panel targeting genes and regions ofinterest using Illumina DesignStudio™ software, a free, easy-to-use,
online tool that provides optimized coverage (Figure 4). DesignStudio
produces TruSeq Custom Amplicon Low Input designs with anaverage of > 94% in silico coverage across all gene sets. Illumina
Concierge services offer support for probe design, functional
evaluation of custom panels, and increasing target coverage.†
TheTruSeq Custom Amplicon Low Input assay features simple,streamlined library preparation that is automation friendly. It can becompleted in 6.5 hours, with only 3 hours of hands-on time. TheTruSeq Custom Amplicon Low Input Library Prep Reference Guidecontains everything a first-time user needs, including an overview ofthe protocol, tips and techniques, detailed protocol steps, kit contents,
and a list of user-supplied consumables.

Data calculations on file. Illumina, Inc. 2015.
† For more information on Illumina Concierge services, contact an Illumina representative.

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

Application Note: Cancer Genomics
features a modular design that allows users to install and update
individual analysis modules as needed, which generate simple reports
for various sequencing applications.
In addition, sequencing data generated with the MiniSeq System
can be instantly transferred, stored, and analyzed in the BaseSpace®
Computing Environment (Cloud-based or Onsite). BaseSpace
Applications (Apps) provide expert-preferred data analysis tools in an
intuitive, click-and-go user interface designed for informatics novices(Figure 6). These Apps support a range of common sequencing data
analysis needs such as alignment, variant calling, and more. The
BaseSpace ecosystem provides one of the largest collections ofcommercial and open-source analysis tools currently available.
Figure 4: Custom Probe Design — Researchers can use DesignStudio tovisualize targeted genomic regions and attempted amplicons to assess design
coverage and score.

VariantStudio enables rapid filtering, identification, and annotationof disease-associated variants from sequencing data (Figure 7).
An easy-to-use application, VariantStudio allows for flexible report
generation that summarizes and annotates disease-relevant variants ina structured format.

Sequencing on the MiniSeq System
Whether using target enrichment or amplicon generation methods
for library preparation, after sample libraries are prepared they canbe easily sequenced on the MiniSeq System (Figure 5). It integrates
clonal amplification and sequencing into a fully automated process ona single instrument. This eliminates the need to purchase and operate
expensive, specialized equipment.
The MiniSeq System features load-and-go operation and an intuitive
user interface that provides simple, step-by-step guidance througheach stage of the sequencing run. It takes less than 5 minutes to loadand set up a MiniSeq System. Sequencing runs can be completedin ≤ 24 hours. MiniSeq reagent kits are available in Mid-Output andHigh-Output formats, allowing optimization of study designs based onread-length, sample number, and output requirements.
Figure 6: BaseSpace Dashboard—The BaseSpace Environment features anintuitive, click-and-go user interface to empower any researcher to perform theirown informatics.

Figure 5: MiniSeq System — The MiniSeq System harnesses the latest
advances in SBS chemistry and an easy, integrated workflow.

Simplified Data Analysis and Bioinformatics
Data analysis with the MiniSeq System requires no informatics
expertise or command-line experience. It features Local Run Manager
software, an onboard system for creating a run, monitoring status, andautomated sequencing data analysis post-run. Local Run Manager

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

Application Note: Cancer Genomics

Figure 7: VariantStudio—VariantStudio software features an intuitive user interface that enables easy data analysis and exploration, without require informatics
expertise. It aggregates information from a broad range of sources into a single database for comprehensive annotation of genomic data. Flexible report generation
summarizes and annotates results.

Demonstrated Workflow - TruSight Cancer
Library Preparation
The TruSight Cancer Oligo Box and the TruSight Rapid Capture Library
Prep Kit were used to prepare libraries. DNA samples were extracted
from blood, and 50 ng of input DNA were used for each sample
in the assay. TruSight Cancer libraries were prepared following theprotocol included in the TruSight Rapid Capture Sample Prep Guide.
Libraries were quantified and normalized before sequencing on theMiniSeq System.

Sequencing on the MiniSeq System
Sample libraries were loaded onto the MiniSeq instrument along withthe reagent cartridge and flow cell. Automated clonal amplification andpaired-end sequencing with a 2 × 100 bp-cycle read was carried out
without any further user intervention, taking 16 hours.

Data Analysis - Alignment and Variant Calling
Sequence data was analyzed by the BWA Enrichment App in
BaseSpace (Figure 8). After demultiplexing and FASTQ file generation,
the software uses the Burrows-Wheeler Aligner (BWA) to align thereads against the hg19 homo sapiens reference genome to create
BAM files. The Genome Analysis Toolkit (GATK) was then used toperform variant analysis for the target regions specified in the manifest
file. The output of GATK are VCF files, which are text files that contain
SNPs, indels, and other structural variants. Summary tables weregenerated to report on enrichment, variant calling, coverage, insertfragment length, and duplicates.

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

Figure 8: TruSight Cancer Data Analysis in BaseSpace—Summary table
generated by the BWA Enrichment App in BaseSpace. Run metrics (alignedreads, aligned bases, and enrichment) are shown.

Results and Discussion
The TruSight Cancer Sequencing Panel provides ≥ 99% of bases
covered at 20×, which provides for high confidence in variant calling(Table 1). It also enables ≥ 99% aligned reads and ≥ 77% padded readenrichment for specific and accurate sequencing (Table 1).
The Single Nucleotide Polymorphism database (dbSNP) is a
public-domain archive for a broad collection of simple genetic
polymorphisms.10 As most SNVs (single nucleotide variants) and indels(insertions or deletions) are found in the dbSNP database, TruSight
Cancer sequencing data produces highly relevant research dataand increases the discovery power for identifying new predisposition
genetic variants (Figure 9).

Application Note: Cancer Genomics
Table 1: Coverage Summary for the TruSight Cancer Sequencing Kit
Mean Region
Coverage Depth

Uniformity of Coverage(% > 0.2× mean)

Target Coverage(1×)

Target Coverage(10×)

Target Coverage(20×)

Target Coverage(50×)

11068×21465×

94.5%

99.9%

99.4%

99.1%

98.6%

95.2%

100.0%

99.6%

99.5%

3

99.1%

1355×

91.7%

99.7%

99.2%

99.0%

98.5%

4

1248×

97.4%

100.0%

99.8%

99.7%

99.4%

5

1129×

97.4%

100.0%

99.9%

99.6%

99.4%

6

1262×

91.5%

99.8%

99.3%

99.0%

98.4%

SampleAggregate summary (BWA enrichment) of a representative run on the MiniSeq System with 6 samples using the TruSight Cancer Sequencing Kit.

Figure 9: TruSight Cancer Variant Summary—The TruSight Cancer Sequencing Panel enables accurate and specific sequencing, provides high confidence in variant
calling, and increases discovery power.

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

Application Note: Cancer Genomics

Summary
The MiniSeq System delivers a clear, complete, cost-effective toolset
for germline screening of predisposition genomic variants linked tocancer. Researchers can choose proven Illumina assays developedwith expert-selected content and following industry guidelines. Or they
can work with Illumina scientists to develop their own custom panels.
With the MiniSeq System, labs of any size can examine multiple
cancer-predisposing alterations with lower cost, faster turnaroundtime, and lower tissue requirements compared to CE/Sanger
sequencing and PCR-based genotyping methods.

Learn More
To learn more about targeted cancer sequencing, visit: www.illumina.
com/applications/cancer/research/cancer-dna-sequencing/targetedcancer-seq.html.

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

Ordering Information
ProductCatalog No.

MiniSeq System

SY-420-1001MiniSeq High Output Kit (75 Cycles)

FC-420-1001

MiniSeq High Output Kit (150 Cycles)

FC-420-1002

MiniSeq High Output Kit (300 Cycles)

FC-420-1003

MiniSeq Mid Output Kit (300 Cycles)

FC-420-1004

TruSight Cancer

Catalog No.

TruSight Cancer Sequencing Panel
Includes oligos sufficient for 4 enrichments and up to48 samples

FC-121-0202

TruSight Cancer MiniSeq Bundle
Includes oligos, library prep panel, and 2 MiniSeq High
Output Kits (300 Cycles), sufficient for 48 samples

20005612

TruSight Rapid Capture (1 index, 8 samples)

FC-140-1101

TruSight Rapid Capture (2 indexes, 8 samples)

FC-140-1102

TruSight Rapid Capture (4 indexes, 16 samples)

FC-140-1103

TruSight Rapid Capture (24 indexes, 48 samples)

FC-140-1104

TruSight Rapid Capture (24 indexes, 96 samples)

FC-140-1105

TruSight Rapid Capture (96 indexes, 288 samples)

FC-140-1106

TruSeq Custom Amplicon Low Input

Catalog No.

TruSeq Custom Amplicon Low Input Kit (96 samples)

FC-134-2001

TruSeq Custom Amplicon Low Input Kit (16 samples)

FC-134-2002

TruSeq Custom Amplicon Index Kit

FC-130-1003

Application Note: Cancer Genomics

References
1.

Rahman N. Integrating germline cancer predisposition gene testing into
routine clinical practice - The Mainstreaming Cancer Genetics programme[AACR abstract 4705]. Cancer Res. 2014;74(19suppl):4705.
2. Münz M, Ruark E, Renwick A, et al. CSN and CAVA: variant annotation tools
for rapid, robust next-generation sequencing analysis in the clinical setting.
Genome Med. 2015;7(76):1-8.
3. Wimberger P, Benet-Pages A, Schubert S, et al. Identification of ERCC2 asnovel susceptibility gene for hereditary breast and ovarian cancer [ASCOabstract 1512]. J Clin Oncol. 2015;33(suppl):1512.
4. Sykorova V, Dvorakova S, Vcelak J, et al. Search for new genetic
biomarkers in poorly differentiated and anaplastic thyroid carcinomas using
next generation sequencing. Anticancer Res. 2015;35(4):2029-2036.
5. Shendrue J, and Hanlee J. Next-generation DNA sequencing. Nat Biotech.
2008;26:1135-1145.
6. Schuster SC. Next-generation sequencing transforms today’s biology. NatMethods. 2008;5(1):16-18.
7. Tuononen K, Maki-Nevala S, Sarhadi VK, et al. Comparison of targetednext-generation sequencing (NGS) and real-time PCR in the detection ofEGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded
tumor material of non-small cell lung carcinoma-superiority of NGS. Genes,Chromosomes and Cancer. 2013;52(5):503-511.
8. Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J
Biomed Biotechnol. 2012;2012:251364.
9. Ross MG, Russ C, Costello M, et al. Characterizing and measuring bias insequence data. Genome Biol. 2013;14:R51.
10. Sherry ST, Ward MH, Kholodov M, et al. dbSNP: the NCBI database ofgenetic variation. Nucleic Acids Res. 2001;29(1):308-311.

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

Application Note: Cancer Genomics

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For Research Use Only. Not for use in diagnostic procedures.
© 2016 Illumina, Inc. All rights reserved. Illumina, MiniSeq, BaseSpace, TruSight, TruSeq, DesignStudio, and the pumpkin orange color are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 770-2015-044 Current as of 21 May 2016