Prep are Lib rary | Seq uence | Analyze Data

AmpliSeq™ for Illumina Oncology Panels on theiSeq™ 100 System
Proven amplicon chemistry meets industry-leading sequencing technology for cancer-related
targeted resequencing applications.
HighlightslHighly accessible sequencing solution
Low cost, easy to implement, NGS systeml

Relevant gene content for cancer researchExpertly selected, cancer-focused contentlHigh analytical sensitivity with low DNA input
Accurate variant detection as low as 5% allele frequency,using 1–100 ng input DNA or RNAlValidated workflow for easy implementation
Streamlined, comprehensive workflow with minimal hands-ontime and automatic variant calling

Introduction
Next-generation sequencing (NGS) has transformed cancer research,
enabling variant detection in numerous genes in a single assay. By
focusing on genes related to a particular disease or pathway, targeted
sequencing offers the ability to conduct NGS on smaller platforms,
while also providing the advantage of higher depth of coverage ontargeted genes. The increased analytical sensitivity that comes withhigher coverage may be particularly important for detection of lowfrequency variants in heterogenous tumor tissues.
The iSeq 100 System was designed to execute small-scale NGS
applications. With immediate access to run various types ofexperiments on the benchtop, the iSeq 100 System provides
autonomy and flexibility to small laboratories. The system is well suitedfor targeted sequencing applications that focus on diseaserelated genes.

This application note demonstrates the performance of the iSeq 100
System with four distinct targeted resequencing panels, each
designed with proven amplicon chemistry to focus on different aspectsof cancer research:
AmpliSeq for Illumina Cancer Hotspot Panel v2 is designed todetect somatic mutations across the hotspot regions of 50 genes withknown associations to cancer. Starting with as little as 1 ng DNA fromhigh-quality DNA, or DNA from formalin-fixed, paraffin-embedded(FFPE) tissues, the panel enables analytically sensitive mutation
detection within multiple cancer types, including lung, colon, breast,ovarian, melanoma, and prostate. The low-input requirement enables
use with precious samples.
AmpliSeq for Illumina Focus Panel was developed for biomarker
analysis in both DNA and RNA concurrently. The panel enables highanalytical sensitivity for mutation detection in 52 genes with known
relevance to solid tumors, including lung, colon, breast, ovarian,melanoma, and prostate. The Focus Panel allows quick and accurate
assessment of genomic variation for translational and clinicalresearch studies.
AmpliSeq for Illumina BRCA Panel is a DNA sequencing panel that
detects somatic and germline mutations across all exonic regions andthe flanking intronic sequences of the BRCA1 and BRCA2 genes.
BRCA1 and BRCA2 are tumor suppressor genes that have been
implicated in an increased risk for breast and ovarian cancers whencarrying specific mutations.1 Understanding BRCA status within thetumor may be a factor when researching potential therapies.2

Figure 1: Simple, integrated workflow—Illumina offers integrated library preparation, sequencing, and automated data analysis, creating a streamlined workflow thatcan go from DNA/RNA input to data in 25 hours.

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

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Prep are Lib rary | Seq uence | Analyze DataAmpliSeq for Illumina Immune Response Panel provides detectionof biomarkers associated with different leukocyte subsets, antigen
presentation, checkpoint pathways, and tumor progression. Startingwith 1-100 ng input RNA, the Immune Response Panel enables quick
and accurate analysis of 395 genes involved in tumor–immune systeminteractions, and evaluation of biomarkers that can be used fordeveloping new immunotherapy treatments.

High operational efficiency
The integrated workflow (Figure 1) includes AmpliSeq for Illumina PCRbased library preparation, Illumina sequencing by synthesis (SBS) NGS
technology, and automated analysis with BaseSpace™ Sequence
Hub or Local Run Manager.

Reproducibility
The iSeq 100 System was designed to provide affordable access toNGS, while also providing the recommended depth of coverage forhigh-confidence variant calling (≥ 500× mean coverage). To
demonstrate high performance of AmpliSeq chemistry on the iSeq
100 System, the same DNA sample was analyzed on the iSeq 100,MiniSeq™, and MiSeq™ Systems for each of the AmpliSeq for
Illumina oncology panels (Table 1). For gene expression analysis
using the AmpliSeq for Illumina Immune Response assay,
reproducibility between samples was demonstrated by read countcomparison performed by the RNA Amplicon App (Figure 2).

The Ampliseq for Illumina panels were optimized to provide highquality, accurate data with low requirements for sample input. The
library prep workflow requires less than 1.5 hours of hands-on-time,
providing an efficient, comprehensive assay. The assay is also
designed to obtain high coverage of targeted genes using as little as 1
ng of nucleic acid input. The low input requirement, combined withthe ability to analyze hundreds of gene simultaneously, can be asignificant benefit for labs with limited samples.

Simple, comprehensive workflow
The AmpliSeq for Illumina workflow is comprehensive and can be easily
integrated into laboratory operations, going from DNA or RNA to data inapproximately 25 hours (Figure 1). AmpliSeq for Illumina PCR-based
library preparation can be completed in as little as 5 hours. Following
library prep, sequencing with the iSeq 100 System is highly automated,
requiring ~ 5 minutes hands-on time for loading of samples into the flow
cell and starting the run. Data can be analyzed on the instrument withLocal Run Manager software, or streamed automatically toBaseSpace Sequence Hub for more advanced analysis using the DNA
Amplicon App or the RNA Amplicon App.
Figure 2: Reproducibility with the AmpliSeq for Illumina Immune Response
Panel—Reproducibility of gene expression is confirmed using the RNA Amplicon
App in BaseSpace Sequence Hub. A read count comparison for two replicates ofthe same lung tumor sample is shown.

Table 1: DNA analysis reproducibility between the iSeq 100, MiSeq, and MiniSeq Systems
AmpliSeq panel

Cancer Hotspot v2

Focus

BRCASystem

SNV precision

SNV recall

Uniformity

Specificity

Percent ontarget/aligned aiSeq 100

1.0000 +/-0

1.0000 +/-0

100 +/-0

93.5 +/-0.5

84.7 +/-0.7

3230

MiniSeq1.00001.000099.0

94.4

85.413950

Mean coverage

MiSeq

0.98611.0000

100

94.7

85.9

7190

iSeq 100

1.0000 +/-00.9458 +/-0.017

99.3 +/-0.2

89.5 +/-0.7

81.5 +/-0.91640

MiniSeq

0.92861.000098.9

91.583.17120

MiSeq

0.94001.000099.091.8

83.62890

iSeq 100

1.0000 +/-00.9002 +/-0.013

100 +/-092.2 +/-0.2

81.3 +/-0.35500

MiniSeq

0.89101.000010092.8

81.922900

MiSeq

0.89101.0000100

93.1

82.39860

One replicate was analyzed on the MiSeq and MiniSeq Systems. The same sample was then analyzed with multiple replicates on the iSeq 100 System (n = 6 forCancer Hotspot v2, n = 3 for Focus, and n = 3 for BRCA) with +/- values indicating standard deviation between replicates.
a. Percent on-target is the ratio of number of bases within a target region to total number of bases output by the sequencer.

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

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Prep are Lib rary | Seq uence | Analyze Data

Reliable, accurate performance

Learn more

Combining the high coverage of targeted sequencing with theperformance of the iSeq 100 System provides cancer researchers withhigh confidence for accurate variant detection. Using AmpliSeq
chemistry to enrich for relevant targets, followed by Illumina
sequencing chemistry, variant calling as low as 5% is possible with truevariants distinguishable from background noise (Figure 3).

To learn more about the iSeq 100 System, visit
www.illumina.com/systems/sequencing-platforms/iseq.html
To learn more about AmpliSeq for Illumina sequencing panels, visit
www.illumina.com/products/by-brand/ampliseq.html

Ordering information
Order AmpliSeq for Illumina products online at www.illumina.com.

Figure 3: Distribution of observed variant frequencies for DNA with lowfrequency variants versus blank DNA—A DNA standard (HD729) with ninesingle-nucleotide variants of expected variant allele frequencies (VAF) of 4-5% wasanalyzed with the AmpliSeq for Illumina Cancer Hotspot Panel v2. Shown are thenumber of times these variants were detected at different frequencies, using boththe HD729 DNA standard and a blank DNA standard (NA12877) known to lackthese variants. There are 48 observations for each variant, half from NA12877 andhalf from HD729. Each DNA standard was sequenced using two runs of 12
replicates each.

User-friendly analysis and reporting

System and reagent kits

Catalog no.

iSeq 100 Sequencing System

20021532

iSeq 100 E1 Reagent

20021533

iSeq 100 E1 Reagent 4 Pack

20021534

Library prep

Catalog no.

AmpliSeq for Illumina Cancer Hotspot Panel

20019161

AmpliSeq for Illumina Focus Panel

20019164

AmpliSeq for Illumina BRCA Panel

20019168

AmpliSeq for Illumina Immune Response Panel

20019169

AmpliSeq for Illumina Library PLUS (24 reactions)

20019101

AmpliSeq for Illumina Library PLUS (96 reactions)

20019102

AmpliSeq for Illumina Library PLUS (384 reactions)

20019103

AmpliSeq for Illumina CD Indexes Set A (96 indexes, 96samples)

20019105

References1.

BRCA1 and BRCA2: Cancer Risk and Genetic Testing Fact Sheet - NationalCancer Institute. www.cancer.gov/about-cancer/causesprevention/genetics/brca-fact-sheet#q1. Accessed November 29, 2017.

2.

Approved Drugs > FDA approves olaparib tablets for maintenance treatment
in ovarian cancer.
www.fda.gov/drugs/informationondrugs/approveddrugs/ucm572143.htm.
Accessed January 12, 2018.

Illumina sequencing systems offer the option to connect toBaseSpace Sequence Hub, the Illumina genomics computingenvironment for sequencing data analysis and management.
Researchers can securely store, analyze, archive, and sharesequencing data. The DNA Amplicon App and the RNA Amplicon
App are designed to perform numerous analyses at the click of abutton, such as variant calling, differential expression analysis, orsample comparison (Figure 2). Raw data outputs are provided, as wellas user-friendly, focused reports.

Summary
With Illumina SBS chemistry and the AmpliSeq for Illumina oncology
panels, the iSeq 100 System provides flexible, affordable access toexpert-defined content for comprehensive investigation of cancerrelated targets in the genome and transcriptome. The optimized
amplicon sets provides comprehensive coverage of regions known tobe frequently mutated in several areas of cancer research. WithAmpliSeq for Illumina panels, hundreds of genes can be analyzed in asingle assay. Combined with the iSeq 100 System, these NGS-based
solutions are highly accessible to small independent laboratories.

Illumina, Inc. • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com© 2018 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information,
see www.illumina.com/company/legal.html. Pub. No. 770-2017-035-A QB 5625

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

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