Data Sheet: Sequencing

TruSeq® Amplicon - Cancer Panel
Rapidly detect hundreds of critical cancer mutations with the fastest and easiest multiplexed
amplicon assay optimized for the MiSeq® system.
Highlights

Comprehensive Screening for FFPE Samples• Most relevant cancer loci in a single comprehensive panel
Sequence hundreds of cancer hotspots from selected genes
such as BRAF, KRAS, and EGFR
• Shortest time to project completion
Go from genomic DNA to fully analyzed data in two days
• Unprecedented amplicon and sample multiplexing
Generate the highest quality and most accurate data with theability to reliably detect mutations below 5% frequency

The TruSeq Amplicon - Cancer Panel provides pre-designed,
optimized oligonucleotide probes for sequencing mutational hotspots
in > 35 kilobases (kb) of target genomic sequence. Within in ahighly multiplexed, single-tube reaction, 48 genes are targeted with
212 amplicons. Table 1 contains a complete list of the oncogenes
included in the panel.
The TSACP is uniquely suited for detection of somatic mutations inFFPE samples. Prior to amplicon preparation, a simple qPCR-basedTable 1: TSACP Cancer-Related Genes

Introduction
The TruSeq Amplicon - Cancer Panel (TSACP) is a highly multiplexedtargeted resequencing assay for detecting somatic mutations
across hundreds of mutational hotspots in cancer genomes. TSACP
provides a streamlined workflow, including a quality control assay for
DNA from formalin-fixed, paraffin-embedded (FFPE) samples, simple
bead-based sample normalization, automated cluster generationnd paired-end sequencing, and on-instrument data analysis(Figure 1). Leveraging the long paired-end read capability, speed,and high data quality of the MiSeq system, entire projects can now
be accomplished in days instead of months. TSACP enables highly
sensitive mutation detection within important genes, including BRAF,KRAS, and EGFR. Mutations in these genes are linked to manycancers, including melanoma, colorectal, ovarian, and lung cancer.
The unique ability of this assay to screen precious FFPE samples forthese important variants will unlock a wealth of genomic informationfrom many tumor types.

ABL1

EGFR

GNAS

MLH1

RET

AKT1

ERBB2

HNF1A

MPL

SMAD4ALKERBB4HRAS

NOTCH1

SMARCB1

APC

FBXW7

IDH1

NPM1

SMO

ATM

FGFR1

JAK2

NRAS

SRC

BRAF

FGFR2

JAK3

PDGFRA

STK11

CDH1

FGFR3KDRPIK3CATP53

CDKN2A

FLT3KITPTEN

VHL

CSF1R

GNA11KRAS

PTPN11CTNNB1GNAQ

MET

RB1Cancer-related genes represented in the TSACP. For a full list oftarget regions, see the manifest file1 (MyIllumina login required).

Figure 1: TSACP Workflow
Pre-screen Samples

FFPE QC Kit

Assay Biochemistry

TruSeq Amplicon - Cancer Panel

Sequencing and Analysis

Automated Sequencing and Variant Calling

The TruSeq Amplicon - Cancer Panel is a comprehensive assay for examining the most relevant cancer mutations, even in challenging FFPE samples.
With the shortest time to project completion, researchers can go from genomic DNA to fully analyzed data in less than two days with the MiSeq system.

Data Sheet: Sequencing
Figure 2: FFPE Screening Improves Assay Performance
4Delta Ct

32
10-1Samples
DNA extracted from 18 FFPE samples was screened with the Illumina
FFPE QC Kit prior to amplicon prep. ∆Ct was calculated by thedifference between the Ct number of each sample and of the control
DNA (not shown). Samples that generated successful ampliconsequencing data are indicated by blue bars; those that did not areshown by purple bars. Using a cutoff value of 2, the assay outcomeof this particular library preparation was 100%.

Table 2: Specificity and Uniformity Across Samples

Sample%Specificity%Uniformity

Variant
Type% Allele
Frequency

Control

93.5

94.0

NANA

1

91.2

94.4

Indel

29.2

2

93.9

91.5

SNV

2.95

3

93.5

91.5

SNV

2.90

4

91.6

94.9

Indel

18.9

5

93.1

91.2SNV8.70

693.9

91.6

SNV

40.8

792.3

92.6

SNV37.1

890.3

93.5

SNV39.5

993.5

92.1

SNV

7.80

Nine FFPE samples representing multiple cancer types were screenedusing the TruSeq Amplicon - Cancer Panel. Insertion/deletion (indels)and single nucleotide variants (SNVs) were called with high specificity
and uniformity.

DNA quality control step allows sample pre-screening. This up front
sample quality control step accurately predicts assay performancefrom fragmented DNA, reducing time and reagent costs (Figure 2).
The TruSeq Amplicon assay chemistry begins with hybridizationof the pre-mixed, optimized oligonucleotide probes upstream anddownstream of the regions of interest. Each probe includes a target
capture sequence and an adapter sequence used in a subsequent
amplification reaction. A proprietary extension-ligation reaction
extends across the region of interest, followed by ligation to unite thetwo probes. This creates a new template strand and gives the assay
excellent specificity. Extension-ligation templates are PCR amplified
and two unique sample-specific indices are incorporated. The final
reaction product contains amplicons that are ready for sequencing.
An integrated bead-based normalization procedure allows simple
volumetric library pooling, avoiding laborious qPCR-based
quantification methods. Pooled amplicon libraries can be loaded
directly onto the MiSeq system without additional processing.

Unprecedented Multiplexing, Excellent Data
With the ability to combine hundreds of amplicons per sample
and up to 96 samples per MiSeq run, the TSACP provides anunprecedented level of sample multiplexing, while providing
excellent specificity and uniformity. An example TSACP experiment
was performed following the workflow described in Figure 1 using
2 × 150 bp MiSeq reads. Representative uniformity data, withpercentage of bases at least 0.2× the mean sequencing depth,
are shown for nine cancer samples plus one control (Table 2).
Specificity values, defined as the percent of sequenced bases
passing filter and aligning to the desired target regions, are also
shown in Table 2. Excellent coverage uniformity and specificity
were obtained, with > 91% of bases covered at > 0.2× of the mean
coverage and > 90% of reads on target.
TSACP maintains excellent coverage, even at high read depths.
In another experiment (Figure 3A), two runs with twenty pooled
samples gave > 90% uniformity and specificity. A single sample
sequenced at > 27,000× provided similar results, demonstrating
consistently high specificity and uniformity at extremely high readdepths. In Figure 3B, one sample chosen from the Run 1 poolof twenty samples showed > 95% of bases are covered at 500×depth, enabling low frequency variant detection. With highly
specific amplicon targeting, combined with the most accurate
TruSeq sequencing chemistry, TSACP lets researchers detectmutations below 5% frequency with high confidence (Figure 4).

Summary
In a single experiment, researchers can access the most content
of any commercially available amplicon cancer panel, including
critical cancer-related genes such as BRAF, KRAS, and EGFR.
The TruSeq Amplicon - Cancer Panel delivers confidence andsimplicity with unparalleled specificity and uniformity.

Data Sheet: Sequencing
Figure 3: High Specificity and Uniformity

Figure 4: Low Frequency Variant Detection

AARun 1Run 2Run 3

Number ofSamples

2020

1MiSeq Output(Gb)

1.8

2.0

1.9

Mean Depth

1,366

1,410

27,01993.7

94.5

90.7% Uniformity

96.2

96.2

94.4

Amplicon
Dropouts*

000

2000
1500
1000
500
01000

2000

3000

4000

5000

Position along Amplicon

B
100*Dropouts are defined as amplicons having no sequencing coveragewhen the sample achieves a mean sequencing depth of > 1000×.

B

7550

The TSACP
25 detected a 2.95% variant (A to T SNV) in the BRAF gene froma colon cancer sample, with a variant score of Q40, or 99.99% confidence(6,048× coverage).

100

Percentage of Bases Covered

2500

0Variant Quality Score% Specificity

3000

Fold Coverage

Parameter0800Reference
1.

60
40
2000200

400

600
Read Depth

800

1000

A Three TSACP experiments- two with 20 pooled samples and one
with a single sample. Excellent assay performance is shown, with > 90%
specificity and uniformity, with zero dropouts across the pooled Runs 1
and 2. Similar output, specificity, and uniformity are obtained with thesingle sample (Run 3), sequenced at an extremely high depth.

B Coverage plot from one sample included in Run 1. The percent ofbases covered plotted against the read depth shows that > 80% of bases
are covered at 1000×, and ~95% are covered at 500× read depth.

500 1000 1500 2000 2500 3000 3500 4000 4500 5000
Position along Amplicon

TruSeq Amplicon Cancer Panel Manifest File at MyIllumina.com

Data Sheet: Sequencing
TSACP Performance Specifications
Parameter

Details

Panel Size

35 kb

Content

212 amplicons

Amplicon Size

170–190 bp

DNA Input

250 ngAssay Time< 7 hrs, 2.5 hrs hands-on

Enrichment Specificity> 85%Coverage Uniformity> 85% at 0.2× mean

Dropouts*0*Dropouts are defined as amplicons having no sequencing coveragewhen the sample achieves a mean sequencing depth of > 1000×.

Ordering Information
Kit

Catalog No.

Illumina FFPE QC Kit

WG-321-1001

TruSeq Amplicon - Cancer Panel(96 samples)

FC-130-1008

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

FC-130-1003

MiSeq Reagent Kit (300-cycles - PE)

MS-102-2002

Optional: TruSeq Index Plate Fixture
and Collar Kit (2 Each)

FC-130-1007

Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
FOR RESEARCH USE ONLY
© 2012, 2014 Illumina, Inc. All rights reserved.
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Pub. No. 770-2012-009 Current as of 24 July 2015