Extracting useful information from liquid biopsy samples using NGS Circulating nucleic acids have emerged as important biomarkers for cancer diagnostics and the detection of various clinical conditions. Utilizing liquid biopsy as an alternative to invasive and at times inaccessible tumor biopsy may enable access to critical information about disease composition and progression. Translational genomics research is utilizing circulating, cell-free DNA (cfDNA) to monitor disease based on specific tumor profiles. To research this valuable information, it is critical to use next-generation sequencing (NGS) to detect variants in low levels of cfDNA from blood samples.

Characteristics of cfDNA: Isolation and Quality Assessment

Isolation of cfDNA from liquid biopsy samples requires careful and timely extraction of cfDNA from plasma within 72 hours. Subsequently, it is important to perform a qPCR-based method such as ALU repeat assay to accurately determine the concentration and quality of sample DNA (Hao T. et. al. 2014).

9 6.4 - 20 0.32

The ALU repeat assay was used to evaluate the integrity score for n=10 cfDNA samples. For each sample, 10 mL of blood was collected in Streck BCT® tubes and plasma was separated within 24 hours. cfDNA was isolated on a PerkinElmer chemagic™ 360 system. Note, integrity score of cfDNA ratio range is 0.3-0.5 and cellular DNA score is at 1.0.

Number of Samples Range of Total Yield (ng) Median Integrity Score (ALU247/115)

Average Peak Size on the Bioanalyzer

In addition to the qPCR-based method to determine relative abundance of amplifiable fragments, characterization of cfDNA from plasma using electrophoresis-based methods is important to confirm a defining peak at ~165 bp.

The coverage plot for PCR-free libraries made from 100 ng of high molecular weight genomic DNA (top-left) compared to 10 ng cfDNA (top-right) indicates even coverage by Accel-NGS® 2S kits. The GC bias inherent within the cfDNA sample is responsible for reduced representation of AT-rich sequences in this library. It is important to note, PCR-amplification could contribute to a shift in coverage (bottom-left). Insert size for all reads in cfDNA is consistent with the expected range of 165 bp (bottom-right).

The single-tube workflow includes two brief incubations to generate the multiplex amplicon targets and add a unique combination of Illumina-compatible indexed adapters, creating up to 96 uniquely-indexed libraries for multiplexing on a single sequencing run.

Comparison of cfDNA to High Molecular Weight Cellular DNA

HMW100 ng PCR-free

10 ng + PCR

cfDNA 10 ng PCR-free

Insert Size for All Reads

cfDNA

Liquid Biopsy Application Note

Accel-Amplicon™ Technology: An All-in-One Amplicon Solution

Targeted Sequencing of Clinical Oncology Research SamplesLongitudinal endometrial study by John Martignetti and Peter Dottino, Icahn School of Medicine at Mount Sinai, for tracking of somatic mutations in tumor/normal pairs in addition to collected cfDNA samples from two time-points.

The libraries were sequenced to an average coverage of 8000X.

CHR:POS

0 0 0.6 1.02:212244718

0 0100 1.9 1.612:253616460 0100 0.5 1.112:406886950 0100 0.7 2.012:115108136

% A Background

% B Background

% C Background

1% A into 10 ng B

1% A into 10 ng C

0 012:25361074 1.6 1.90 012:25361142 1.1 0.9

23 15,000 97.5 + 1.4% 97.8 + 1.4%

The Accel-Amplicon 56G Oncology Panel was used to assess the germline and somatic variant profile of 23 tumor/normal paired samples. Following qPCR input quantification using the ALU115 assay, 10 ng of input DNA was used per sample and libraries were sequenced using MiSeq® v2 chemistry. Bases on target is the percentage of aligned bases on target and coverage uniformity is the percentage of amplicons covered greater than 20% of the mean.

Number of Samples Average Coverage Coverage Uniformity On Target

The representative table shows variant profile in samples from normal, tumor and corresponding cfDNA collected from two time-points. The Accel-Amplicon 56G Oncology Panel was used to prepare libraries from 10 ng of input DNA per sample. The libraries were sequenced using MiSeq v2 chemistry.

Sample Ref Tumor(%) cfDNA t=1(%)

cfDNA t=2(%)Gene Alt Normal

1 G 3.5 0.8 3.2EGFR p.Ser715Ile T Not Detected2 PIK3CA p.His1047Arg CA C Not Detected 16.0 1.0 5.03 TP53 p.Leu252fs TGATGGTGA C Not Detected 28.0 0.4 78.0

Limit of Detection of Pre-Verified cfDNA variants in Pan-Cancer genes determined by Accel-NGS 2S Hyb DNA Library KitTo assess the limit of detection of the Accel-NGS 2S Hyb kit, DNA samples from two individuals with different ethnic backgrounds were used to prepare libraries. The DNA from Sample A was pre-screened for point mutations using the 56G primer sub-set of the Accel-Amplicon 56G Oncology Panel v2 and was spiked into Samples B and C at 1% final concentration. The libraries were prepared and hybridized to IDT xGen® Lockdown® Pan-Cancer probes and SNPs were detected within this panel.

To determine if SNPs present at 1% allele frequency could be detected, 1% of cfDNA sample (A) with a unique ethnic background was spiked into two 10 ng cfDNA samples (B and C) of different ethnic backgrounds. SNPs at 100% frequency unique to sample A could be detected near the expected 1% allele frequency in the spiked-in samples.

1% Spike in

C B

1% Spike in

A

100100100

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© 2017, Swift Biosciences, Inc. The Swift logo and Accel-Amplicon are trademarks and Accel-NGS is a registered trademark of Swift Biosciences. Chemagic is a trademark of PerkinElmer Inc. MiSeq is a registered trademark of Illumina, Inc. xGen and Lockdown are registered trademarks of Integrated DNA Technologies, Inc. 17-1766, 10/17

Low Sequencing Read Duplication Rates from Low Input cfDNALibraries generated from the Accel-NGS 2S Hyb DNA Library Kit utilize inputs as low as 1-10 ng cfDNA with low percent duplication rates without compromising coverage as shown in the table below. In this experiment, cfDNA was extracted using the PerkinElmer chemagic 360. The libraries were generated from as little as 1 ng and 10 ng of cfDNA and hybridized using IDT xGen Lockdown Pan-Cancer Panel probes.

Input % Aligned % Duplication Mean Bait Coverage

% Covered ≥ 1X

% Covered ≥ 20X

% on Target

10 ng

98 0.1 50x 99 92 87

98 0.1 49x 99 95 85

98 0.1 50x 99 93 88

1 ng

97 17 37x 99 87 8597 15 39x 99 88 8697 16 38x 99 90 8397 11 41x 99 88 86

Visit www.swiftbiosci.com for easy ordering.

Ordering InformationProduct Name Reactions Catalog No.

Accel-Amplicon 56G Oncology Panel v2 48 AL-56248

Accel-Amolicon Comprehensive TP53 Panel 48 AL-53048

Accel-Amplicon EGFR Pathway Panel 48 AL-51048

Accel-Amplicon Sample_ID Panel 48 AL-50048

Accel-NGS 2S Hyb DNA Library Kit 24 23024

Accel-NGS 2S Hyb DNA Library Kit 96 23096

PEG NaCl Solution 96 90196

Low EDTA TE 96 90296