A Guide to NGS Applications Using cfDNA SamplesOverview__Liquid+Biopsy... · Introducing the Swift...

A Guide to NGS Applications Using

cfDNA Samples

Swift Snapshot

• Swift Biosciences

– Develops and commercializes NGS

library kits

– Compatible with all NGS platforms

• Illumina®

• Ion Torrent™

• Pacific Biosciences

– Developed on broad IP portfolio

• Leader in molecular biology

– Fundamentally different from all commercially available kits

– Enables better performance across an expanding set of applications

• Whole genome sequencing and hybridization enrichment

• Bisulfite sequencing (WGBS)

• Amplicon sequencing for screening applications

Downtown Ann Arbor, MI

Introducing the Swift Portfolio for cfDNA

Product Application Technical Advantage Benefits

Accel-NGS® 2S Liquid biopsy for hyb

capture or WGS

• 90% library conversion rate for

cfDNA

PCR-free input down

to 10 ng

Accel-

Amplicon™

Liquid biopsy for somatic

mutations

• Single tube assay (< 2 hrs.)

• > 100s of primer pairs in one

tube

10 ng input for 1-5%

mutation detection

Accel-NGS

Methyl-Seq

Methylation sequencing

using bisulfite-converted

DNA

• Generates a library post-

bisulfite treatment

• 10-100X higher recovery

Sensitive detection

of hypomethylation

in cancer

Accel-NGS 1S

Plus

Compatible with single-

stranded DNA

• Allows the detection of

damaged or nicked cfDNA

Conversion of

ssDNA and dsDNA

Outline

• Introduction to cfDNA

– Application of liquid biopsy

– cfDNA extraction from plasma

– Low DNA input requirements

– Concentration and integrity of cfDNA

• Accel-NGS® 2S DNA Library Kits

– 90% library conversion for cfDNA samples

– PCR-free library prep from 10 ng of cfDNA

– Compatibility with multiple hybridization capture technologies

• Accel-Amplicon™ Panels

– Performance with Horizon’s Quantitative Multiplex Reference Standard

– Liquid biopsy samples experimental design

• Accel-NGS Methyl-Seq DNA Library Kits

– Out performs other kits

– Detecting genome-wide hypomethylation

Introduction to cfDNA

Bettegowda, C., et al Sci. Trans. Med., (2014) vol6

Limitations for Using cfDNA to Detect Cancer

Burden

• Over 75% of samples had

detectable cancer mutations from

17 different cancer types

• Lower levels of detection correlated

with tumor types that had some type

of physical barrier (blood brain)

• Frequency of detection of cancer

burden within cfDNA increased with

disease severity

Stage I II III IV

Bettegowda, C., et al Sci. Trans. Med., (2014) vol6

cfDNA Extraction from Plasma

• Validated blood collection into Streck Cell-Free DNA BCT® that preserves

cfDNA and prevents lymphocyte lysis

• Validated manual extraction using Qiagen QIAamp® circulating nucleic acid kit

and automated extraction using Perkin Elmer chemagicTM system

Sample mL of Plasma ng/mL of Plasma

1 2.5 6.3

2 5.0 4.3

3 3.8 4.4

4 4.0 10.5

5 3.0 6.7

6 4.5 7.1

7 4.5 2.6

8 4.5 2.9

Normal (5) Avg. 3.5 Avg. 3.5

Average results obtained from manual extraction

Low DNA Input Requires a High Conversion

Rate

Conversion Rates

Library Molecules

The number of genome copies available for detection by sequencing is impacted

by low input DNA quantities and the conversion rate of DNA fragments

constructed into library molecules.

cfDNA inputs range from 1-30 ng input

1 ng = 330 genome copies

30 ng = 9,900 genome copies

Accel-NGS® 2S Kits can achieve 90% library conversion for

cfDNA samples.

Achieving a 1% Limit of Detection from Low

DNA Input

cfDNA inputs range from 1-30 ng extracted from 10 mL blood draw

1 ng = 330 genome copies

1% allele frequency = 3 mutant copies





Swift’s Accel-NGS® 2S Kits and Accel-Amplicon™ Panels can

achieve 1% mutant allele detection from 10 ng cfDNA input.

Concentration and Integrity of cfDNA

• cfDNA is ~ 165 bp in size

• High molecular weight cellular DNA can contaminate cfDNA samples

• Swift library kits include primers for a qPCR assay designed for Alu

repeat sequences from human gDNA at two different sizes

For more information, see our Instructional Manual: “Quantification and

Quality Assessment of Human DNA Samples”

qPCR quantification

(Alu Amplicons)

115 bp

247 bp

Total DNA Concentration

HMW Cellular DNA Concentration

(cfDNA + HMW Cellular DNA Contamination)

DNA Integrity Index = Alu247/Alu115

T.B. Hao in the British Journal of Cancer (2014) 111, 1482-1489

Accel-NGS® 2S DNA

Library Kits

Technology for dsDNA

Accel-NGS® 2S Plus Library Kit:How Improved Performance Is Achieved

Dephosphorylation of both the 3’ and 5’ termini

• Reduces chimera formation

• Improves ligation

Repair both the 3’ and 5’ bases

• Eliminating any damaged bases

Sequential ligation of P7 and P5 adapters

prevents adapter dimer formation

• Eliminates need for adapter titration

• P7 adapter is attached to the repaired 3’ termini

• P5 adapter ligation to the repaired 5’ termini

Optional PCR

• PCR-free: inputs down to 10 ng

• With PCR: inputs down to 10 pg

Equivalent adapter ligation efficiency at all input levels

PCR-free Library Prep from 10 ng of cfDNA

• Up to 90% library conversion:

• 10 ng input (91 fmol) = PCR-free library yield of 4.3 nM (82 fmol)

• Sample integrity: qPCR Alu247/Alu115 ratio = 0.4

• PCR-free sequencing eliminates read duplicates and provides more uniform

coverage of AT-/GC-rich sequences

# PE Reads % Mapped to Human

Genome

Estimated Library Size Chimera Dimers

69,853,401 99.5% 1.2 X 109 2.6% 0.02%

Size Distribution

Median

insert size:

169 bp

0.010

0.100

Nu

mb

er

of

rea

ds

pe

r b

as

e a

t e

ac

h c

hro

mo

so

me

no

rma

lize

d

to m

ap

ab

ilit

y

chromosomes

Mapability and read length, Li W. Freudenberg, J.; Front Genet 2014 Nov

Superior Coverage Uniformity from cfDNA

WGS

Two cfDNA samples were prepared PCR-free with the Accel-NGS® 2S PCR-free DNA Library Kit

and sequenced on a HiSeq® 2500 Rapid Run with v3 chemistry. Analysis was performed using a

custom pipeline using BWA as the aligner and enrichment metrics were collected using Picard tools.

Sequencing Metrics Sample A Sample B

Yield 1.8 nM 3.8 nM

Total Reads 238,230,712 254,136,768

% Aligned 99.4% 99.4%

Average Genome Coverage 14.6X 15.6X

% Genome Missing 1.9% 2.0%

% Genome Covered ≥ 5X 99.6% 99.7%



% Duplication 0.04% 0.08%

Median Insert Size 172 bp 168 bp

Accel-NGS® 2S Hyb Library Kit:Compatibility with Multiple Hyb Capture Technologies

Compatible with cfDNA samples

Simple with bead protocol

Broad input range 10 pg-1 µg

Repairs both 5’ and 3’ termini to enhance ligation

efficiency

Increased library complexity

Balanced coverage of AT-/GC-rich regions.

A variety of indexing kits allow for compatibility with

multiple hybridization capture technologies: Agilent

SureSelectXT and SureSelectXT2, NimbleGen™

SeqCap™ EZ, and IDT xGen® Lockdown® Probe

Limit of Detection for Accel-NGS® 2S Hyb Kit

• To assess the limit of detection of Accel-NGS 2S Hyb, 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 Accel-

Amplicon™ 56G Oncology Panel.

• Sample A was spiked into Samples B and C at 1% final concentration.

• Once libraries were prepared, they were hybridized to IDT xGen® Lockdown®

Pan-Cancer probes and SNPs were detected within this panel.

cfDNA Spike-in to Determine Limit of Detection

for the Accel-NGS® 2S Hyb Kit

• 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. Libraries were

sequenced to an average coverage of 8000X.

cfDNA input with xGen® Pan-Cancer Panel

CHR: POS%A

Background

%B

Background

% C

Background

1% A into

10 ng B

1% A into

10 ng C

2: 212244718 100 0 0 0.6 1.0

12: 25361074 100 0 0 1.6 1.9

12: 25361142 100 0 0 1.1 0.9

12: 25361646 100 0 0 1.9 1.6

12: 40688695 100 0 0 0.5 1.1

12:115108136 100 0 0 0.7 2.0

Accel-NGS 2S® Hyb Kit:Low Duplication Rates from Low Input cfDNA

• cfDNA extracted using the PerkinElmer chemagicTM 360

• Libraries generated from as little as 1 ng and 10 ng of cfDNA

• IDT xGen® Lockdown® Pan-Cancer Panel

Hybridization Enrichment of 1 ng cfDNA

Swift Libraries

• Pooled normal human plasma DNA (Innovative Research Inc.)

• 1 ng input, 10 PCR cycles, and library yield ~1 µg

HGSC Standard Library

• NA12878, 500 ng input

Developed by Donna Muzny,

Harsha Doddapaneni, Qingchang

Meng, and Richard Gibbs

Coverage

Library Input Total

MB

Dup.

Rate

Avg.

Coverage

Reads on Hit

Target/Buffer

Targets

Hit

Buffer

Aligned

Reads

Target

Aligned

Reads

> 1X > 20X > 40X

Swift

(Avg. 4

Samples)

1 ng 10,325 20% 93X 55.94% 99.75% 9.79% 46.15% 99.63% 96.53% 89.15%

HGSC

Standard

Library

500 ng 12,925 3% 152X 76.44% 99.65% 14.84% 61.60% 99.35% 97.61% 95.44%

Accel-Amplicon™ Panels

Targeted NGS Panels

Accel-Amplicon™ Technology

Simple, Single-Tube Workflow

10 ng of input from genomic DNA and cfDNA was used to generate libraries with the

Accel-Amplicon 56G Oncology Panel. The coverage uniformity, as the percentage of the

bases covered at least 20%, 30%, 40%, or 50% of the average depth, was determined

across two sample types. The percentage of reads on target was > 95% for all sample

types.

56G Coverage Uniformity Across

Sample Types

A complete solution offering primer pairs, indexing, and

sequencing adapters all in a single kit.

75

80

85

90

95

100

gDNA cfDNA

Co

vera

ge u

nif

orm

ity

0.2x mean 0.3x mean 0.4x mean 0.5x mean

Genomic DNA

Multiplex PCR

90 minutes

Indexing Step

20 minutes

+

Dual-Indexed Amplicon Libraries

Accel-Amplicon™: Available Panels

Targeted sequencing to screen for either germline or somatic variants

Amplicon Panel Description Single Tube

Comprehensive TP53 Comprehensive coverage of all exons ✔

56G Oncology Panel Hotspot coverage of 56 cancer-related genes ✔

EGFR Pathway Panel Hotspot coverage of 4 EGFR pathway genes ✔

Sample_ID Panel 95 high MAF SNPs to generate a unique genetic

fingerprint for every sample

✔

Custom Panels Customer-driven content ✔

Coming Soon:

BRCA1 & BRCA2 Comprehensive coverage of all exons ✔

CFTR Complete exonic coverage of coding regions ✔

Pharmacogenomics 170 genes involved in drug metabolism ✔

Accel-Amplicon™ 56G Oncology Panel v2

• Single-tube oncology panel with 2-hour workflow

• 263 amplicons sized 92-184 bp (avg. 138 bp) for compatibility with cfDNA and FFPE

• 104 Sample_ID amplicons spiked in at a low percentage (4%) for genetic

fingerprinting

• Coverage of hotspots and contiguous regions of 56 clinically-relevant, oncology-

related genes

• Total target size of 23.7 kbTable of Genes and Number of Amplicons Per Gene

ABL1 5 CSF1R 2 FBXW7 6 GNAS 2 KIT 14 NPM1 1 STK11 5

AKT1 2 CTNNB1 1 FGFR1 2 HNF1A 4 KRAS 3 NRAS 3 SMAD4 10

ALK 2 DDR2 1 FGFR2 4 HRAS 2 MAP2K1 5 PDGFRA 4 SMARCB1 4

APC 9 DNMT3A 1 FGFR3 6 IDH1 1 MET 6 PIK3CA 11 SMO 5

ATM 19 EGFR 9 FLT3 4 IDH2 2 MLH1 1 PTEN 14 SRC 1

BRAF 2 ERBB2 4 FOXL2 1 JAK2 2 MPL 1 PTPN11 2 TP53 21

CDH1 3 ERBB4 8 GNA11 2 JAK3 3 MSH6 4 RB1 12 TSC1 1

CDKN2A 2 EZH2 1 GNAQ 2 KDR 9 NOTCH1 3 RET 6 VHL 3

█ Contiguous, overlapping coverage is included for APC, ATM, EGFR, FBXW7, FGFR3,

HNF1A, KIT, MSH6, PIK3CA, PTEN, SMAD4, and TP53.

█ Comprehensive coding exon coverage is included for TP53.

56G Performance with Horizon’s Quantitative

Multiplex Reference Standard

The Accel-Amplicon™ 56G Oncology Panel consistently detected validated variants at the expected frequency in replicates by five different users

from 10 ng of the Horizon Diagnostics Quantitative Multiplex DNA Reference Standards HD701. The variants were called by LoFreq 2.1.1

(Genome Institute of Singapore) and GATK HaplotypeCaller (Broad Institute). When examining sporadic variants among the 10 replicates, the

majority of background variants were present at less than 0.6%. No sporadic variants greater than 0.6% were detected.

Gene AA Change CHR POS REF ALT Expected Allele

Frequency

Detected Allele

Frequency (N=10)

Standard

Deviation

EGFR G719S 7 55241707 G A 24.5 23.8 1.5

PIK3CA H1047R 3 178952085 A G 17.5 17.5 1.3

KRAS G13D 12 25398281 C T 15.0 15.0 1.8

NRAS Q61K 1 115256530 G T 12.5 13.4 1.2

BRAF V600E 7 140453136 A T 10.5 9.9 0.3

KIT D816V 4 55599321 A T 10.0 10.3 1.1

PIK3CA E545K 3 178936091 G A 9.0 8.5 1.1

KRAS G12D 12 25398284 C T 6.0 6.6 1.2

EGFR L858R 7 55259515 T G 3.0 2.7 0.5

EGFR ΔE746-A750 7 55242465-

55242479

Del15bp 2.0 1.4 0.5

EGFR T790M 7 55249071 C T 1.0 1.0 0.3

Accel-Amplicon™ Sample_ID Panel

• SNP Specific Sample_ID Panel

– Generate a genetic fingerprint to track samples within and between studies

– Minimum 1 in 90k discrimination between samples when using a subset of 24

of the 104 amplicons

– Based upon the Genome Medicine publication: Pengelly, RJ et al (2013)

• http://www.genomemedicine.com/content/5/9/89

• 104 Total Amplicons

– 95 for exonic SNPs (all 95 SNPs are covered by most exome panels)

– 9 specific sex ID amplicons

• Can be run as stand-alone panel or combined with

Accel-Amplicon gene panels

– Verification that multiple samples are derived from the same individuals

– When analyzing distinct samples from the same individual (i.e., plasma,

metastatic, normal, tumor compared to normal adjacent, etc.)

Sample_ID Panel as a Spike-In for 56G

cluster

5000x

200x

Sequencing Depth

per Amplicon

Flow CellPCR Reaction

Somatic

Mutation Detection

Germline

Variant Detection

56G

Sample_ID

Experimental Design

Isolate cfDNA with

QIAamp® Circulating

Nucleic Acid kit

5 ng 10 ng

Healthy control blood, n = 5

(Streck Cell-Free DNA BCT)

Tumor bearing blood, n = 8

(Streck Cell-Free DNA BCT®)

Accel-Amplicon™ 56G Oncology

Panel library construction

Accel-NGS® Methyl-Seq

Liquid Biopsy Samples:Tumor Compared to Normal cfDNA Samples

SAMPLE PATHOLOGY

1 Fallopian tube high-grade papillary serous carcinoma pT3c N1 with 2 nodes involved by

micrometasasis

2 5 cm ovarian ‘borderline’ serous content (cancer-like)

3 Recurrent pT2, pN0 mammary carcinoma, 2.15 cm

4 pT1/pN1 pancreatic adenocarcinoma with neoadjuvant therapy

5 Metastatic colon cancer to the liver (previously treated)


7 Colon-cancer, non-resectable Adenocarcinoma T4a by imaging

8 Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary

Sequencing cfDNA and Matching Tumor

(FFPE) Samples

Sample Cancer Type Gene Hg19

Coordinate

Amino Acid

Change

% Mutant in

FFPE Normal

Adjacent

% Mutant in

FFPE Tumor

% Mutant in

cfDNA

1 Fallopian Tube

Adenocarcinoma

TP53 chr17:757708

5

E285K 0 48 0

TP53 chr17:757848

8

D148H 0 0 5

2 Ovarian

Cystadenofibroma

BRAF chr7:1404531

36

V600E 0 23 1

3 Mammary

Carcinoma

PIK3CA chr3:1789520

85

H1047R 0 17 0

TP53 chr17:757848

8

D148H 0 0 9

8 Metastic

Colorectal

Adenocarcinoma

PIK3CA chr3:1789360

91

E545K 0 23 11

APC chr5:1121755

76

Q1429* 0 20 5

TP53 chr17:757957

5

Q38* or

intron

0 21 14

KRAS chr12:253982

81

G13D 0 22 5

Accel-NGS® Methyl-Seq

DNA Library Kit

For Bisulfite-Converted DNA

Workflow Comparison for Bisulfite Converted

Samples

Accel-NGS® Methyl-Seq Outperforms Other Kits

Accel-NGS

Methyl-Seq

Traditional

Workflow

Random Primer

Approach

Input (ng) 1 1000 50

# PCR Cycles 10 10 10

Yield (nM) 7 15 20

nM Output Per ng Input 7 0.015 0.4

Bias Low Low High

cfDNA Sequencing to Detect Cancer Burden:A Global Biomarker for Cancer

PNAS, vol 110, no 47 (2013) pp18761-18768

Experimental Design

Isolate cfDNA with

QIAamp® Circulating

Nucleic Acid kit

5 ng 10 ng

Healthy control blood, n = 5

(Streck Cell-Free DNA BCT)

Tumor bearing blood, n = 8

(Streck Cell-Free DNA BCT®)

Accel-Amplicon™ 56G Oncology

Panel library construction Accel-NGS® Methyl-Seq

Detecting Genome-Wide Hypomethylation from

10 Million Reads with Accel NGS® Methyl-Seq

• 5 ng of cfDNA from 5 healthy controls and

sample 8 (Metastatic colorectal

adenocarcinoma with liver metastasis.

• Circos plot represents hypomethylation

status on chromosomes 1-22 between.

• Percent hypomethylation calculated by

comparing the Methylation Density in 1

Mb genome bins to healthy controls.

• Red is hypomethylated (> 3 SD lower

than normal mean MD) and green is

comparable to normal.

• Bins assigned hypomethylated if the MD

was > 3 SD lower than the healthy MD

average.

Liquid Biopsy Samples:Tumor Compared to Normal cfDNA Samples

SAMPLE PATHOLOGY

1 Fallopian tube high-grade papillary serous carcinoma pT3c N1 with 2 nodes involved by

micrometasasis


3 Recurrent pT2, pN0 mammary carcinoma, 2.15 cm

4 pT1/pN1 pancreatic adenocarcinoma with neoadjuvant therapy

5 Metastatic colon cancer to the liver (previously treated)


7 Colon-cancer, non-resectable Adenocarcinoma T4a by imaging

8 Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary

Sequencing cfDNA and Matching Tumor

(FFPE) Samples

Sample Cancer Type Gene Hg19

Coordinate

Amino Acid

Change

% Mutant in

FFPE Normal

Adjacent

% Mutant in

FFPE Tumor

% Mutant in

cfDNA

1

Fallopian Tube

Adenocarcinoma

TP53 chr17:757708

5

E285K 0 48 0

TP53 chr17:757848

8

D148H 0 0 5

2Ovarian

Cystadenofibroma

BRAF chr7:1404531

36

V600E 0 23 1

3

Mammary

Carcinoma

PIK3CA chr3:1789520

85

H1047R 0 17 0

TP53 chr17:757848

8

D148H 0 0 9

8

Metastic

Colorectal

Adenocarcinoma

PIK3CA chr3:1789360

91

E545K 0 23 11

APC chr5:1121755

76

Q1429* 0 20 5

TP53 chr17:757957

5

Q38* or

intron

0 21 14

KRAS chr12:253982

81

G13D 0 22 5

Summary

• Accel-NGS® 2S DNA Library Kits– 90% library conversion

– Superior coverage uniformity with cfDNA WGS

– Compatibility with multiple hybridization capture technologies

• Accel-Amplicon™ Panels– Performance with Horizon’s Quantitative Multiplex Reference Standard

– Liquid biopsy samples experimental design

• Accel-NGS Methyl-Seq DNA Library Kits– Accel-NGS Methyl-Seq outperforms other kits

– Detecting genome-wide hypomethylation

THANK YOUwww.swiftbiosci.com

© 2016, Swift Biosciences, Inc. The Swift logo and Accel-Amplicon are trademarks and Accel-NGS is a registered

trademark of Swift Biosciences. Illumina and HiSeq are registered trademarks of Illumina, Inc. Ion Torrent is a trademark

of Thermo Fisher Scientific Inc. Cell-Free DNA BCT is a registered trademark of Streck, Inc. QIAamp is a registered

trademark of QIAGEN. Chemagic is a trademark of PerkinElmer Inc. SureSelectXT and SureSelect XT2 are products of

Agilent Technologies. NimbleGen and SeqCap are trademarks and xGen and Lockdown are registered trademarks of

Roche NimbleGen Inc. 16-1041 08/16

A Guide to NGS Applications Using cfDNA SamplesOverview__Liquid+Biopsy... · Introducing the Swift...

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