Digital DNA-seq Technology: Targeted Enrichment for Cancer Research

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Mutational analysis using QIAGEN’s QIAseq® panels and Sample to Insight® NGS solutions

Raed Samara, PhDGlobal Product Manager, QIAGEN

QIAseq Targeted NGS for Cancer Research, 10.10.2016

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Legal disclaimer


• QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.

• For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.

http://www.qiagen.com/

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Precision medicine: Right drug, right patient, right time and dose


“One size fits all” does not work

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Mutations

AGCTCGTTGCTCAGCTCReference genome

AGCTCGTTGCTCAGCGTTCInsertion

AGCTC---GCTCAGCTC

Deletion

Indels Copy number variations

T

G

CA

T GA

C

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DNA variants for precision medicine


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Actionable DNA variants for precision medicine


Mutations

AGCTCGTTGCTCAGCTCReference genome

AGCTCGTTGCTCAGCGTTCInsertion

AGCTC---GCTCAGCTC

Deletion

Indels Copy number variations

Only a handful of mutations are actionable

Actionable DNA Variant BRAF V600E

EGFR E746-750+ Kinase domain mutation

HER2

Disease Melanoma Lung adenocarcinomas IDC-Breast cancer

Therapy Vemurafenib (PLX4032) Erlotinib / Gefitinib Trastuzumab

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How many?

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EGFR (L858R)

KRAS (G12C)

+

Response rates of >70% in patients with

non-small cell lung cancer treated with either erlotinib or

gefitinib

Poor response rate in patients with non-small

cell lung cancer treated with either erlotinib or gefitinib

KRAS25%

EGFR

23%

EML4-ALK6%

BRAF3%

PIKC3A3%

MET2%

ERBB2

1%

MAP2K10.4%

NRAS

0.2%

Un-know

n37%

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Precision medicine for lung cancer


Current lung cancer biomarker landscape

• How many mutations to test for?

• How to test for these mutations

◦ Sequential testing

◦ Parallel testing

Adapted from: Govindan, R. et al. (2012). Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150, 1121–34.

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Attribute /Parameter

Information level

Cost per sample

Coverage achieved

DNA input

No. of samples multiplexed

Whole Genome

Sequencing

3 x 109 bps

$5000

30x

1 µg

1

Whole Exome

Sequencing

5 x 107 bps

$2000

100x

100–200 ng

2

Targeted DNA

Sequencing

6 x 104 bps

$200

1000x

10 ng

96

Benefits of Targeted DNA Sequencing:

More relevant data

More cost effective

Detect low-frequency mutations

Lower DNA requirements

Higher multiplexing capabilities

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Targeted DNA sequencing delivers accurate information required for precision medicine

Clinical utility requires targeted analysis

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• Well-defined content

• Small target size

• More reads per sample

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Why choose targeted DNA sequencing?


Targeted DNA sequencing limits the genes or targets to be sequenced

Features Benefits

• Examine variants that matter

• Multiplex many samples to save money

• Detect low frequency variants

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Sample Insight

The principle of targeted enrichment is to simultaneously sequence millions of small DNA fragments that represent the region of interest

gDNA

Variants Report:

KRAS G12DEGFR T790MIDH1 R132H

KRASEGFRIDH1

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Sample isolation

Library construction & targeted enrichment

NGS run Data analysis Interpretation

Targeted DNA Sequencing (TDS)


Shrink the genome

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Why choose PCR-based targeted enrichment?


• Offers specificity that beats capture-based approaches

Features Benefits

• Lets you use sequencing capacity on regions targeted by the panel, with minimal off-target sequencing

• Lets you achieve more uniform enrichment for more sequencing efficiency

It delivers unmatched specificity and uniformity (compared to capture-based methods)

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Sample Insight

• Turnaround time, and limited amounts of DNA

• Uniformity of enrichment

• Coverage of GC-rich regions

• Platform-dependent challenges

• Data processing & variant calling

• Isolation of high-quality DNA samples

• Quantification of amplifiable (not total) amounts of DNA

• Clinical & biological interpretation of data

RS

Sample QC Library QC

Variantconfirmation

Sample isolation



Sample to Insight: Integrated universal targeted NGS workflow


To overcome NGS challenges

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Inability to detect low-frequency mutations

Inefficient enrichment and sequencing of

GC-rich regions

PCR and sequencing errors• Limits sensitivity and accuracy of calling low-frequency variants

o Doesn’t let you confidently call variants down to 1% variant allele frequency (VAF)

Suboptimal uniformity of

enrichment and sequencing

Suboptimal, GC-rich region-incompatible PCR chemistry• Limits comprehensiveness of panel coverage

o Doesn’t let you efficiently sequence clinically-relevant genes such as CEBPA or CCND1 – or clinically-relevant regions such as TERT promoter

Conventional PCR protocols and two-primer amplicon design• Increases variability in coverage across targeted genomic regions

o Causes you to over-sequence to accommodate the under-sequencedo Doesn’t let you call variants in low-depth regions

Mainly due to inferior PCR amplification approaches

Challenges of current DNA targeted sequencing approaches


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DNA

dsDNA

PCR amplification & sequencing

PCR and sequencing errors

The necessary evil: PCR amplification


PCR amplification is required for target enrichment, but…

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5 reads OR library fragments that look exactly the same. Cannot tell whether they represent:

1. 5 unique DNA molecules, or

2. Quintuplets of the same DNA molecule (PCR duplicates)

Conventional targeted DNA sequencing

EGFR exon 21

Quantification based on non-unique reads does not reflect quantities of original DNA molecules

Challenges of conventional targeted DNA sequencing


PCR duplicates limit accurate quantification

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EGFR exon 21

*

Variant calling based on non-unique reads does not reflect the mutational status of original DNA molecules

Challenges of conventional targeted DNA sequencing


A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is:

1. A PCR or sequencing error (artifact) / false positives, or

2. A true low-frequency mutations

PCR and sequencing errors (artifacts) limit variant calling accuracy

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• Proprietary PCR chemistry to enrich even GC-rich regions

• Primers based on single primer extension (SPE) approach for enhanced uniformity

Panel box (kit)

QIAGEN’s solutions to overcome challenges of targeted NGS


QIAseq targeted DNA panels

• Molecularly-barcoded library adapters to incorporate unique molecular indices (UMIs).

Index box (kit)

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With the QIAseq targeted DNA panels, variant detection is done by analyzing unique DNA molecules instead of total reads

Overcoming current challenges


Current approach ChallengesHow QIAseq targeted DNA panels overcome challenges of current approaches

• Conventional targeted DNA sequencing for variant detection

• PCR and sequencing errors • UMIs that enable digital sequencing to correct for PCR and sequencing errors

• Inefficient sequencing of GC-rich regions

• Proprietary chemistry to efficiently sequence GC-rich regions

• Suboptimal uniformity of enrichment and sequencing

• SPE-based primer design to increase uniformity

For optimal variant detection

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TATCGTACAGAT(12 nucleotides long)

Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness

What is a UMI (molecular barcode)?


Tag (barcode) to identify unique DNA molecules

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DNA

dsDNA

TATCGTACAGAT

Molecularly-barcoded adapter Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness

PCR amplification & sequencing

Correct for PCR duplicates & errors

How are UMIs incorporated?


Ligate molecularly-barcoded adapters to unique DNA molecules before amplification

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5 unique DNA molecules

since 5 molecular barcodes are detected

Quintuplets of the same DNA molecule (PCR duplicates)

since 1 molecular barcode is detected

UMI

Digital sequencing with UMIs

UMIs before any amplification

Achieve accurate quantification with molecular barcodes


Count and analyze single original molecules (not total reads) = digital sequencing

5 reads OR library fragments that look exactly the same. Cannot tell whether they represent:

1. 5 unique DNA molecules, or

2. Quintuplets of the same DNA molecule (PCR duplicates)


EGFR exon 21

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False variant is present in some fragments carrying the same UMI

True variant is present in all fragments carrying the same UMI

UMI

UMIs before any amplification

* *****

Digital sequencing with UMIs

Achieve accurate variant calling with molecular barcodes



EGFR exon 21

*A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is:

1. A PCR or sequencing error (artifact) / false positives, or

2. A true low-frequency mutations

Count and analyze single original molecules (not total reads) = digital sequencing

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Sample isolation


NGS run Data analysis InterpretationSample Insight

Panels and molecularly-

barcoded adapters

Barcode-aware variant calling

pipeline

QIAseq targeted DNA panels: Sample to Insight


Panels, molecularly-barcoded adapters and data analysis algorithms

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Specifications of QIAseq targeted DNA panels


DNA input As little as 20 ng DNA

Primer multiplexing level 11,500 / 9600 primers (Catalog / Custom DNA)

Number of primer pools 1

Enrichment technology SPE-based with molecularly-barcoded adapters

Amplicon size Average 150 bp

Sample multiplexing level 384 (Illumina), 96 (Ion Torrent)

Total workflow time 8–9 hours

Number of libraries per sample 1

Sequencer compatibility Illumina and Ion Torrent platforms

Variant allele frequency called 1%

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End repair and A tailing

Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes)

5’5’

5’

5’

MB

MB

Adapter

5’

IL-FRSP

Add GSPs and UP*

5’IL-U SIP

Add indexes and UP*

Universal PCR amplificationSample indexing and amplification

Sequencing-ready library

MB: Molecular barcodeRSP: Region-specific primerFP: Forward primerUP: Universal primerSIP: Sample index primer

AA

5’MB

Target enrichment by SPE5’

*Preceded by bead cleanup

Lib quant*

Enzyme-based random DNA fragmentation

DNA

5’5’

1.5

Day

s

QIAseq Targeted DNA Panel: Workflow (Illumina®)

QIAseq Targeted NGS for Cancer Research, 10.10.2016 25

Sample to Insight

QIAseq Targeted DNA Panel: Workflow (Ion Torrent™)

QIAseq Targeted NGS for Cancer Research, 10.10.2016 26

End repair and A tailing

Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes)

5’5’

5’

5’

MB

MB

Adapter

5’

LT-FRSP

Add GSPs and UP*

5’LT-U P1

Add indexes and UP*

Universal PCR amplificationSample indexing and amplification

Sequencing-ready library

MB: Molecular barcodeRSP: Region-specific primerFP: Forward primerUP: Universal primerP1: P1 primer

AA

5’MB

Target enrichment by SPE5’

*Preceded by bead cleanup

Lib quant*

Enzyme-based random DNA fragmentation

DNA

5’5’

1.5

Day

s

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1. Exonic regions of genes plus 10 bases to cover intron / exon junctions

2. Mix of type of coverage 1 (for tumor suppressor genes) and HotSpots for oncogenes

3. SNPs

4. Full chromosome



PanelVariant (Cat) number

Number of genes

Number of primers

Type of coverage

Breast cancer panel DHS-001Z 93 4831 1

Colorectal cancer panel DHS-002Z 71 2929 1

Myeloid Neoplasms panel DHS-003Z 141 5887 1

Lung cancer panel DHS-005Z 72 4149 1

Actionable solid tumor panel DHS-101Z 23 651 2

BRCA1 and BRCA2 panel DHS-102Z 2 223 1

BRCA1 and BRCA2 Plus panel DHS-103Z 6 348 1

Pharmacogenomics panel DHS-104Z 39 146 3

Mitochondria panel DHS-105Z Chromosome M 222 4

Inherited diseases panel DHS-3011Z 298 11,579 1

Comprehensive cancer panel DHS-3501Z 275 11,311 1

List of panels

Types of coverage:

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List of panels

PanelVariant (Cat) number

Panel size (bases)

Specificity (reads with primers, %)

Uniformity (0.2x mean baseMT, %)

Breast cancer panel DHS-001Z 370,942 96.47 99.84

Colorectal cancer panel DHS-002Z 215,328 90.39 99.79

Myeloid Neoplasms panel DHS-003Z 436,672 95.31 99.71

Lung cancer panel DHS-005Z 318,059 97.3 99.91

Actionable solid tumor panel DHS-101Z 15,160 90.48 99.85

BRCA1 and BRCA2 panel DHS-102Z 16,405 99.59 100

BRCA1 and BRCA2 Plus panel DHS-103Z 25,590 99.46 99.92

Pharmacogenomics panel DHS-104Z 3313 93.43 99.34

Mitochondria panel DHS-105Z 16,570 99.72 99.08

Inherited diseases panel DHS-3011Z 838,627 97.29 99.21

Comprehensive cancer panel DHS-3501Z 836,670 97.42 199.76

Uniformity and specificity are defined based on NA12878 tests

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Extended and Custom

What is the list of your targets?

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Extended panels

Custom panels

• Extend the contents of an existing cataloged panel• Turnaround time = 14 days

• Bioinformatically target any gene(s) or genomic region(s) within the human genome

• Turnaround time = 14 days

Customized panels


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CEBPA

GC content

Coverage

GC content

Coverage

CCND1

The proprietary PCR chemistry used in the QIAseq targeted DNA panels enables efficient coverage of regions high in GC content

Comprehensive coverage of GC-rich regions


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830 kb region was enriched from 20 ng of NA12878 DNA with Comprehensive Cancer Panel. Library was constructed for sequencing on a MiSeq, with 2600x read depth. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 98% at 0.3x of mean coverage.

Unmatched uniformity


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Benefits of QIAseq targeted DNA panels


Feature Benefit

Low DNA input (as low as 20 ng DNA) Preserve sample

High primer multiplexing capability (up to 10,000 primers) Detect a large number of DNA variants

Single pool of primers Easier sample handling

SPE-based target enrichment Flexibility in primer design

Small amplicons (average size 150 bp) Generate relatively small library fragments to maintain compatibility with fragmented DNA (FFPE and ctDNA samples)

High sample multiplexing (up to 384 samples) Increased sample throughput to decrease sequencing costs

Automation-friendly workflow Streamline operations for high throughput

Molecular barcode-aware variant caller Confidently call low-frequency mutations

Suite of complementary data analysis tools Save resources

Affordable per-sample cost Save $$$

All required reagents (including beads) in 2 kits Simplified logistics & ordering

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This online GenomeWeb seminar focused on the design of a large cohort study for assessing breast cancer risk and how using an innovative digital sequencing approach is able to solve the previously unmet challenges of this type of NGS study design.

Fergus J. Couch, PhD

Professor and Chair Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic

https://genomeweb.webex.com/genomeweb/lsr.php?RCID=30c8e3fe698b7feca1ed79ac117fbed0

Application: Large cohort study for breast cancer risk


Sequencing 60,000 samples




Sample to Insight

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Sample to Insight: Integrated universal targeted NGS workflow


To overcome NGS challenges

Sample Insight

• Turnaround time, and limited amounts of DNA

• Uniformity of enrichment

• Coverage of GC-rich regions

• Platform-dependent challenges

• Data processing & variant calling

• Isolation of high-quality DNA samples

• Quantification of amplifiable (not total) amounts of DNA

• Clinical & biological interpretation of data

Sample QC Library QC

Variantconfirmation

Sample isolation



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Barcode-aware variant caller has been developed

Caller is available on the cloud

In conjunction with molecular barcodes incorporated in the workflow, the caller can confidently call low-frequency variants (down to 1% variant allele frequency, “VAF”)

Variant caller will do the following:• Mapping

• Alignment

• Molecular barcode counting

• Variant / calling

• Variant / annotation – variants based on public databases

Data analysis with barcode-aware variant caller – overview


For QIAseq targeted DNA panels

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FASTQ or BAM files are uploaded into cloud-based data analysis portal

The following inputs are needed (by customer):• Set up file

• Panel used

• File laneso 1-lane (MiSeq/HiSeq/NextSeq concatenated)o 4-lane (NextSeq individual lane files)

Inputs

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Outputs

Summary file• Stats

o Specificityo Uniformityo Molecular barcode counts

• Variantso Frequencyo Annotations

VCF

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Actionable solid tumor

Disease-specific Comprehensive

DetectionDiscovery

MultiplexingTarget size

Custom & extended

Pan

els

App

licat

ions

Spe

cific

atio

ns

Clinical research Translational & discovery research

Whole Exome SeqWhole Genome Seq

Targeted DNA sequencing: robust detection, limited discovery

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Why choose targeted DNA sequencing?


Detect known variants & discover novel variants

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Thank you for attending today’s webinar!Contact QIAGENCall: 1-800-426-8157

Email: [email protected]

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Questions?

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Thank you for attending


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Healthcare

Digital DNA-seq Technology: Targeted Enrichment for Cancer Research