Advanced NGS Data Analysis & Interpretation- BGW + IVA: NGS Tech Overview Webinar Series Part 3

Sample to Insight

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Mutational analysis using QIAGEN’s GeneRead panels and Sample-to-Insight NGS solutions

Raed Samara, PhD

Part 3Data AnalysisInterpretation

Sample to Insight

NGS part 3: Targeted NGS for Cancer Research 2

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/

Sample to Insight

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

‘One size fits all’ does not work


Sample to Insight

Mutations

AGCTCGTTGCTCAGCTCReference genome

AGCTCGTTGCTCAGCGTTCInsertion

AGCTC---GCTCAGCTC

Deletion

Indels Copy number variations

T

G

CA

T GA

C

DNA variants for precision medicine

RS NGS part 3: Targeted NGS for Cancer Research 4

Sample to Insight

Mutations

AGCTCGTTGCTCAGCTCReference genome

AGCTCGTTGCTCAGCGTTCInsertion

AGCTC---GCTCAGCTC

Deletion

Indels Copy number variations

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

Actionable DNA variants for precision medicine

Only a handful of mutations are actionable


Sample to Insight


How many?


Sample to Insight

• How many mutations to test for?• How to test for these mutations?

o Sequential testingo Parallel testing

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

Adapted from Govindan et al., Cell 150 (2012)

Precision medicine for lung cancer

Current lung cancer biomarker landscape

KRAS25%

EGFR

23%

EML4-ALK6%

BRAF3%

PIKC3A3%

MET2%

ERBB2

1%

MAP2K10.4%

NRAS

0.2%

Un-know

n37%


Sample to Insight


Targeted DNA sequencing delivers accurate information required for precision medicine

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:

Clinical utility requires targeted analysis

More relevant data

More cost effective

Detect low-frequency mutations

Lower DNA requirements

Higher multiplexing capabilities


Sample to Insight

Why choose targeted DNA sequencing?

• Well-defined content

• Small target size

• More reads per sample

• Examine variants that matter

• Multiplex many samples to save money

• Detect low frequency variants

Features Benefits

Targeted DNA sequencing limits the genes or targets to be sequenced


Sample to Insight

Targeted DNA sequencing (TDS)

Shrink the genome

Sample Insight

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

gDNAVariants Report

KRAS G12DEGFR T790MIDH1 R132H

KRASEGFRIDH1


Sample isolation

Targeted enrichment

Library construction NGS run Data

analysis Interpretation

Sample to Insight

NGS part 3: Targeted NGS for Cancer Research

Sample-to-Insight: Integrated universal targeted NGS workflow

11

To overcome NGS challenges

Sample Insight

Turnaround time, and limited

amounts of DNA

Library yield Coming soon

Data processing & Variant calling

Isolation of high-quality DNA

samples

Quantifying amplifiable (not

total) amounts of DNA

Clinical & Biological

interpretation of data

RS

Sample isolation

Targeted enrichment



Sample QC Library QC

Variantconfirmation

Sample to Insight

Why choose multiplex PCR-based targeted enrichment?

• Offers specificity that beats capture-based approaches

• Offers uniformity that beats capture-based approaches

• Use sequencing capacity on regions targeted by the panel, with minimal off-target sequencing

• Achieve more uniform enrichment for more sequencing efficiency

Features Benefits

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


Sample to Insight

GeneRead DNAseq Panels V2

Multiplex PCR-based enrichment of gene(s) or genomic region(s)

Builds on our 10-year experience in designing PCR assays

GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2

Collection of primer pairs dispended into primer pools/tubes. Primers correspond to the targeted

region(s) (Gene 1 and Gene 2, as example)

Primer pair; amplicon


Sample to Insight

Gene 1 Gene 2

Primer design algorithm


Multiplex PCR-based enrichment of gene(s) or genomic region(s)

PCR Chemistry

Primer separation algorithm

GeneRead DNAseq Panel V2(sets of primer pools/tubes)

GeneRead DNAseq PCR Kit V2


Sample to Insight


Product description

Sample Insight

ACCAGTGAC

TATAGCTAG

GTCCTATTG

CCGGTGTAC

Variants Report

Ref: AAGTCTCase: AACTCT

GeneRead DNAseq Panel V2

GeneRead DNAseq PCR Kit V2

DNA

Amplicons

Library

Reads

GeneRead DNAseq panels enrich genomic targets by generating amplicons (small ds DNA fragments) through PCR


Sample isolation

Targeted enrichment



Sample to Insight

Clinically- and biologically-focused panels

Cataloged panels; bench-tested Type Panel name

Solid TumorClinically Relevant Tumor

Tumor Actionable Mutations

Hematologic malignancies Myeloid Neoplasms

Tissue Specific

Breast Cancer

Colorectal Cancer

Liver Cancer

Lung Cancer

Ovarian Cancer

Prostate Cancer

Gastric Cancer

Cardiomyopathy

Gene(s)-specific BRCA1 and BRCA2

Comprehensive

Cancer Predisposition

Comprehensive Cancer

Carrier testing

Cancer Genome Census

ClinVar

Largest collection of pre-designed panels suitable for a wide range of needs


http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/

Sample to Insight

Application-specific amplicon design

Builds on >10 years in assay development

G12

Deep (redundant) tiling amplicon designDense overlap amplicon design

Sparse overlap amplicon design

Primers Amplicons


Sample to Insight

Customized panels

Custom and Mix-n-Match

1 No competitor offers bench-tested gene designs2 Fastest on the market

What is the list of your targets?


GeneRead Mix-n-Match

GeneReadCustom

• Access to 570 bench-tested gene designs1

• Fixed specifications• Turnaround time = 3 days2

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

• Flexible specifications• Turnaround time = 14 days2

Sample to Insight

The power of Mix-n-Match pool of validated genes

Consistent performance00

4X00

8X01

3X50

1X00

2X00

3X00

6X50

1X00

6X01

3X50

1X00

1X00

2X00

4X00

6X00

7X00

8X01

3X50

1X00

1X00

6X00

7X01

3X50

1X00

1X00

3X00

6X00

7X01

3X50

1X00

1X00

8X01

3X50

1X00

1X00

2X00

3X00

4X00

6X00

7X00

8X01

3X50

1X

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

Experimental Coverage UniformityMET NRAS TP53PDGFRA PIK3CA PTEN RB1 RET


Sample to Insight

Why choose GeneRead panels workflow for targeted enrichment?

• Small target sizes

• Uses Multiplex PCR

• Achieve a high degree of coverage depth

• Low DNA input material

• Quick turnaround time

• No physical removal of primer sequences

• Platform-independence

• Pool amplicons before library construction

• Integrated workflow

• Multiplex many samples to save money

• Achieve higher specificity and uniformity than other approaches1

• Make more confident calls to detect low frequency variants 4

• Sequence low-yielding samples such as FFPE and cfDNA samples

• Go from sample to insight quickly

• Reduce false positives and negatives2

• Achieve 100% specificity and 100% sensitivity3

• Use one panel for your different sequencers

• Generate one library per sample to save money

• Sequence low-quality samples

Features Benefits

GeneRead DNAseq panels address many NGS challenges

1. Mamanova et al. 2010. Nature Methods, 7(2):111 2. Satya and DiCarlo. 2014. BMC Genomics, 15:10733. Heydt et al. 2015. BMC Cancer, 15:291 4. Helen Fernandes, AMP 2014


Sample to Insight


Achieve better variant calling accuracy to reduce false negatives


Sample to Insight


Achieve 100% specificity and 100% sensitivity through better variant calling accuracy

The sensitivity of the AmpliSeq panel on the MiSeq™ (Illumina) was only 93%. Using this panel, four of the exon 11 mutations (p.M552_K558del, p.M552_V559del, K550_K558del, c.1648-5_1672del) could not be detected as these mutations were at the amplicon boundaries and primer binding sites.


Sample to Insight


Integrated workflow to optimally-sequence low-quality samples

The GeneRead workflow enables the optimal sequencing of low-yielding and low quality FFPE samples

Data presented by Helen Fernandes (Cornell University) at AMP 2014

AmpliSeq workflow GeneRead workflow

Group # of samples

DNA Quant Library quality & quantity DNA Quality Library quality &

quantity

Qubit Agilent QuantiMIZE qPCR

1 30

2 3

3 6 X

XSequenced optimallyNot sequenced optimally


3

Sample to Insight

Compatibility with major sequencing platforms

Same panel for different platforms

IlluminaQIAGEN

Ion

GeneRead® DNAseq Panels V2

HiSeq 2500MiSeq

NextSeq

PGMProton

Confirm variants on different platforms using the same panel


Sample to Insight


To overcome NGS challenges

Sample Insight

Turnaround time, and limited

amounts of DNA

Library yield Coming soon

Data processing & Variant calling

Isolation of high-quality DNA

samples

Quantifying amplifiable (not

total) amounts of DNA

Clinical & Biological

interpretation of data


1

Sample isolation

Targeted enrichment



Sample QC Library QC

Variantconfirmation

Sample to Insight


Bioinformatics solutions

Sample

Insight

Upstream Analysis ‘Primary’ ‘Secondary’ ‘Tertiary’


Sample Prep

AssayData

Sequence-Level

Statistics

Biology of Interest(Genes,Variants,

etc.)

Annotation & Comparative(Statistical Analysis)

Annotation & Biological

Interpretation

Sample to Insight

CLC Biomedical: Flexible workflows tailored for end-users

Lock key parameters of workflow to standardize processing

Identify Variants Example

Sample Prep

Plug-in for GeneRead DNAseq targeted panels


Assay Data

Sequence- Level

Statistics

Biology of Interest

Annotation & Comparative

Analysis

Annotation & Comparative Interpretation

Sample to Insight

IVA: Rapid prioritisation and annotation of variants

Examine a single sample or compare multiple samples


Sample to Insight

Examine evidence and references

• Variant Findings• ACMG Classification Rules• Variant Views


Sample to Insight

Why choose targeted DNA sequencing?

Detect known & discover novel variants

AM & CR

Disease-specific Comprehensive

DetectionDiscovery

MultiplexingTarget size

Custom & Mix-n-Match

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 discoveryAM: actionable mutations panel; CR: clinically relevant panel


Sample to Insight

Novel mutation discovery by targeted sequencing

Gene panels can facilitate the discovery of novel mutations

Notably, this study facilitated the identification of BRCA2 Thr9976, which is the strongest genetic association in lung cancer reported so far. For a smoker carrying this variant (2% of the population), the risk of developing lung cancer is approximately doubled, which may have implications for identifying high-risk ever-smoking subjects for lung cancer screening. Additionally, future study of the effects of PARP inhibition in smokers with lung cancer carrying BRCA2 Thr9976 may be warranted.


Sample to Insight

Clinical value of targeted sequencing with targeted panels

More is not necessarily better; in some cases, less is better


Sample to Insight

GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to identify mechanisms of drug resistance in cancer

• EGFR-mutant lung cancer is a subtype of non–small cell lung cancer (NSCLC) that exhibits sensitivity to EGFR TKIs such as erlotinib and gefitinib1

• However, acquired resistance develops after a median of 9–14 months1

• The most common mechanism of TKI resistance is a second-site mutation (T790M) in the EGFR kinase domain, which can be detected in >50% of biopsies done after resistance develops2

1Mok, T.S. et al. N. Engl. J. Med. 361, 947–957 (2009)

2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011)

Landscape of resistance mutations in NSCLC

2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011)


Sample to Insight

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GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to Identify mechanisms of drug resistance in cancer

Power of liquid biopsy: Predict resistance before a solid biopsy is available

• AZD9291 is an oral, irreversible, mutant-selective EGFR TKI developed to have potency against tumors bearing EGFR activating mutations (for example, L858R or exon 19 deletion) in the presence of the T790M mutation

• Can we identify potential mechanisms of resistance to AZD9291 before the availability of resistance biopsy specimens?

• Use GeneRead targeted NGS panel to screen cell-free plasma DNA (cfDNA) for potential resistance mutations by comparing pretreatment and post-disease progression plasma specimens from a phase I clinical trialo cfDNA serves as a liquid biopsy to monitor disease progression by examining the landscape of

mutations before a resistance solid biopsy is available

May 2015

RS

Sample to Insight


Liquid biopsy approach

Collect 10-20 cc blood (pre-treatment (Baseline) and post progression)

Prepare plasma

Isolate cfDNA using the QIAmp Circulating Nucleic Acid Kit

Enrich targets (20 genes) using the GeneRead DNAseq Lung Cancer panel

Construct libraries

Ultra-deep sequencing (30,000x) on a HiSeq (2 × 100 bp)


Sample to Insight


Liquid biopsy: Proof of concept

Targeted NGS identified an acquired T→A mutation (green) in 1.3% of reads, encoding an EGFR C797S mutation.


Sample to Insight


Liquid biopsy: EGFR C797S mutation in progression/resistance to AZD9291 (TKI)

NGS of baseline and progressiontumor biopsies (top) confirmed the acquired C797S mutation detected with plasma NGS (bottom).

Plasma NGS detects the same T→A C797S mutation (green) found in the tumor and additionally detects a second G→C mutation encoding the C797S mutation (blue).


Sample to Insight

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Advanced NGS Data Analysis & Interpretation- BGW + IVA: NGS Tech Overview Webinar Series Part 3

Healthcare

Transcript of Advanced NGS Data Analysis & Interpretation- BGW + IVA: NGS Tech Overview Webinar Series Part 3