Ngs part ii 2013

Sample & Assay Technologies

Next Generation Sequencing for Cancer Research

Vikram Devgan, Ph.D., MBADirector, R&D

[email protected]

Sample & Assay Technologies Welcome to the three-part webinar series

Next Generation Sequencing and its role in cancer biology

Webinar 1: Next-generation sequencing, an introduction to technology and applications

Speaker: Quan Peng, Ph.D.

Webinar 2: Next-generation sequencing for cancer research Speaker: Vikram Devgan, Ph.D., MBA

Webinar 3: Next-generation sequencing data analysis for genetic profiling Speaker: Ravi Vijaya Satya, Ph.D.

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Agenda

1. Introduction to Next Generation Sequencing in Cancer Research

2. Need for change

3. QIAGEN solutions: sample-to-result experience

Sample & Assay Technologies Cancer: A Disease of Genome

T

G

CA

Variation

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Sample & Assay Technologies Genetic Variations

Mutations

AGCTCGTTGCTCAGCTC Reference genome

AGCTCGTTGCTCAGCGTTC Insertion

AGCTC---GCTCAGCTC Deletion

Indels Copy number variation

Structural variation

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Challenge in Treating Cancer:

� Every tumor is different� Every cancer patient is different

Sample & Assay Technologies Cancer: Facts

� In 2008 over 12.7 millions new cases were diagnosed across the planet and approximately 7.6 million cancer death occurred

� In 2030, these numbers will rise to an expected 21.4 millions new cases and 13.2 million cancer death………..

If our ability to prevent, diagnose and treat cance r doesn't improve

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Sample & Assay Technologies Need of Cancer Research

� Identify new cancer susceptibility genes

� Identify mutations that drive cancer progression

� Assist in developing new diagnostic tests and targeted therapies

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Science 339, 1546 (2013);Bert Vogelstein et al.Cancer Genome Landscapes


e.g. lung cancer e.g. lung cancer

Need: Patient stratification / Personalized medicine


KRAS25%

EGFR23%

EML4-ALK6%

BRAF3%PIKC3A

3%MET2%

ERBB21%

MAP2K10.4%

NRAS0.2%

Unknown37%

Current lung cancer biomarker landscape

� Multiple mutation tests � If tested sequentially

– Not adequate tissue sample– Takes time to rule out individual mutations

Need tool to conduct simultaneous genetic testing for multiple oncogenic drivers

EML4-ALK Crizotinib

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


Population screening for high risk preventable disorders

� ~0.25% of US women (375,000) carry a mutation in BRCA1/2

� At very high risk of breast and ovarian cancer

– 85% lifetime breast cancer risk

– 25-50% lifetime ovarian cancer

� Knowledge of risk allows prevention

Need: Public health

Sample & Assay Technologies Need of Cancer Research

Cancer susceptibility genes

Resistance marker identification

Causative mutations for multigenic diseases

Patient stratification for clinical trails

Tumor sub-typing

Biomarker identification

Population screening for high risk preventable disorders

Personalized treatment

Therapeutic monitoring

Basic Research Clinical Research Public Health

Need a tool to detect multiple mutations in multiple genes with multiple samples

Sample & Assay Technologies Evolution of technologies for mutation detection

ARMS PCR

Liquid bead array

Sequenom MALDI-TOF

Next Generation Sequencing

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Few

Many

Next Generation Sequencing: Multiple Mutation/Multiple Genes/Multiple Samples

Sample & Assay Technologies NGS: the best method for mutation detection

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� High throughput� Test many genes at once

� Cost effective� Drastic decrease in cost of sequencing

� Systematic and unbiased � Detection of all mutation types

� Quantitative� Easy to quantify mutation frequency

Sample & Assay Technologies Challenges in translating NGS to oncogenomics

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� Quantity� Limiting for biopsy specimens

� Purity (genetic heterogeneity)� Cancerous cells may be a minor fraction of total sample� Multiple sub-clones of cancer may be present in one tumor

sample� Genomic alterations in cancer found at low-frequency

� Data analysis � Biologically interpretable data

� Fragmented Workflow� Technologies are not jointly developed

Sample & Assay Technologies QIAGEN solution

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NGS workflow

Library preparation

Target enrichment

NGS run

Data alignment / analysis

Primary sample to purified DNA

DNA preprocessing

Sequencing preparation

1. Accessing single cell genomes for next generation sequencing

2. Target enrichment enabling rare mutation detection and QC

3. Streamlined one-tube library preparation

4. Raw reads to completely annotated variants



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REPLI-g Single Cell Kit

NGS workflow

Library preparation

Target enrichment

NGS run



DNA / RNApreprocessing


� Problem:

� Isolation of NGS compatible DNA quantity from single to few

cells

� Solution:

� REPLI-g Single Cell Kit

– Unbiased amplification of ultra-low DNA amount

Sample & Assay Technologies Technology

� Primers anneal to template DNA

� Phi 29 DNA polymerase moves along the DNA

template strand displacing the complementary strand

� The displaced strand becomes a template for

replication

� Advantages

� High yield of high-molecular-weight DNA

� High fidelity amplification – minimal error rate

� No effect of secondary structure - minimal bias

Multiple displacement based whole gemome amplification

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How REPLI-g Single Cell Kit work

Simple three step protocol

(Yield: 20-40 µg)

Sample & Assay Technologies Comparable NGS results with gDNA & amplified single cells

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� REPLI-g Single Cell technology provide high genome coverage & high fidelity

Sample & Assay Technologies Unbiased amplification with REPLI-g Single Cell Kit

2020

� No allele drop out after REPLI-g Single Cell WGA


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NGS workflow

Library preparation

Target enrichment

NGS run



DNA preprocessing






Sample & Assay Technologies NGS: DNA sequencing

Increase in•Cost•Data complexity

Decrease in•Deep sequencing•Multiplexing •Reimbursements

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Gene Panels

Exome

Genome

Gene Panel Sequencing: Efficient, Effective and Economical method

Sample & Assay Technologies Clinical utility requires targeted analysis

Linking genetic variants with biology

Title, Location, Date 23

Sample & Assay Technologies Shrink the Genome

Focus on Genes of interest relevant to research topic

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Sample Information Analysis Time Coverage Depth Sample Size

Genome 3 x 109 bps 7 days 10X 1 ug

20 Genes 6 x 104 bps 8 hours 1000X 10 ng


Hybridization Hybridization, Ligation + PCR Multiplex PCR

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Which is most suitable target enrichment technology?


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� Workflow:

Library construction(4 hrs)

Hybridization with probes (24-48 hrs)

PCR & Indexing (2 hrs)

� DNA input:1-3 ug

� Time from DNA sample to NGS library:2-3 days

� Workflow:

Hybridization with probe (16 hrs)

Ligation (1 hr)

PCR & Indexing (2 hrs)

� DNA input:200-400 ng

� Time from DNA sample to NGS library:2 days

� Workflow:

PCR amplification (3 hrs)

Library construction (4 hrs)

� DNA input:<100 ng

� Time from DNA sample to NGS library:1 days

Hybridization Hybridization, Ligation + PCR Multiplex PCR

Multiplex PCR technology

(SIMPLE)

(LOW)

(RAPID TAT)

Sample & Assay Technologies GeneRead DNAseq Gene Panel

� Multiplex PCR technology based targeted enrichment for DNA sequencing

� Cover all human exons (coding region + UTR)

� Division of gene primers sets into 4 tubes; up to 1500 plex in each tube

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Genes Involved in Disease

Genes with High Relevance

� Comprehensive Cancer Panel (124 genes)

� Cancer-specific Focused Gene Panels (20 genes)

� Breast cancer

� Colon Cancer

� Gastric cancer

� Leukemia

How Genes on Panels Are Selected

� Clinically/Biologically relevant � Multiple Publically accessible databases� Text mining tools� Manually curated

� Technically relevant� Most frequently mutated genes� Specific feedback from the thought leaders

� Liver cancer

� Lung Cancer

� Ovarian Cancer

� Prostate Cancer

Focus on your Disease of Interest

Sample & Assay Technologies Breast Cancer Gene Panel

� Somatic: All� Germline and Somatic: APC, BRCA1, BRAC2, TP53� With approved FDA inhibitor: BRAF, EGFR, ERBB2

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� Deep sequencing enabling detection of low prevalence mutations

� Multiplex more samples (cost saving)

� Without missing potentially important mutations

Advantages of Focused and Curated Gene Panel

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MiSeq Competitor ACCP (400 genes)

GeneReadLung Cancer Panel (20 genes)

Coverage Depth 1000X 1000X

Number of Samples 1 8

Ion Torrent 316 Chip Competitor ACCP (400 genes)


Median Coverage Depth 87X 944X

Region with >100X coverage 39.0% 91.2%

COSMIC database Competitor ACCP (400 genes)


Lung cancer specific mutations 79.9% 71.4%

Sample & Assay Technologies Gene Read DNASeq Gene Panel

� Simple� Any gene� Any sequencer� Any sample: average amplicon size is 145 bp

� Superior � Wet bench verified gene panels

– High design rate: >90%– High specificity: >85%– High uniformity (0.1X of median coverage depth): >85%

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>10 years of proven expertise in primer design: GeneRead algorithm


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GeneRead DNAseq Custom Panel


Simple Protocol

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GeneRead DNAseq Gene PanelPCR primer mix + control primers

Add GeneRead Mastermixand genomic DNA (20 ng/rxn)

Pool reactions for each sample and purify (QIAquick PCR Purification Kit)

PCR amplification

2 ho

urs


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GeneRead DNASeq Library Quant Array

primer assays forLibrary

Quantification

primer assays for library QC

Serial dilution of DNA standard with primer assays for library quantification

Sample & Assay Technologies GeneRead DNASeq Library Quant Array

Quantification and QC in a single PCR run

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QC Score

1-4 4-8 >8

Results Pass Marginal Fail

Proceed Caution! Do not proceed

GeneRead DNAseq Library Quant Array

Sample & Assay Technologies GeneRead DNASeq Library Quant Array

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� Segregate bad libraries before sequencing; save time and money

An Example

Sample & Assay Technologies DNAseq Panels compatible with NGS platforms

No manipulation of target enrichment protocols needed

IonIllumina

• MiSeq• GA Iix• HiSeq 1000• HiSeq 2000• HiSeq 1500• HiSeq 100

• Torrent PGM• 314• 316• 318

• Proton


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NGS workflow

Library preparation

Target enrichment

NGS run



DNA preprocessing






Sample & Assay Technologies GeneRead DNA Library Prep Kits

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NGS workflow

Library preparation

Target enrichment

NGS run



DNA preprocessing


Library preparation

Problem

� Inefficient, complex and error-prone workflow

Solution

� Combine various enzymatics steps in single tube

� Fast procedure that allows up to 50% time savings

� High yields from minimal amounts of starting material

� Unbiased and high-fidelity amplification mastermix (optional)

� Can be automated on QIAcube

� For Illumina & Ion Torrent platforms


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GeneRead DNA Library Prep Kits

Faster, less variable & more efficient workflow

Sample & Assay Technologies GeneRead Size Selection Kit

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Adapterdimers

Adaptermonomers

� Precise size selection of DNA fragments

� Fast procedure, based on QIAGEN’s proven silica-column

technology

� An easy-to-follow protocol, automatable on the QIAcube

Remove DNA fragments <150 bp

Before size selection GeneRead size selection

Sample & Assay Technologies GeneRead DNA Library Prep Kits

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� High yield of Library DNA

� Uniform coverage distribution

Input DNA as little as 50 ng

Sample & Assay Technologies HiFi Polymerase Comparison: Experimental Design

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Sample & Assay Technologies GeneRead Library Prep Kit

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Low error rate with minimal sequence bias

� High-fidelity amplification

� Greater and more even coverage in GC- and AT- rich areas of DNA


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NGS workflow

Library preparation

Target enrichment

NGS run



DNA preprocessing






Sample & Assay Technologies Data Analysis: Free, Complete and Easy to Use

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Sample & Assay Technologies Results in Multiple Analysis Format

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Run Summary� Specificity� Coverage� Uniformity� Numbers of SNPs and Indels

Summary By Gene� Specificity� Coverage� Uniformity� # of SNPs and Indels


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Features of Variant Report

dbSNP and COSMIC ID

Predicted amino acid change

Effect of SNP Impact of SNP

Link to qPCRsomatic mutation

assay

� SNP detection� Indel detection

Sample & Assay Technologies Genetic variant analysis in FFPE lung adenocarcinoma samples

Experimental Design

gDNA isolated from 3 FFPE lung adenocarcinoma and one FFPE normal lung samples

GeneRead Lung Cancer Gene Panel was used to enrich 20 genes

Library preparation, quantification and sequencing

QIAGEN NGS Data Analysis Web Portal

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Run Summary

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Sample Tumor Sample-1

Tumor Sample-1

Tumor Sample-1

Normal Sample

Total number of reads 1,629,983 1,892,621 1,851,514 1,625,119

Specificity(Reads on target)

92% 92% 90% 90%

Median Coverage 608 744 703 577

Uniformity(regions with >10% of median coverage)

93% 92% 90% 93%

No. of SNPs 84 72 87 83

No. of Indels 8 11 9 6

Genetic variant analysis in FFPE lung adenocarcinoma samples


� Snap shot of variant report


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Sample & Assay Technologies Streamlined Sample-to-Result Workflow

PyroMark Assays

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Sample & Assay Technologies Genetic variant analysis in FFPE lung adenocarcinoma samples

Experimental Design

gDNA isolated from 3 FFPE lung adenocarcinoma and one FFPE normal lung samples

GeneRead Lung Cancer Gene Panel was used to enrich 20 genes

Library preparation, quantification and sequencing

QIAGEN NGS Data Analysis Web Portal

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Run Summary

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Sample Tumor Sample-1

Tumor Sample-1

Tumor Sample-1

Normal Sample

Total number of reads 1,629,983 1,892,621 1,851,514 1,625,119

Specificity(Reads on target)

92% 92% 90% 90%

Median Coverage 608 744 703 577

Uniformity(regions with >10% of median coverage)

93% 92% 90% 93%

No. of SNPs 84 72 87 83

No. of Indels 8 11 9 6



Variant Filtering

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Detection of low frequency variant in lung adenocarcinoma



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Tumor sample-1 (KRAS c.35G>T 35%)

Mutation analysis of codons 12 of KRAS using PyroMark Q24. Upper Pyrogram show G to T mutation in position 1 of codon 12 of KRAS in lung adenocarcinoma sample. The mutation rate (35%) is similar to the NGS results (38%) confirming the reliability of GeneRead DNAseq Gene Panel. The lower Pyrogram shows normal genotype in normal sample-1.

Normal sample-1(KRAS c.35G>T 0%)

Data Validation: PyroMark Assay

Validation of KRAS:G12V somatic mutation by pyrosequencing assay

Sample & Assay Technologies Streamlined sample-to-result workflow

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PyroMark Assays

Streamlined, standardized and automated sample-to-result workflow

Gene Reader Eco Solution

2013

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Ngs part ii 2013

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