molecular analysis via next-generation sequencing - national

Sample & Assay Technologies

Molecular analysis via next-generation sequencing

NMG Annual Fall Meeting, Toronto, Canada

Raed Samara, PhD, PMP

Global Product Manager

October 21, 2013

Sample & Assay Technologies Legal Disclaimer

Raed Samara, PhD; NMG Conference; 10/21/2013 2

• 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 & Assay Technologies Agenda

3 Raed Samara, PhD; NMG Conference; 10/21/2013

1. Primer on NGS

2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight

3. Application example (Sequencing of FFPE cancer samples)

4. Considerations for using NGS in Molecular Microbiology



1. Primer on NGS




Sample & Assay Technologies What is Next-Generation Sequencing (NGS)?

5

Massively Parallel Sequencing

Instead of sequencing a DNA sequence from

Sequence many small pieces at the same time

Resulting in the original sequence

(with overlaps removed)

.Target DNA is fragmented

Adaptors ligated to fragments

(Library construction)

Clonal amplification of fragments

.Direct step-by-step detection of

each nucleotide base

incorporated during the

sequencing reaction

Starting gDNA

Raed Samara, PhD; NMG Conference; 10/21/2013

Sample & Assay Technologies NGS needs in Molecular Analysis

6

Simple, but integrated workflow

Work with low quality and qualntity DNA samples

Focus efforts on a focused set of genes important to molecular analysis (save

time) + Selective sequencing saves sequencing capacity (save $$$)

Identify low frequency DNA variations

Simple methodology to make variant calls

Sample Insight




1. Primer on NGS




Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges

8

Sample Insight



Three major components in workflow

9

Platform Pre-Analytics

Sample Insight

Software



….. but several steps in each component of the workflow

10

Platform

Sample

Isolation

Target Enrichment

Library Preparation

Library Quantification

NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



Each step has its own challenges

11

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software

DNA Quality Library Quality Platform-specific Data processing Variant calling

Biological Interpretation of

data

Efficiency & Yield


Content AND Coverage,

Specificity & Uniformity


Major challenge: Fragmented workflow

12

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Dedicated products in a complete workflow to overcome challenges of your

Preanalytic DNA NGS workflow

Efficiency & Yield





13

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Efficiency & Yield




Sample & Assay Technologies QIAGEN — the gold-standard in DNA purification

The best DNA quality possible for outstanding NGS results

High-quality purification from virtually all sample types

Sample type Kit

FFPE tissue QIAamp DNA FFPE Kit

Blood QIAamp DNA Blood Mini Kit

Tissues, swabs, CSF, body fluids QIAamp DNA Mini Kit

High-molecular weight DNA MagAttract HMW DNA Kit


Automated on QIAcube:

Consistency and standardization


15

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Efficiency & Yield




Sample & Assay Technologies Three levels of DNA Sequencing

Targeted Exome

Sequencing

(Genes Panels)

Whole Exome

Sequencing

Whole Genome

Sequencing Cost

Data Complexity

Irrelevant data

Starting DNA material

Poor quality reads

Deep sequencing

Multiplexing

Quality of reads

Coverage

Gene Panel Sequencing: Efficient, Effective and Economical method


Sample & Assay Technologies Traditional NGS Workflow

17 Shankar Sellappan, Ph.D. Raed Samara, PhD; NMG Conference; 10/21/2013

Sample & Assay Technologies New NGS Workflow with Targeted Enrichment

18

• Focused on your genes of interest

• Why look at all 20,000

genes in the human

genome when you are

interested in only a few?

• More confidence and deeper

coverage

• Enables deep sequencing to ID

low frequency / rare variants

• Integrated controls to assess

target enrichment

Achieve more sensitive variant

detection with 1 additional step

80 ng

Shankar Sellappan, Ph.D. Raed Samara, PhD; NMG Conference; 10/21/2013

Sample & Assay Technologies Shrink the Genome

Focus on Genes of interest relevant to disease

19

Sequencing Information Cost per

sample

Coverage No. of

samples

multiplexed

DNA input

Genome 3 x 109 bps - 30X N/A 1µg

Exome 5X 107 bps $2000 100X 1 500 ng- 1µg

30 Genes 6 x 104 bps $200 1000X 12 80 ng

* 7.5 GB sequencing capacity


Sample & Assay Technologies Targeted Exome Enrichment: Principles

Multiplex PCR-enabled enrichment of gene of interest

20

Exon 1 Exon 2 Exon 1 Exon 2 Exon 1 Exon 2

gDNA

GOI 3 GOI 2 GOI 1

BRAF Akt1 ErbB2

Each exon of

GOI is targeted

by several

overlapping

primers

Targeted Exome Enrichment allows you to focus on you GOI

The entire collection

of primers is divided

into 4 tubes, each

containing non-

adjacent primer sets

Contains a set

of endogenous

controls (for use

in QC)


Sample & Assay Technologies Targeted Exome Enrichment: Principles

Simple Protocol – 2 hours

21

GOI 3 GOI 2 GOI 1

BRAF Akt1 ErbB2

Exon 1 Exon 2 Exon 1 Exon 2 Exon 1 Exon 2

Isolated

gDNA

GeneRead DNASeq

Master Mix

GeneRead DNASeq

Panels

Total of 4

PCR

reactions


Template for

Clonal

Amplification

Pooled

Amplicons Prepare for sequencing

(Library Preparation)

Wet-bench verification – we confirm that primers work

All of the amplicons of your GOI in ONE tube

Sample & Assay Technologies Performance metrics

High coverage, specificity, and uniformity


Panel 1 (6 genes): Custom GeneRead

DNAseq Gene Panel

Panel 2 (20 genes): Human Lung Cancer

GeneRead DNAseq Gene Panel

Panel 3 (124 genes): Human Comprehensive

Cancer GeneRead DNAseq Gene Panel

Specific

ity (

% o

n-t

arg

et re

ads)

Design coverage (≥90%) Specificity (on-target reads; ≥85%)

Coverage uniformity

(≥85% with 0.1X of median sequencing depth)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Panel 1 (6 genes) Panel 2 (20 genes)

Panel 3 (124 genes)

% C

overe

d b

y d

esig

n

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


Panel 3 (124 genes)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


Panel 3 (124 genes)

Covera

ge u

niform

ity

Sample & Assay Technologies Biologically/Clinically relevant gene panels

GeneRead DNAseq Gene Panels

Comprehensive Cancer Panel

Disease Focused Gene Panels

Breast cancer

Colon Cancer

Gastric cancer

Leukemia

Onco-hematology (coming soon)

Cardiomyopathy (coming soon)

Inheritance disease Panel (coming soon)

Actionable Mutation Cancer Panel (coming soon)

Clinically-relevant Mutation Tumor Panel (coming soon)

Cancer Predisposition Panel (coming soon)

Custom Content

ANY GENE or COLLECTION OF

GENES in Human Genome

23

Liver cancer

Lung Cancer

Ovarian Cancer

Prostate Cancer Cancer Genome Census

ClinVar


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


24

GeneRead DNAseq Custom Panel

Custom Content

ANY GENE or COLLECTION OF

GENES in Human Genome


Sample & Assay Technologies Breast Cancer Gene Panel

Description


Sample & Assay Technologies Breast Cancer Gene Panel

Somatic: All

Germline and Somatic: APC, BRCA1, BRAC2, TP53

With approved FDA inhibitor: BRAF, EGFR, ERBB2


Sample & Assay Technologies Advantages of Targeted Exon Sequencing

Lower Cost:

– Cost of whole genome sequencing is still high

Biology:

– Disease associated genetic varaiants are limited to a

small number of genes

Results:

– Enable deep sequencing, ideal to identify or discover low

frequency variants

Clinical Clinical research



28

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software

DNA Quality Efficiency &

Yield Library Quality Platform-specific

Data processing Variant calling


data




Sample & Assay Technologies Library Preparation


End Repair Adapter ligation

Size selection & Clean up

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Biological Insight

Pooled

amplicons

Sequencing-

ready

amplicons


30

GeneRead DNA Library Prep Kits


Automated on QIAcube:

High reproducibility

No handling variability

Fast procedure that allows

up to 50% time savings

Faster, less variable & more efficient workflow

Sample & Assay Technologies GeneRead DNA Library Prep Kits

31

High yield of Library DNA

Uniform coverage distribution

Input DNA as little as 50 ng


Sample & Assay Technologies Automated Spin column-based Size Selection

Before size selection

Adapter dimers

Adapter monomers

Proven silica-technology delivers precise size selection

Efficient removal of small DNA fragments and adapter-dimers

No tedious bead handling or risk of ethanol carry-over

Can be automated on the QIAcube


Clean up your library before sequencing

Spend your sequencing capacity on amplicons not adapters

GeneRead size selection


33

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Efficiency & Yield




Sample & Assay Technologies Library Quantification & Qualification

Why quantify & qualify your library?

Too much template

Mixed signals and un-resolvable data

Too little template

Reduced sequencing coverage

Reduced read depth

Empty runs

Increase cost per run

Wasted time

Save money and time by running libraries of adequate

quality ONLY

Simple out-of-the-box solution to:

Quantify to ensure uniform starting concentrations of libraries

Qualify to ensure high quality reads

GeneRead DNAseq


Array


Sample & Assay Technologies GeneRead DNAseq Library Quantification Array

Workflow: Quantification and QC in a single PCR run

QC score QC result Proceed?

1–4 Pass Yes

4–8 Marginal With Caution

>8 Fail No

Enables consistent results with every NGS run


Sample & Assay Technologies GeneRead DNASeq Library Quantification Array

36

An Example

Segregate bad libraries before sequencing; Save money and time



37

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Efficiency & Yield




Sample & Assay Technologies DNAseq Panels compatible with NGS platforms

No manipulation of target enrichment protocols needed

Ion Illumina

• MiSeq

• GA Iix

• HiSeq 1000

• HiSeq 2000

• HiSeq 1500

• HiSeq 100

• Torrent PGM

• 314

• 316

• 318

• Proton



39

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software





data

Efficiency & Yield



Data Analysis: Free, Complete and Easy to Use


Primary level

Secondary level

Sample & Assay Technologies Results in Multiple Analysis Format

41

Run Summary Specificity

Coverage

Uniformity

Numbers of SNPs and Indels

Summary By Gene Specificity

Coverage

Uniformity

# of SNPs and Indels


Primary data analysis


42

Features of Variant Report

dbSNP and

COSMIC ID

Predicted amino

acid change

Effect of SNP Impact of SNP

Link to qPCR

somatic mutation

assay

SNP detection

Indel detection


Secondary data analysis


43

Platform

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Pre-Analytics

Sample Insight

Biological Insight

Software



data

Efficiency & Yield




Sample & Assay Technologies Fast biological Interpretation with up-to-date scientific knowledge


Tertiary data analysis

Sample & Assay Technologies Verify your NGS results

Complete Systems downstream of NGS

45

Product

Line

Technology

Detection of

RT2 Profiler

SYBR Green qPCR

mRNA

qBiomarker

ARMS qPCR

Somatic Mutations

PyroMark

Pyrosequencing

Mutations, Variants, and Methylation

Complete workflow: Sample-to-Insight solutions


Microbial

ARMS qPCR

Microbes

Sample & Assay Technologies Microbial DNA qPCR Multi-Assay Kits

28 focused kits around the pathogenicity of a specific microbe

46

Bartonella henselae Pathogenicity

Bordetella pertussis Pathogenicity

Brucella spp Pathogenicity

Campylobacter jejuni Pathogenicity

Chlamydia trachomatis Pathogenicity

Clostridium difficile Pathogenicity

Clostridium perfringens Pathogenicity

C. diphtheriae Pathogenicity

Enterococcus faecalis Pathogenicity

EHEC Pathogenicity

Haemophilus influenzae Pathogenicity

Helicobacter pylori Pathogenicity

Legionella pneumophila Pathogenicity

Listeria monocytogenes Pathogenicity

Mycobacterium spp Pathogenicity

Neisseria meningitidis Pathogenicity

Pseudomonas aeruginosa Pathogenicity

Salmonella enterica Pathogenicity

Shigella dysenteriae Pathogenicity

Strepto. agalactiae Pathogenicity

Strepto. pneumoniae Pathogenicity

Streptococcus pyogenes Pathogenicity

Vibrio cholerae Pathogenicity

Yersinia enterocolitica Pathogenicity

Yersinia spp Pathogenicity

Laboratory Rodent Testing

MRSA: Methicillin-resistant S. aureus

SHV Antibiotic Resistance Genes


Sample & Assay Technologies Microbial DNA qPCR Arrays

Profile or Identify the presence of microbial DNA with any array

Identification experiment answers the following question:

Are any of these microbes or genes present in the sample?

Must be compared against a known negative sample.

Answers are Yes or No.

Profiling experiment answers the following question:

Have the amounts of any of these microbes or genes changed?

Must be compared against a reference sample.

Answers are fold change.


Sample & Assay Technologies Microbial DNA qPCR Array content

14 Arrays

Antibiotic Resistance Genes

Bacterial Vaginosis

Biodefense

Food testing: Dairy

Food testing: Meat

Food testing: Poultry

Food testing: Seafood

Food testing: Vegetable

Intestinal infections

Respiratory Infections

Sepsis

Urinary Tract Infections

Vaginal Flora

Water Analysis




1. Primer on NGS




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

Detection of Mutations in FFPE Lung Adenocarcinoma Samples

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


Question: What are the mutations present in 3 similar Lung Cancers?

Experimental Design


Run Summary

51

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



52

Detection of low frequency variant in lung adenocarcinoma

Genetic variant analysis in FFPE lung adenocarcinoma samples


• Low-quality DNA samples (FFPE): ~1% DNA good enough quality for analysis

• Focus efforts on a focused genes: Save time and money

• 20 Gene Panel on 20 Samples = 1.6 GB of data (WG analysis = 150-200 GB)


53

Data validation: PCR Assay

0

1

2

3

4

5

6

Tumor Sample-1

Tumar sample-2

Tumor Sample-3

Normal Sample

Ct

valu

e

Validation of EGFR:C499Y mutation by qBiomarker Somatic Mutation Assay

EGFR c.1496G>A (p.C499Y)



54

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




1. Primer on NGS




Sample & Assay Technologies Considerations for using NGS in Molecular Microbiology

How will you isolate DNA?

How will you amplify and purify DNA?

Will you be targeting the 16S rRNA or a different gene?

How big is the genome?

How much coverage do you need?

How much data do you need?

Which chemistry/instrument to use?

Which reference genome to use?


Sample & Assay Technologies Summary

57

Sample

Isolation

Target Enrichment

Library Preparation


NGS

Run

Data

Analysis

Sample Insight

Biological Insight


• Isolate high quality DNA to ensure successful sequencing runs

• Enrich for your GOI to enable deeper and more confident sequencing

• Achieve higher library yields to generate higher coverage

• Quantify libraries to achieve consistent results

• Qualify libraries to save money

• Identify variants easily to save time

• Use a comprehensive knowledge database to gain biological insight


Molecular analysis via next-generation sequencing

NMG Annual Fall Meeting, Toronto, Canada

Raed Samara, PhD, PMP

Global Product Manager

October 21, 2013

molecular analysis via next-generation sequencing - national

Documents