Advance Your Genomic Research Using Targeted ......Marilou Wijdicks International Product Manager...

Advance Your Genomic Research Using Targeted

Resequencing with SeqCap EZ Library

For Life Science Research Only. Not for Use in Diagnostic Procedures.

Marilou Wijdicks

International Product Manager – Research

Why Perform Targeted Resequencing?

Focus Your Research on Relevant Regions

• Genetic Diseases: identify mutations that correlate to disease or contribute

to genetic risk.

– Follow up of GWAS, re-sequence of disease associated regions

– Exome sequencing for discovery of mutations in rare diseases

– Candidate genes panels involved in disease pathways

• Cancer: identify somatic mutations involved in tumorigenesis or metastasis,

discover germ line mutations that contribute to cancer risk.

– Focus on exome or cancer gene panel for comprehensive coverage of

known and rare mutations

– Deep sequencing of cancer related regions to identify rare mutations

present in heterogeneous tumor tissues

Exome Capture Comparison Study - Stanford

Sept 25, 2011

• Study compared the performance of three capture technologies:

– NimbleGen SeqCap EZ Exome Library v2.0

– Agilent SureSelect Exome

– Illumina TruSeq Exome

• Comparison of single nucleotide variant (SNV) detection between

exome and whole genome sequencing

Clark et al., Performance comparison of exome DNA sequencing technologies (2011)

Nature Biotechnology Published online 25 September 2011 doi:1038/nbt.1975

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.1975.html


“…NimbleGen SeqCap EZ Exome demonstrate(s) the

highest efficiency target enrichment, able to adequately

cover the largest proportion of its targeted bases with the

least amount of sequencing.”

Percentage bases covered with >10X coverage

with 80M reads

NimbleGen 96.8%

Agilent 89.6%

Illumina 90.0%


Efficiency and Coverage





b. Indels detected in bases targeted

c. Indels detected in RefSeq coding region


Indel Detection

“In shared and RefSeq regions, NimbleGen had the highest

sensitivity for detecting indels because of higher average

read depth.” Clark et al., Performance comparison of exome DNA sequencing technologies (2011)





Comparison to Whole Genome Sequencing




“…we found that exome sequencing can identify variants that

are not evident in WGS (Whole Genome Sequencing)

because of greater base coverage after enrichment.”


Exome Capture Comparison Study – BGI

Sept 28, 2011

“…the NimbleGen platforms showed better uniformity of coverage

and greater genotype sensitivity at 30-100 folds sequencing depth.”

Asan et al., Comprehensive comparison of three commercial human whole-exome capture platforms (2011)

Genome Biology Published online 28 September 2011, 12:R95

http://genomebiology.com/2011/12/9/R95/abstract

• Study compared the performance of three capture technologies:

• NimbleGen SeqCap EZ Exome Library v1.0

• NimbleGen Sequence Capture 2.1M Array

• Agilent SureSelect Exome


Exome Capture Comparison Study – FINN

Sept 28, 2011

Sulonen et al., Comparison of solution-based exome capture methods for next generation sequencing (2011)

Genome Biology Published online 28 September 2011, 12:R94


“In our data, libraries captured with NimbleGen kits aligned more

accurately to the target regions. NimbleGen SeqCap v2.0 most

efficiently covered the exome with a minimum coverage of 20x....”

• Study compared the performance of two capture technologies:

• NimbleGen SeqCap EZ Exome Library v2.0

• Agilent SureSelect Exome


NimbleGen SeqCap EZ Products

From small custom gene panel to Exome

• Unique design process with up to 2.1 million long DNA probes to

ensure high target coverage and good capture uniformity.

• Optimized protocols for Illumina and Roche 454 Sequencing

systems

SeqCap EZ Choice Library

– Capture any regions from 100kb – 50Mb.

– Available in convenient kit sizes: 12, 24, 48, 96, 384, and 960

reactions.

SeqCap EZ Exome v2.0 Library

– Superior coverage due to empirical rebalancing

– Targeting 44 Mb human genomic regions, covering coding

exons from ~ 20K unique genes from RefSeq and CCDS, and

710 miRNA genes.

NimbleGen SeqCap EZ Exome Products

New 64Mb Exome




systems

SeqCap EZ Exome Library v2.0




710 miRNA genes.

SeqCap EZ Exome Library v3.0 New!



exons from ~ 20K unique genes from RefSeq, CCDS,

Ensembl, Vega, Gencode, and miRBase.

SeqCap EZ Workflow

Genomic DNA Library Preparation Hybridization Capture and Washing Amplification and QC Sequencing

Target

Regions

Amplify DNA and

Enrichment QC

Prepare with Next-Gen

Sequencing Adaptors

Sequence DNA

on a Next-Gen

Sequencer

SeqCap EZ

(Solution Capture)

Capture any targets from 100kb – 50Mb with 2.1 million long oligos in solution

SeqCap EZ Choice Probe Design

More Probes, Better Capture

Target Region

Target Region

Up to 55,000 (120mer)

probes/baits tiled across the

region.

Simple Tiling Design (Agilent)

NimbleGen Sequence Capture Design Up to 2,100,000 (50-105mer)

probes selected for the region

using special algorithm.

Significantly more probes, 38X.

Benefits:

•Higher density tiling

•Redundancy to reduce risk

•Ability to move probes for

better uniformity

Good depth Average depth Poor depth

Exon 1 Exon 2 Exon 3

$

Minimum data required

NimbleGen SeqCap EZ Library

Reduce Sequencing Costs – Capture Uniformity


$

$






$

$

$





NimbleGen SeqCap EZ Exome Products

New 64Mb Exome




systems

SeqCap EZ Exome Library v2.0




710 miRNA genes.

SeqCap EZ Exome Library v3.0 New!



exons from ~ 20K unique genes from RefSeq, CCDS,

Ensembl, Vega, Gencode, and miRBase.

NimbleGen SeqCap EZ Exome Library v3

Achieve the most comprehensive coverage of coding regions

88.00%

90.00%

92.00%

94.00%

96.00%

98.00%

100.00%

RefSeq Vega Gencode Ensembl CCDS miRBase

SeqCap EZ Exome v3 SureSelect TruSeq

Coverage of Coding Exons

How does SeqCap EZ Exome Library v3.0

compare?

Gigabases of input sequence

Coverage of raw sequence

How does SeqCap EZ Exome v3.0 compare?

Multiplex SeqCap EZ – Illumina TruSeq Library

library prep

capture

sequencing

(pre-cap multiplexing)

(post-cap multiplexing)

(GAIIx or HiSeq2000)

ADAPTERS INDEXE

D

INDEXE

D

Precap Multiplexing with Illumina TruSeq Libraries

Balanced distribution of indexed reads

Million

Read

• 8 Samples (4 tumor/normal pairs)

• Each capture reaction was sequenced with 1 lane on HiSeq (2x100bp paired-

end)

SeqCap EZ Exome Pre Capture Multiplexing

Experimental Summary

Pre Capture vs. Post Capture Multiplexing

Yoruban Trio captured with both 3 plex pre

capture and 3 plex post capture

Pre and post capture multiplex sequenced on

separate Illumina HiSeq lanes

2x100 bp

Exome 7 plex Pre Capture Multiplexing

3 Exome captures with 7 samples pre captured

in each capture

Each replicate sequenced on one Illumina HiSeq

lane

2x100 bp

Protocols for use with Illumina TruSeq Libraries Preparation Kit can be found at nimblegen.com


Balanced index reads distribution compared to post capture

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

NA19238, postcap

NA19239, postcap

NA19240, postcap

NA19238, precap

NA19239, precap

NA19240, precap

One Lane of HiSeq v3 sequenced at 100 bp reads and subsampled to 75 Million

Reads


Post Capture -

3 samples

Pre Capture –

3 samples

% On Target ~72% ~71%

Average Coverage ~50x ~46x

Median Coverage ~40x ~38x

% bases at > 10 coverage ~93% ~93%

% PCR Duplicates ~1% ~2.5%

% Sensitivity ~97% ~97%

% Specificity ~99% ~99%

One Lane of HiSeq v3 sequenced at 100 bp reads and subsampled to 75 Million

Reads


Balanced distribution of indexed reads raw reads

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

NA19238

NA19239

NA19240

One Lane of HiSeq v3 sequenced at 100 bp reads and raw reads/not

subsampled


Av. raw reads 7 plex pre capture on 3 separate HiSeq

lanes

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Pre Capture (ave 7 plex)



One Lane of HiSeq v3 sequenced at 100bp reads and raw reads/not subsampled


Pre Capture

– 3 samples

Pre Capture -

7 samples

% On Target ~71% ~72%

Average Coverage ~75x ~33x

Median Coverage ~60x ~26x

% bases at > 10

coverage

~95% ~87%

% PCR Duplicates ~4% ~2.5%

% Sensitivity ~98% ~93%

% Specificity ~99% ~98%

One Lane of HiSeq v3 sequenced at 100 bp reads and raw reads/not

subsampled


Coverage depth for 3 plex pre capture multiplexing and one lane of Illumina HiSeq

Coverage depth

Perc

en

t co

ve

rag

e

Automated SeqCap EZ Workflow Using the

Caliper Sciclone NGS Workstation

• Optimized scripts written for

the SeqCap EZ workflow

• Capability to load tips as

needed based on number of

samples

• 24 deck locations for more

walk-away time

• Small size to fit on bench top

• Fully enclosed system for

contamination protection

• Process 288 samples per

week

NimbleGen SeqCap EZ Exome v.2.0

Automation vs. Manual

Chr 22

Sequence Coverage from Automated Protocol on Sciclone

Sequence Coverage from Manual Protocol

Target Exons

HapMap sample NA12762 was captured with either automated protocol or

manual protocol with NimbleGen SeqCap EZ Exome v2.0.

IMPORTANT NOTICE

Intended Use

Unless explicitly stated otherwise, all Roche NimbleGen products

and services referenced in this presentation are intended for the

following use:

For Life Science Research Only.

Not for Use in Diagnostic Procedures.

454, 454 SEQUENCING, GS JUNIOR, NIMBLEGEN and SEQCAP are

trademarks of Roche.

All other product names and trademarks are the property of their respective

owners

© 2011 Roche NimbleGen, Inc.

We Innovate Healthcare

Advance Your Genomic Research Using Targeted ......Marilou Wijdicks International Product Manager...

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