advance your genomic research using targeted ......marilou wijdicks international product manager...
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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%
Exome Capture Comparison Study - Stanford
Efficiency and Coverage
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
b. Indels detected in bases targeted
c. Indels detected in RefSeq coding region
Exome Capture Comparison Study - Stanford
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)
Nature Biotechnology Published online 25 September 2011 doi:1038/nbt.1975
http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.1975.html
Exome Capture Comparison Study - Stanford
Comparison to 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
“…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
http://genomebiology.com/2011/12/9/R94/abstract
“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
• 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 Exome Library v2.0
– 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.
SeqCap EZ Exome Library v3.0 New!
– Superior coverage due to empirical rebalancing
– Targeting 64 Mb human genomic regions, covering coding
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
Exon 1 Exon 2 Exon 3
$
$
Minimum data required
NimbleGen SeqCap EZ Library
Reduce Sequencing Costs – Capture Uniformity
Good depth Average depth Poor depth
Exon 1 Exon 2 Exon 3
$
$
$
Minimum data required
Good depth Average depth Poor depth
NimbleGen SeqCap EZ Library
Reduce Sequencing Costs – Capture Uniformity
NimbleGen SeqCap EZ Exome Products
New 64Mb 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 Exome Library v2.0
– 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.
SeqCap EZ Exome Library v3.0 New!
– Superior coverage due to empirical rebalancing
– Targeting 64 Mb human genomic regions, covering coding
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
SeqCap EZ Exome Pre Capture Multiplexing
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
SeqCap EZ Exome Pre Capture Multiplexing
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
SeqCap EZ Exome Pre Capture Multiplexing
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
SeqCap EZ Exome Pre Capture Multiplexing
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)
Pre Capture (ave 7 plex)
Pre Capture (ave 7 plex)
One Lane of HiSeq v3 sequenced at 100bp reads and raw reads/not subsampled
SeqCap EZ Exome Pre Capture Multiplexing
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
SeqCap EZ Exome Pre Capture Multiplexing
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.
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