Advance Your Genomic Research Using Targeted Resequencing with SeqCap EZ Library Marilou Wijdicks International Product Manager Research For Life Science Research Only. Not for Use in Diagnostic Procedures.
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
Exome Capture Comparison Study - Stanford Efficiency and Coverage Percentage bases covered with >10X coverage with 80M reads NimbleGen 96.8% Agilent 89.6% Illumina 90.0% 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. 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 Indel Detection b. Indels detected in bases targeted c. Indels detected in RefSeq coding region 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 we found that exome sequencing can identify variants that are not evident in WGS (Whole Genome Sequencing) because of greater base coverage after enrichment. 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 BGI Sept 28, 2011 Study compared the performance of three capture technologies: NimbleGen SeqCap EZ Exome Library v1.0 NimbleGen Sequence Capture 2.1M Array Agilent SureSelect Exome 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
Exome Capture Comparison Study FINN Sept 28, 2011 Study compared the performance of two capture technologies: NimbleGen SeqCap EZ Exome Library v2.0 Agilent SureSelect Exome 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... 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
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 Capture any targets from 100kb 50Mb with 2.1 million long oligos in solution Target Regions Prepare with Next-Gen Sequencing Adaptors SeqCap EZ (Solution Capture) Amplify DNA and Enrichment QC Sequence DNA on a Next-Gen Sequencer Genomic DNA Library Preparation Hybridization Capture and Washing Amplification and QC Sequencing
SeqCap EZ Choice Probe Design More Probes, Better Capture Simple Tiling Design (Agilent) Target Region Up to 55,000 (120mer) probes/baits tiled across the region. NimbleGen Sequence Capture Design Target Region 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
NimbleGen SeqCap EZ Library Reduce Sequencing Costs Capture 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 Good depth Average depth Poor depth $ $ 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
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 100.00% 98.00% 96.00% 94.00% 92.00% 90.00% 88.00% 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 ADAPTERS library prep (pre-cap multiplexing) capture INDEXE D INDEXE D (post-cap multiplexing) sequencing (GAIIx or HiSeq2000)
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 pairedend)
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 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 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 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 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 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 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
Percent coverage SeqCap EZ Exome Pre Capture Multiplexing Coverage depth Coverage depth for 3 plex pre capture multiplexing and one lane of Illumina HiSeq
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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