Genetic Analysis Platform. Wendy Winckler, Ph.D. October 7, 2010

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1 Genetic Analysis Platform Wendy Winckler, Ph.D. October 7, 2010

2 Whole Genome Arrays Custom Content Genotyping Genetic Analysis Platform: Technologies to enable genomic Gene Expression discovery Technology Development

3 GAP Genotyping Whole genome Candidate genes Validation Candidate genes Fingerprinting Illumina OmniExpress, Omni1-Quad, Omni 2.5M Affymetrix 6.0, Axiom 700,000 2,500,000 SNPs Illumina Golden Gate Infinium iselect ,000 SNPs Illumina BeadXpress SNPs Sequenom HME, iplex 1-36 SNPs

4 Variant Spectrum in Human Disease Sequencing Rare variants Large effect size Effect Next gen GWAS arrays Low frequency variants Intermediate effect size 1st gen GWAS arrays Common variants Small effect size Rare MAF Common

5 Whole Genome: Illumina Omni Roadmap Content Source HapMap Phase 1 HapMap Phase 3 1,000 Genomes Project Future GWAS Products Array Products Human1M-Duo Human660-Quad HumanHap500 HumanHap M 5M Data Points per Sample 1M 550K 660K 317K Current Projected

6 SNPs: 700,000 1,100,000 1,250,000 2,500,000 2,500,000 5,000,000 CNV: No Yes Yes Yes Yes Yes ETA: Now Now Now Now Early 2011 Early 2011 MAF > 5% MAF > 2.5% MAF > 1%

7 Illumina Multi-use Sample Kit 750 ng native DNA Multi-use sample prep ng 200 ng 200 ng

8 Whole Genome Platform: Affymetrix SNP 6.0 Axiom Current arrays: CEU, ASI, custom* *Select from database of 7.4 million SNPs

9 Whole Genome Genotyping at Broad

10 Samples to Genotypes: Overview Sample Intake Genotyping Data Review Data Analysis Follow-up Sample quantitation Plating Fingerprinting Fingerprint comparison Call Rate cutoff HapMap concordance Replication Illumina Omni1-Quad Capacity : >2,000/ wk Fine mapping Variant discovery All phases coordinated by a Project Manager

11 GWAS Sample Process Workflow 1) Sample Kit to Investigator 2) Samples shipped to Broad 3) Biological Samples Platform Registration -provides 2D bar code and LIMS tracking HapMap 6) Plate for whole genome scan 5) Fingerprinting 4) Quantify Concentration -randomize on phenotype Pre-Scan Filter 2: 1) Gender mismatch 2) Mendelian inheritance (if pedigree available) Pre-Scan Filter 1: Exclude samples w/ insufficient DNA

12 Data quality checks Sequenom 24 SNPs Illumina Remove poorly performing loci Repeat samples <97% projectspecific call rate Fingerprint Comparison Call Rate HapMap Concordance

13 GWAS Follow up Studies Genotype SNPs in an extended panel of samples Replication Fine mapping Type CNVs in an extended panel Validation Replication Directed sequencing Sequence candidate region in a panel of reference samples to survey common variation Sequence candidate genes in many cases/controls for rare variation

14 Illumina Infinium: iselect Custom Array Custom Infinium assays for 3, ,000 SNPs Disease consortia arrays CVD Metabochip Immunochip Neuro/cognitive traits

15 Low- to mid-plex Custom Genotyping Illumina GoldenGate Up to 3,000-plex custom assays Illumina BeadXpress Up to 384-plex custom assays Sequenom HME: 1 to 7-plex custom assays iplex: 1 to 36-plex custom assays Fingerprinting: 24-plex iplex assays

16 Custom CNV on Nanostring Copy number CNV #1 CNV #2 Probe 1 Probe 2 Probe 3 Select up to 200 regions Multiple probes per CNV No amplification; digital counting 200 ng DNA input Data courtesy of Nanostring

17 Custom Agilent CGH 60-mer tiled probes for accurate copy number characterization New option to include SNPs on same array Allele specific copy number and UPD Haplotypes Choose from 28M predesigned CN probes and 65,000 SNPs Formats: 1x1M, 2x400K, 4x180K, 8x60K Can purchase as few as 2 custom slides

18 Targeted Resequencing Whole Exome Custom Hybrid Selection Genome Sequencing Platform: Illumina Genome Sequencing Platform: Illumina PCR: ~<2000 amplicons GAP PCR pipeline: Illumina 454

19 GAP Expression Profiling Whole genome Low throughput, custom: sensitive digital counting High throughput, custom: screening Illumina Ref 12, Ref 8, WG 6 Affymetrix GeneTitan, HT, cartridge Agilent standard, custom 18,000 48,000 transcripts Nanostring Standard panels custom transcripts Luminex L1000 (CMAP panel) custom transcripts

20 Nanostring Gene Expression Custom gene expression Multiplex up to 800 targets No amplification Low sample input (100ng RNA) mirna expression 100ng RNA input No amplification Current throughput: 144 samples per week

21 GAP Tech Dev/Special Projects Purpose: Dedicated team that has both crossplatform expertise and bandwidth to undertake Special Projects and pilot new technologies Goals: Actively pursue new technologies and protocols Work with platform managers to establish a robust process, validate, and implement new production technologies Design, execute, and troubleshoot special projects that do not fit into typical GAP workflows

22 Please contact us For help with project design When planning for grants If you have ideas about new ways to apply a GAP technology or new capabilities that you would like to see in GAP With any questions Wendy Winckler Director winckler@broadinstitute.org Andrew Crenshaw Asst Director, Operations crenshaw@broadinstitute.org George Grant Asst Director, Informatics ggrant@broadinstitue.org

23 GAP intranet website Provide Broad community with easy reference for GAP s technologies, informatics, project initiation and contact information iwww.broadinstitute.org/gap

24 Broad s Genetic Analysis Platform GAP Informatics Boris Boukhgalter Jason Carey Michael Dinsmore Nina Lapchyk Pei Lin Huy Nguyen Howard Rafal Greg Rushton Dennis Ryan Ellen Winchester George Grant Project Management Maegan Harden Robb Onofrio Molly Milinazzo Production and Tech Dev Diane Gage Melissa Parkin Wendy Brodeur Jody Camarata Mike DaSilva Kunsang Gualtsen Ryan McGinty Jordan Sullivan Teni Minnetyan Karina Klonoski Liuda Ziaugra Tony Rachupka Michelle Redman Cherylyn Smith Katie Larkin Amy Camargo Supriya Gupta Brad Taylor Prapti Pokharel Dave Murchison Scott Donovan Andrew Crenshaw

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