Genetic Analysis Platform - Broad Institute · Genetic Analysis Platform: Technologies to enable...

Genetic Analysis Platform

Wendy Winckler, Ph.D. October 7, 2010

Genetic Analysis Platform: Technologies to enable genomic

discovery Technology Development

Custom Content Genotyping Whole Genome Arrays

Gene Expression

GAP Genotyping

Illumina BeadXpress 48-384 SNPs

Sequenom HME, iPLEX 1-36 SNPs

Illumina Golden Gate Infinium iSelect

384 – 200,000 SNPs

Illumina OmniExpress, Omni1-Quad, Omni 2.5M Affymetrix 6.0, Axiom

700,000 – 2,500,000 SNPs

Whole genome

Candidate genes

Validation Candidate genes Fingerprinting

Variant Spectrum in Human Disease

Common variants Small effect size

Low frequency variants Intermediate effect size

Rare variants Large effect size

MAF

Effe

ct

Rare Common

1st gen GWAS arrays

Next gen GWAS arrays

Sequencing

Whole Genome: Illumina Omni Roadmap HapMap Phase 1 HapMap Phase 3 1,000 Genomes Project

Future GWAS Products

Human1M-Duo

Human660-Quad

HumanHap500

HumanHap300

5M

2.5M

1M 660K 550K

317K Current Projected

Arr

ay P

rodu

cts

Dat

a P

oint

s pe

r Sam

ple

Content Source

MAF > 5% MAF > 2.5% MAF > 1%

SNPs:

CNV:

ETA:

700,000

No

Now

1,100,000

Yes

Now

1,250,000

Yes

Now

2,500,000

Yes

Now

2,500,000

Yes

Early 2011

5,000,000

Yes

Early 2011

Illumina Multi-use Sample Kit

Multi-use sample

prep

-80°

200 ng

750 ng native DNA

200 ng 200 ng

Whole Genome Platform: Affymetrix

SNP 6.0 Axiom

Current arrays: CEU, ASI, custom* *Select from database of 7.4 million SNPs

Whole Genome Genotyping at Broad

Samples to Genotypes: Overview

Sample Intake

Genotyping Data Review

Follow-up Data Analysis

Sample quantitation

Plating

Fingerprinting

Illumina Omni1-Quad

Capacity : >2,000/ wk

Fingerprint comparison

Call Rate cutoff

HapMap concordance

Replication

Fine mapping

Variant discovery

All phases coordinated by a Project Manager

1) Sample Kit to Investigator 2) Samples shipped to Broad

GWAS Sample Process Workflow 3) Biological Samples Platform Registration

5) Fingerprinting 4) Quantify Concentration

Pre-Scan Filter 1: Exclude samples w/ insufficient DNA

Pre-Scan Filter 2: 1)  Gender mismatch 2)  Mendelian inheritance (if

pedigree available)

-provides 2D bar code and LIMS tracking

6) Plate for whole genome scan HapMap

-randomize on phenotype

Data quality checks

HapMap Concordance

Sequenom Illumina

24 SNPs

Fingerprint Comparison

•  Remove poorly performing loci

•  Repeat samples <97% project-specific call rate

Call Rate

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

Illumina Infinium: iSelect Custom Array

•  Custom Infinium assays for 3,000-200,000 SNPs

•  Disease consortia arrays –  CVD –  Metabochip –  Immunochip –  Neuro/cognitive traits

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

Custom CNV on Nanostring

Select up to 200 regions Multiple probes per CNV No amplification; digital counting 200 ng DNA input

Cop

y nu

mbe

r CNV #1 CNV #2

Probe 1

Probe 2

Probe 3

Data courtesy of Nanostring

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

Targeted Resequencing

GAP PCR pipeline: Illumina

454

Genome Sequencing Platform: Illumina Whole Exome

Custom Hybrid Selection

PCR:

~<2000 amplicons

Genome Sequencing Platform: Illumina

GAP Expression Profiling

Luminex

L1000 (CMAP panel) 1-500 custom

transcripts

Illumina Ref 12, Ref 8, WG 6 Affymetrix GeneTitan, HT, cartridge

Agilent standard, custom

18,000 – 48,000 transcripts

Whole genome

High throughput, custom: screening

Nanostring

Standard panels 1-800 custom transcripts

Low throughput, custom: sensitive digital counting

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

GAP Tech Dev/Special Projects

•  Purpose: Dedicated team that has both cross-platform 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

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

GAP intranet website •  Provide Broad community with easy reference for GAP’s

technologies, informatics, project initiation and contact information

iwww.broadinstitute.org/gap

Broad’s Genetic Analysis Platform 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 Project Management Maegan Harden Robb Onofrio Molly Milinazzo

GAP Informatics Boris Boukhgalter Jason Carey Michael Dinsmore Nina Lapchyk Pei Lin Huy Nguyen Howard Rafal Greg Rushton Dennis Ryan Ellen Winchester George Grant