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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 [email protected]
Andrew Crenshaw Asst Director, Operations [email protected]
George Grant Asst Director, Informatics [email protected]
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
