Benefits to validating GWAS studies with custom genotyping
Daniel Peiffer, PhDSr. Product Manager
Genotyping Applications
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Cycle of discovery and validation
Custom genotyping applications
Application in study design
Overview of Illumina technologies
Overview
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Next-Gen SequencingIdentifying Variants
Low Density PanelsBarcoding &
QCing Samples
Targeted resequencing
GWAS ArraysConfirming
Variants
Low Density Arrays
Deploying Variants
Custom Validation Arrays
Re-Confirming Variants
RNA-sequencing
Presenter
Presentation Notes
Discuss utility of each step as outline for entire process Low density panels as a new step gaining traction low density at end as a translational step to diagnostic and therapeutic panels ref omni discussion from Jen/Carsten at GWAS step
Common Considerations When Designing a Custom GT Project
Complexity of disease / focus of study
Breadth of information currently known on disease / focus
Sample size targeted for adequate power of study
Availability (value) of samples
Consistency of sample preparation and identification
Availability of funding
Timelines driving project
Presenter
Presentation Notes
All of these contribute to how the entire study is designed and what level of info is available for expanding the follow up into validation and new discovery. Walk through each point to describe how it impacts the study deisgn.
• 1 - 384-plex• GoldenGate Assay• ASPE Assay• Cost effective solution for QC and screening
VeraCode®
• 48 samples – up to 4-plex• Open platform, supports all chemistries and real-time PCR applications,
including HRM, GEX, SNP genotyping, viral load and SBS library validation• +/- 0.1oC temperature uniformity, 1 copy sensitivity• 12”x12”x13”, 30 lbs
Eco™ Real-Time PCR
System
Presenter
Presentation Notes
Walk through each one, can give a little more info than what is actually here
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200,0003,000 60,800
Infinium iSelect®HD Custom Genotyping
24x1 12x1
Presenter
Presentation Notes
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Researchers incorporate some or all of the following into their study design:
Key hits from initial GWAS
SNPs with compelling ρ trends from previous studies (if done)
SNPs from GWAS arrays in LD blocks around hit regions
SNPs in regions of interest (e.g., HLA, MHC, suspected gene regions)
Wild card SNPs of interest
Loci for copy number variation information
Custom Genotyping Applications:Replication / Validation / Candidate Gene Region
Presenter
Presentation Notes
The meat of the presentation Yes doing key hits is necessary but there is so much extra value in expanding the set to include more info. Pubs = few recent examples. 100’s alone on ILMN tech.
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Core Service labs like CIDR* have begun implementing barcoding as a standard process prior to running GWAS or large custom genotyping projects
Findings: – Avg. savings on a 3,000-sample GWA study was $40,000 (excluding labor)
*ASHG 2009 Poster: Impact of sample pretesting in a high through-put genotyping facility. B. Marosy, C. Boehm, B. Craig, J.Romm, C. Oncago, M. Zilka, M. Adams-Carr, Y. Osimokun, K. Hetrick, H. Ling, E. W. Pugh, K. F. Doheny CIDR/GRCF-IGM, JHU-SOM, Baltimore, MD.
Presenter
Presentation Notes
What is the value of sample QC. Walk through example of how this has helped CIDR and others like it Be respective and aware that not all labs will do this, or have the resources to afford it or feel it necessary but speak to value of cost savings plus additional value of labor saved etc.
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Custom Genotyping Applications:Implementing LIMS in High Throughput Processing
Track samples and reagents through the entire process
Reduce sample to sample variability
Reduce sample handling and processing errors
Dramatically increase throughput
Tracking QC
Workflow Management Reporting
LIMS
Presenter
Presentation Notes
Very brief slide for most audiences. Speak to increased accuracy and decreased variability if labs have high value samples and/or high throughput needs
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Rat Citrus
Wheat HoneyBee
Armadillo
Cacao
Rhesus
Cat
Canola
Rice
Atlantic Cod
LettuceFalse
Brome
SheepSwine
HorseCattle
Human
PeachPoplar
Rye grass
Turkey
Soy BeanBarleyCitrus
Cotton Rapeseed Spruce
MouseCornPine
Chicken
Dog
TomatoAtlantic Salmon Grape
Vine
Sweet Sorghum
P. falciparum
Zebra Finch
Zebra Fish
Applications
Custom Genotyping on the Infinium AssayThe right content lets you capture more biology
Genome-wide selection
Genetic Prediction
SNP discovery
Commercial Agriculture screening panels
Targeted follow-up studies after GWAS
Targeted disease panels
Targeted rare variant panels
Sample QC and tracking panels
Consumer Genomics
Forensics
Presenter
Presentation Notes
Moving away from genome-wide association studies, I’d like to now switch gears and tell you about Illumina’s portfolio for focused and custom genotyping products. As you can see in the collage of pictures, Illumina has extensive experience designing custom arrays for a variety of human and non-human species. Some of the major applications researchers come to us for include: ‘read the list of apps from the slide’ Our custom gt offerings span a very dynamic range of densities: from 200K all the way down to 48-plex
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Example of Focused Genotyping Panels - Ag
BovineSNP50/BovineHD– Developed in collaboration with USDA – Beltsville, University of Missouri, and
University of Alberta
CanineSNP20/CanineHD– Developed in collaboration with the LUPA consortium
– ~170K validated SNP probes derived from the CamFam2.0 assembly
EquineSNP50 – Developed in collaboration with: International Equine Genome Mapping
Workshop and the Morris Animal Foundation's Equine Genome Consortium
PorcineSNP60– Developed in collaboration with Int’l Porcine SNP Consortium (Martien
Groenen: Wageningen Univ)
OvineSNP50– Developed in collaboration with the International Sheep Genomics Consortium
(ISGC)
MaizeHD– Developed in collaboration with Pioneer, Syngenta, USDA, and Trait Genetics
Presenter
Presentation Notes
Here are a few example of how our customers are using the iSelect platform to create 100% customizable content for use in cardiovascular disease, cattle and dog typing etc…These panels all started as custom iSelect projects and were later commercialized and made available to the public. This platform is very popular and customers are using it for both human and non-human CVDSNP55 Developed in collaboration with the Institute of Translational Medicine and Therapeutics at the University of Pennsylvania, the Broad Institute and the National Heart, Lung and Blood Institute’s Candidate-gene Association Resource Over 55,000 SNPs in candidate genes selected for vascular disease phenotypes Allow researchers to access approximately 2,100 genes believed to be involved in myocardial infarction, heart failure, stroke, insulin resistance, metabolic disorders, dyslipidemia and inflammation Consortium plans to analyze over 120,000 samples from large population studies and clinical trials Multi-sample format (12x1), Call Rates greater than 99% Shipping in Q1 BovineSNP50 Currently Shipping Developed 50,000 SNP Illumina iSelect® assay USDA-ARS Beltsville Agricultural Research Center: Bovine Functional Genomics Laboratory and Animal Improvement Programs Laboratory University of Missouri University of Alberta USDA-ARS US Meat Animal Research Center Over 54,000 robust SNPs per assay (including 23K+ from the Genome Analyzer, 12K+ from Bovine HapMap, etc.) Have plans to genotype >30,000 animals for multiple projects Over 105m head of cattle in the US alone - SNP Genotyping used for selective breeding to determine elements of beef production such as “marbling quality” (518 SNPs associated with marbling), and dairy production Goals of Initial Research - SNPs used to map QTLs and aid in selective breeding of cattle - Gene discovery for better meat and milk production and quality - Discover elements responsible for disease, growth, and development - Assist modeling of human disease - Study the history of domestication of cattle - Explore evolution of genomes under strong selection CanineSNP20 Currently Shipping Develop 20,000 SNP Illumina iSelect® assay Over 22,000 validated SNP probes derived form the CanFam 2.0 assembly (Broad’s Dog Genome Sequencing Project) Have plans to genotype over 4,000 animals for multiple projects EquineSNP50 The EquineSNP50 genotyping BeadChip is currently in development and due to launch Q1 this year. Developed in collaboration with the International equine Genome Mapping workshop and the Morris Animal Foundation’s equine Genome Consortium and with content derived from Broad Institute’s Equine Genome Sequencing project, this array promises to be highly informative across equine breeds and ideal for genome-wide investigation of genetic variation in horses.
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Examples of Focused Genotyping Panels - Human
HumanCVD– Developed in collaboration with the Institute of Translational Medicine and Therapeutics at
the Univ. of Pennsylvania, the Broad Institute, and the NHLBI
– Candidate diseases include myocardial infarction, heart failure, stroke, insulin resistance, metabolic disorders, dyslipidemia and inflammation
Human Cardio-Metabo– Developed in collaboration with Broad, University of Michigan, Sanger
– Interrogates loci of interest for researchers studying the genetics of the Cardiovascular and Metabolic disease in humans
Presenter
Presentation Notes
CVDSNP55 Developed in collaboration with the Institute of Translational Medicine and Therapeutics at the University of Pennsylvania, the Broad Institute and the National Heart, Lung and Blood Institute’s Candidate-gene Association Resource Over 55,000 SNPs in candidate genes selected for vascular disease phenotypes Allow researchers to access approximately 2,100 genes believed to be involved in myocardial infarction, heart failure, stroke, insulin resistance, metabolic disorders, dyslipidemia and inflammation Consortium plans to analyze over 120,000 samples from large population studies and clinical trials Multi-sample format (12x1), Call Rates greater than 99% Shipping in Q1 You’ll have to wing it on Metabo, but I think you will be just fine... CanineSNP20 Currently Shipping Develop 20,000 SNP Illumina iSelect® assay Over 22,000 validated SNP probes derived form the CanFam 2.0 assembly (Broad’s Dog Genome Sequencing Project) Have plans to genotype over 4,000 animals for multiple projects
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The Right Technology Enables Truly Novel Discovery
Cont
ent
Den
sity
Time from Content Design to Analyzed Results
Months
Targeted Re-Sequencing
Array Genotyping Multi-plex PCRMass Spec
Hig
hLo
w
Structural Variations, SNP Discovery
Fine mapping hit regionsSNPs in LD, wild card
Top Hits
Weeks
Presenter
Presentation Notes
First reference Jen (or Carsten’s) swoosh slide Second talk about levels of content Third talk about technologies and time to make use of it Allude to Eco for PCR. Again speak to value of additional content as move to array GT (ref iSelect, GG, etc.) really point out length of time for TRS but that you do get a lot more information
Consistency of sample preparation and identification
Timelines driving project
Availability of funding
Speed Results
Presenter
Presentation Notes
Really phrase it that the customer needs to evaluate all technologies and what will fit with their needs First talk to left side – so how do you choose a product. Funding and time really the key drivers But if available you get so much more from additional content Second speak to right side..other components we didn’t talk about as much LIMS/automation – brief mention that not all technologies can integrate these and depending on needs could be an issue DQ – spend more time here. Mention ILMN tech is know for its DQ Mention mass spec has issues that result in high re-queue rates and this will impact time and cost Mention that several companies have tried to increase throughput and plex levels of Taqman. Some have been more successful than others. Design Support – spend more time here -talk about ADT and all the support ILMN offers. Mention not the same level of support for all technologies - Can allude to some companies providing technology while custom design support is through an alternate vendor (i.e. fludigm), and that we will work as closely as needed.
