Introduction to Genotyping Technologies

Introduction to Genotyping
Technologies
Andrew Crenshaw
Asst. Director, Operations
Genetic Analysis Platform
September 8, 2011
Overview
•  Whole Genome Microarrays
•  Custom Microarrays
•  Custom “low-plex” genotyping
•  CNVs
•  Analysis tools
Genotyping Platforms
Whole
genome
Candidate
genes
Low plex
genotyping
Illumina OmniExpress, Omni1-Quad, Omni 2.5M, 2.5S, 5M
Affymetrix 6.0, Axiom
500,000 – 5,000,000 SNPs
Illumina Golden Gate
Infinium iSelect, Axiom
384 – 2,300,000 SNPs
Illumina BeadXpress
Fluorescence probe based Systems
Fluidigm
Sequenom MassARRAY
1 – 384 SNPs
What are historical uses of Whole Genome
Microarrays?
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Genome wide association studies (GWAS)
Linkage analysis
Meta-analysis
Standard off the shelf arrays with little room for
customization
–  Technologies available:
•  Illumina “OMNI” arrays
•  Affymetrix Axiom, 6.0, CytoScan
Illumina OMNI Family
Omni
Express*
Omni1Quad
Combination of
High-throughput
common SNPs,
array with industryCNVs, and content
proven quality
from 1kGP.
MAF > 5%
Omni1S-8
Omni2.5-8 Omni2.5S-8*
Takes researchers
from Omni1/
Express to 2.5M
Covers both
common and rare
SNP content
from the 1kGP
MAF >2.5%
~2.5M additional
markers providing
rare 1kGP content
Omni5Quad*
Includes the
markers from
both the 2.5 &
2.5S
MAF > 1%
* Available as semi-custom products
Affymetrix products
SNP 6.0
Axiom
Current arrays: “Hypothesis-free”
CEU, ASI, CHB, PanAFR, custom*
*Select from database of 11 million SNPs
Genotyping Platforms
Whole
genome
Candidate
genes
Low plex
genotyping
Illumina OmniExpress, Omni1-Quad, Omni 2.5M, 2.5S, 5M
Affymetrix 6.0, Axiom
500,000 – 5,000,000 SNPs
Illumina Golden Gate
Infinium iSelect, Axiom
384 – 2,300,000 SNPs
Illumina BeadXpress
Fluorescence probe based Systems
Fluidigm
Sequenom MassARRAY
1 – 384 SNPs
Custom Microarrays
–  Usually uses same technology as whole genome
microarrays, i.e. no new capital equipment or analysis
methods
–  Flexible in amounts of SNPs interrogated
–  Usually high amount of initial investment (>500 arrays
for initial order)
–  From design to delivery around 6-10 weeks
–  Technologies available:
•  Illumina I-Select
•  Affymetrix Axiom myDesign
Illumina Infinium: iSelect Custom Array
•  Custom Infinium assays for
3,000-1,000,000 beadtypes*
•  12 or 24 sample array format
•  Consortia arrays
–  CVD
–  Metabochip
–  Neuro/cognitive traits
–  Immunochip
–  Exome
Affymetrix: Axiom myDesign,
GeneChip Universal Tag Array
•  Axiom
–  TG 1,500 - 500,000 SNPs
–  WG 500,000 – 2,600,000
SNPs
–  Select from a proprietary
database of SNPs
•  GeneChip
–  3K, 5K, 10K, 25K, or 75K
size arrays
–  Molecular Inversion Probe
Technology
Genotyping Platforms
Whole
genome
Candidate
genes
Low plex
genotyping
Illumina OmniExpress, Omni1-Quad, Omni 2.5M, 2.5S, 5M
Affymetrix 6.0, Axiom
500,000 – 5,000,000 SNPs
Illumina Golden Gate
Infinium iSelect, Axiom
384 – 2,300,000 SNPs
Illumina BeadXpress
Fluorescence probe based Systems
Fluidigm
Sequenom MassARRAY
1 – 384 SNPs
Custom “Low plex” Genotyping
–  Common uses: Validation, Extension, Candidate Gene,
Identity determination
–  Panels can consist of SNPs, In/Dels, CNV
–  Typically new designs therefore chance of failure
•  Illumina - Golden Gate, BeadXpress, VeraCode
•  Sequenom MassARRAY
•  Fluorescence probe based Systems
– Beckman – GenomeLab SNPStream
– Millipore – Ampliflour
– Life Technologies – BioTrove/TaqMan/Stokes
– Fluidigm – SNPType (other assays)
Illumina - Golden Gate, BeadXpress,
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Golden Gate
–  384-3,072 beadtypes
BeadXpress
–  48-384 beadtypes
VeraCode
–  1-48 beadtypes
Can be successful with multiple
DNA types
Labor intensive (3 day process)
Becomes more cost effective the
more samples you run
Sequenom MassARRAY
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Single base extension assay
interrogated on a MALDI-TOF
mass spectrometry
HME
–  1 to 7-plex assays
iPLEX
–  1 to 40-plex custom assays
Fluorescence probe based
Systems
•  PCR based technologies in conjunction with flourescence
resonance energy transfer (FRET)
•  Usually Single-plex assays with using arrays to achieve
high throughput multi-plexing
•  Relatively low DNA input (5-60ng)
TaqMan
Companies offering FRET based
assays
Millipore – Amplifluor
http://www.millipore.com/
Life Technologies – TaqMan
BioTrove/Stokes
http://www.invitrogen.com/site/us/en/
home/brands/taqman.html?
Fluidigm – SNPType
BioMark
http://www.fluidigm.com/
Custom “Low plex” Genotyping
Technology
Benefits
Drawbacks
Illumina BeadXpress
>95% assay conversion
rate
High minimum sample
number (500-1000s)
>>99% call rate and
accuracy
6-12 week assay design/
synthesis
Less sensitive for rare
variants and mutations
Sequenom MassARRAY
Short turn around from
design to data
Sensitive to 1% MAF
Lower assay design and
pass rate
Low multiplexing
Higher cost per genotype
FRET technologies
Simple workflow
Various protocols can be
used to overcome
difficult areas
Need additional
technology to increase
efficiency and lower
costs
Single-plex without
technology
Custom Copy Number Variants
(CNVs)
Whole
Genome
Mid-size
targeted
Low-plex
Agilent CGH, Affymetrix 6.0, Nimblegen
500,000 – 2,300,000 CNVs
Infinium iSelect
3,072– 2,300,000 CNVs
NanoString
200-400 CNVs
FRET based Systems
1-48
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
Affymetrix 6.0 & BirdSuite
Fawkes
Copy number
Custom CNV on Nanostring
CNV #1
CNV #2
Probe 1
Probe 2
Probe 3
Select up to 400 regions
Multiple probes per CNV
No amplification; digital counting
200 ng DNA input
Investment of at least 192 samples
Data courtesy of Nanostring
Analysis
PLINK
GISTIC
Birdsuite
www.broadinstitute.org
Conclusion
•  Wide selection of technologies available to tailor fit to the
question to be answered
–  Limiting factors
•  Cost
•  DNA quality/quantity
•  Hypothesis driven or confirmation