SNP Genotyping
• Introduction to Fluidigm technology James Miller
• Fluidigm genotyping applications Paul Lacaze
• Case study Jonathan Beesley
Overview
Why SNP genotyping?
Dynamic Array IFC Architecture (96.96)
Technology
Fluid Line
Control Line
Open NanoFlex™ Valve
Fluid Line
Fluid Line
Control Line
Closed NanoFlex Valve
Sample Assay
Close Interface Valve
Load Samples
Sample Assay
Load Assays
Sample Assay
Close Containment Valve
Sample Assay
Mix Sample and Assay
Reaction
96.96
9,216 data points(= 24 x 384)
Dynamic Array™ Integrated Fluidic Circuits
192.24
4,608 data points(= 12 x 384)
23 mL master mix6 µL each 80X assay (576 µL total)24 x 384-well plates8 days
Traditional
genotyping
system
96 samples x 96 SNPs (TaqMan)
240 µL master mix0.625 µL each 80X assay (60 µL total)1 chip4 hours
Fluidigm
System
96 samples x 96 SNPs (TaqMan)
BioMark HD™ Real-Time PCR System
EP1™ System
FC-1™ Cycler-Embedded cycler in the Biomark HD
FC1TM System picture courtesy of CRITFC
Increasing Throughput
Pipette Load PCR
20 min 96.96:192.24:
Scan
< 10 min
Genotyping Workflow
90 min30 min
60 – 100 min30 – 60 min
Text box
Fast preparation, simple workflow
• Minimal hands-on time, pipette steps up front only
• Samples are easily processed in batches of 96 or 192 per day
• PCR reactions separated into individual reaction chambers
with no high multiplexing in tube = efficient reactions
Flexibility
• Assay design service for all species
• Low DNA input required, can accommodate large plant genomes
• Flexible and interchangeable assay design
= change whenever/whatever you want from run to run
Advantages of Fluidigm for SNP Genotyping
1. Many samples
2. Easy workflow and quick time to result
3. Low cost per reaction
4. Flexibility of assay design and selection
When is Fluidigm the best time choice for SNP genotyping?
Genotyping Basics
X
X
Genotyping by PCR
X
Y
Y
Y
X – PCR assay 1 (FAM)
Y – PCR assay 2 (VIC)
FAM
VIC or HEX
Genotyping Basics – Relative Fluorescence
• XX (homozygote)High– FAM Low – VIC or HEX
• XY (heterozygote)Intermediate – FAM Intermediate – VIC or HEX
• YY (homozygote)High– VIC or HEXLow – FAM
Relative
Fluorescence
Cycle
VIC or HEX
FAM
• XX (homozygote)High– FAM Low – VIC or HEX
• XY (heterozygote)Intermediate – FAM Intermediate – VIC or HEX
• YY (homozygote)High– VIC or HEXLow – FAM
Genotyping Basics – Relative Fluorescence
96.96 Genotype Map
Standard Genotyping Chemistry
• TaqMan® Probes (Applied Biosystems)
• Allele-specific probes
• SNPtype™ Assays (Fluidigm)
• Allele-specific primers
• Universal probe = more economical
• Can be designed for any species
Rev
Fwd
STA primer
C
T
Y
SNPtype™ - Allele Specific Primers
ASP = Allele Specific Primer (Fwd)LSP = Locus Specific Primer (Rev)STA = Specific Target Amplification Primer (STA)
SNPType – Allele-Specific Primers
C
C
C
T
T
T
T
ASP1
ASP2
LSP
Allele-specific Forward primers x2
Reverse primer
C
T
C
T
C
Tagged Amplicons
Master mix + fluor/quencher
(SNPType reagent)
C
T
Allelic Discrimination
Rev
Fwd
STA primer
C
T
Y
STA - Specific Target Amplification (PreAmp)
ASP = Allele Specific Primer (Fwd)LSP = Locus Specific Primer (Rev)STA = Specific Target Amplification Primer (STA)
SNPtype Assays(Allele-specific primers)
TaqMan Probes(Allele-specific probes)
SNP1
SNP2
SNPtype Assays vs. TaqMan Probes
Multiplex Primer Pool
(0.2X or 500 nM)
Multiplex PCR Master Mix (Qiagen)
DNA sample(low conc)
14 cycles
Preamplified DNA
1:100 dilution
+ +
Specific Target Amplification (STA)
Specific Target Amplification (STA)
• Improves data quality under conditions of:
• Low and/or variable sample concentration (<10ng/ul)
• Low sample quality (i.e. degradation)
• Carryover of inhibitory compounds from extraction (Example: plant extractions)
Cacao Genotyping: Call RatesgD
NA
STA
SNP 1 SNP 2 SNP 3
66.67% 0.00%70.37%
100.0% 100.0%100.0%
5 copies 10 copies 20 copies
50 copies 100 copies 150 copies
Relative Copies Per Reaction Chamber
Heterozygote Spread
60 ng/µL human genomic DNA =
50 genomic copies/chamber =
25 allelic copies/chamber (2 alleles)
11 41Poisson distribution, mean = 25
# of molecules in chamber
FAM
VIC or HEX
Genotyping Basics – Relative Fluorescence
• XX (homozygote)High– FAM Low – VIC or HEX
• XY (heterozygote)Intermediate – FAM Intermediate – VIC or HEX
• YY (homozygote)High– VIC or HEXLow – FAM
Relative
Fluorescence
Cycle
Standard Genotyping Chemistry
TaqMan® SNPtype™
MechanismAllele-specifichybridization
Allele-specific extension
Primers Locus-specific (2)Sequence-tagged,allele-specific (2) & locus-specific (1)
Probes FAM, VIC FAM, HEX
Labeling Individual ($$$) Universal ($)
Fluorescence GenerationProbe displacement
& cleavageFluorescent amplicon
formation
Fluorescence DetectionReal-time or end point
End point
STA-compatible Yes Yes
Text boxFluidigm custom assay design service
Assay Design Process
Assay Design Report
Assay Design Report
Assay Design Report
• Come shipped in 3 x 96-well plates• ASPs, LSPs, STA
• Make up primer mix (F/R) and STA mix (R/STA) in 96-well plates
• Minimum order 24 assays
• Small size: primer volume sufficient for 150 96.96 IFCs (with STA) and 360 96.96 IFCs (without STA).
SNPType Assays
Text box
96 DNA Samples2.5ul @ 60ng/ul per sample
or 2.5ul diluted STA product
96 SNP Assays
(primer pairs)
Workflow
9,216 genotypes
Pipette Load PCR
20 min 96.96:192.24:
Scan
< 10 min
Genotyping Experimnetal Workflow
90 min30 min
60 – 100 min30 – 60 min
Data Anlaysis
Text box
Text box
Assay Reference Library Manager
Assay List Box
Chip Run Grid
Chip Run Scatter Plot
Master Details
Assay Reference Library (ARL)
The Basics
• Add specific assays to a “database” file
• Use ARL Manager to visualize and maintain the library
• ARL is “training set” for clustering with correct and standard calls
• ARL guides clustering on new chip run
Alaska Department ofFish and Game
Gene ConservationLaboratory
Fishing Season in Bristol Bay, Alaska
Alaska20 km
Bristol Bay
Managers regulate the fishery by opening and closing the fishing districts based on data supported by SNP genotyping.
Time
Labor
Reagent
Project Summary: Salmon Genotyping
• 96.96 IFCs:
• 96 SNPs (TaqMan assays)
• 190 fish / 2 days
• 12 sets of runs in ~3 weeks
• Totals:
• ~24 chips
• ~2,300 samples
• ~221,000 genotypes
• Average Call Rate: 99.3%
Summary
• Identify SNP targets (ie from sequencing, or previous projects)
• Submit sequences for assay design (could be excess)
• Decide on final panel of 48 or 96 assays
• PreAmplify gDNA samples (if necessary)
• Use your panel to genotype very large numbers of samples in short time periods