snp genotyping - millennium science · • introduction to fluidigm technology (michelle) •...
TRANSCRIPT
SNP Genotyping
Michelle Garred MSc, Paul Lacaze PhD
• Introduction to Fluidigm technology (Michelle)
• Running a Fluidigm genotyping project (Paul)
• SNP genotyping by PCR – the basics
• Fluidigm SNPType chemistry
• Assay design process
• Experimental workflow
• Data analysis
• Case study (Simon Southerton)
Overview
Why SNP genotyping?
Technology
Fluid Line
Control Line
Open NanoFlex™ Valve
Fluid Line
Fluid Line
Control Line
Closed NanoFlex Valve
Dynamic Array IFC Architecture (96.96)
Sample A
s
s
a
y
Close Interface Valve
Load Samples
Sample A
s
s
a
y
Load Assays
Sample A
s
s
a
y
Close Containment Valve
Sample A
s
s
a
y
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 mix
6 µL each 80X assay (576 µL total)
24 x 384-well plates
8 days
Traditional
genotyping
system
96 samples x 96 SNPs (TaqMan)
240 µL master mix
0.625 µL each 80X assay (60 µL total)
1 chip
4 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 min
30 min
60 – 100 min
30 – 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
XX
Genotyping by PCR
XY
YY
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 HEX
Low – FAM
Rela
tive F
luore
scence
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 HEX
Low – 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
CC
CT
TT
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 Rates gD
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
Text box Fluidigm 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 Samples 2.5ul @ 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 min
30 min
60 – 100 min
30 – 60 min
Data Anlaysis
Text box
Assay Reference Library Manager
Assay List Box
Chip Run Grid
Chip Run Scatter Plot
Master Details
Alaska Department of
Fish and Game
Gene Conservation
Laboratory
Alaska 20 km
Bristol Bay
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%
• 107 SNPType designs
• Finalized into panel of 96
• inc 3 add ons, 3 re-designs
• Genotyping 1000s of samples
• DNA in 384 well plates dried down
• <10ng/ul PreAmp required
• No running 6 x 96.96 IFCs per day
Aus Case Study – Human Cancer SNP
Genotyping
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
