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

Thank you!

Questions?

Paul Lacaze, PhDField Application Scientistplacaze@mscience.com.au