snp genotyping - millennium science · • introduction to fluidigm technology (michelle) •...

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

Thank you!

Questions?

Paul Lacaze, PhD

Field Application Scientist

placaze@mscience.com.au