SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL...
Transcript of SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL...
![Page 1: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/1.jpg)
SNP Genotyping
![Page 2: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/2.jpg)
• Introduction to Fluidigm technology James Miller
• Fluidigm genotyping applications Paul Lacaze
• Case study Jonathan Beesley
Overview
![Page 3: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/3.jpg)
Why SNP genotyping?
![Page 4: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/4.jpg)
Dynamic Array IFC Architecture (96.96)
![Page 5: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/5.jpg)
Technology
![Page 6: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/6.jpg)
Fluid Line
Control Line
Open NanoFlex™ Valve
![Page 7: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/7.jpg)
Fluid Line
Fluid Line
Control Line
Closed NanoFlex Valve
![Page 8: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/8.jpg)
Sample Assay
Close Interface Valve
![Page 9: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/9.jpg)
Load Samples
Sample Assay
![Page 10: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/10.jpg)
Load Assays
Sample Assay
![Page 11: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/11.jpg)
Close Containment Valve
Sample Assay
![Page 12: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/12.jpg)
Mix Sample and Assay
Reaction
![Page 13: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/13.jpg)
![Page 14: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/14.jpg)
![Page 15: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/15.jpg)
96.96
9,216 data points(= 24 x 384)
Dynamic Array™ Integrated Fluidic Circuits
192.24
4,608 data points(= 12 x 384)
![Page 16: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/16.jpg)
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)
![Page 17: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/17.jpg)
240 µL master mix0.625 µL each 80X assay (60 µL total)1 chip4 hours
Fluidigm
System
96 samples x 96 SNPs (TaqMan)
![Page 18: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/18.jpg)
BioMark HD™ Real-Time PCR System
![Page 19: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/19.jpg)
EP1™ System
![Page 20: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/20.jpg)
FC-1™ Cycler-Embedded cycler in the Biomark HD
![Page 21: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/21.jpg)
FC1TM System picture courtesy of CRITFC
Increasing Throughput
![Page 22: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/22.jpg)
Pipette Load PCR
20 min 96.96:192.24:
Scan
< 10 min
Genotyping Workflow
90 min30 min
60 – 100 min30 – 60 min
![Page 23: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/23.jpg)
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
![Page 24: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/24.jpg)
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?
![Page 25: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/25.jpg)
Genotyping Basics
![Page 26: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/26.jpg)
X
X
Genotyping by PCR
X
Y
Y
Y
X – PCR assay 1 (FAM)
Y – PCR assay 2 (VIC)
![Page 27: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/27.jpg)
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
![Page 28: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/28.jpg)
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
![Page 29: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/29.jpg)
96.96 Genotype Map
![Page 30: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/30.jpg)
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
![Page 31: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/31.jpg)
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)
![Page 32: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/32.jpg)
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
![Page 33: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/33.jpg)
Master mix + fluor/quencher
(SNPType reagent)
C
T
Allelic Discrimination
![Page 34: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/34.jpg)
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)
![Page 35: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/35.jpg)
SNPtype Assays(Allele-specific primers)
TaqMan Probes(Allele-specific probes)
SNP1
SNP2
SNPtype Assays vs. TaqMan Probes
![Page 36: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/36.jpg)
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)
![Page 37: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/37.jpg)
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)
![Page 38: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/38.jpg)
Cacao Genotyping: Call RatesgD
NA
STA
SNP 1 SNP 2 SNP 3
66.67% 0.00%70.37%
100.0% 100.0%100.0%
![Page 39: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/39.jpg)
5 copies 10 copies 20 copies
50 copies 100 copies 150 copies
Relative Copies Per Reaction Chamber
![Page 40: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/40.jpg)
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
![Page 41: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/41.jpg)
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
![Page 42: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/42.jpg)
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
![Page 43: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/43.jpg)
Text boxFluidigm custom assay design service
![Page 44: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/44.jpg)
Assay Design Process
![Page 45: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/45.jpg)
Assay Design Report
![Page 46: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/46.jpg)
Assay Design Report
![Page 47: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/47.jpg)
Assay Design Report
![Page 48: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/48.jpg)
• 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
![Page 49: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/49.jpg)
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
![Page 50: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/50.jpg)
Pipette Load PCR
20 min 96.96:192.24:
Scan
< 10 min
Genotyping Experimnetal Workflow
90 min30 min
60 – 100 min30 – 60 min
![Page 51: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/51.jpg)
Data Anlaysis
![Page 52: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/52.jpg)
Text box
![Page 53: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/53.jpg)
Text box
![Page 54: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/54.jpg)
Assay Reference Library Manager
Assay List Box
Chip Run Grid
Chip Run Scatter Plot
Master Details
![Page 55: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/55.jpg)
Assay Reference Library (ARL)
![Page 56: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/56.jpg)
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
![Page 57: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/57.jpg)
Alaska Department ofFish and Game
Gene ConservationLaboratory
![Page 58: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/58.jpg)
Fishing Season in Bristol Bay, Alaska
![Page 59: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/59.jpg)
Alaska20 km
Bristol Bay
![Page 60: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/60.jpg)
Managers regulate the fishery by opening and closing the fishing districts based on data supported by SNP genotyping.
![Page 61: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/61.jpg)
Time
Labor
Reagent
![Page 62: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/62.jpg)
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%
![Page 63: SA GT Training slides SNP Genotyping PLacaze …...23 mL master mix 6 µLeach 80X assay (576 µL total) 24 x 384-well plates 8 days Traditional genotyping system 96 samples x 96 SNPs](https://reader033.fdocuments.us/reader033/viewer/2022042310/5ed86ad9b7394006ce602824/html5/thumbnails/63.jpg)
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