time-saving, cost-effective snp genotyping strategies from ...tgc.ifas.ufl.edu/tbrt...
TRANSCRIPT
Time-saving, cost-effective SNP genotyping
strategies from LGC Genomics
Erin Steer, Ph.D. – Application Scientist
2
LGC Genomics overview
• Reagents
– DNA extraction kits
– qPCR and SNP genotyping
chemistries
• High throughput automation
– DNA extraction robots
– PCR platforms
• Services
– KASP genotyping
– Array-based genotyping
– Next generation sequencing
Roadblocks to
high-
throughput
genotyping EXPENSIVELABORIOUSPOOR DATA
QUALITY
Our approach
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Automated DNA
extractionAutomated SNP
genotyping• Seeds collected
from seven corn
varieties
• Samples crushed
using
Geno/Grinder
oKtopure & sbeadex
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oKtopure™
• Optimized for sbeadex DNA
extraction kits
• Extract from up to eight 96-well
plates in a single run
sbeadex™
• Magnetic bead nucleic acid
extraction kits
• Compatible with the oKtopure
sbeadex extraction mechanism
6Elution
Lysis
Wash 2 Bind 2
Wash 1Bind 1
sbeadex™
magnetic particle
Throughput capacity on the oKtopure
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Mini kits
• 8 x 96-well
extractions in
1-1.5 hours
• ~4,000
extractions per
8 hour day
• ~1M annually
Maxi kits
• 8 x 96-well
extractions in
~2.5 hours
• ~2,300
extractions per
8 hour day
• ~600K annually
DNA extraction on the oKtopure
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0
5
10
15
20
25
30
35
40
45
#1 #2 #3 #4 #5 #6 #7
Yiel
d (
ng/
µL)
Maize variety
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
#1 #2 #3 #4 #5 #6 #7
26
0/2
80
rati
oMaize variety
Yield Purity
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IntelliQube®
Fully automated PCR setup, amplification and analysis system
Array Tape Consumable Information
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• Array Tape® optimized for use with
IntelliQube®
– Opaque, continuous polymer
strip
– 384-well and 768-well
formats
• Cover Seal
– Optically clear, pressure-
sensitive seal
– Applied automatically to each
array after dispensing
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IntelliQube®
Fully automated PCR setup, amplification and analysis system
Components of a KASP reaction
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Assay Mix Master Mix
Sample DNA
KASP reaction – PCR cycles 1 and 2
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Round 2 - Complement of allele-specific tail sequence generated
Round 1 - Denatured template and annealing components
KASP reaction – PCR cycle 3 and beyond…
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Round 3 – Signal Generation
Running KASP on the IntelliQube
Maize SNP Assay #1 Maize SNP Assay #2
• Core set of 384 validated
KASP assays
> Selected to be evenly distributed
throughout the genome
> Polymorphic markers across fresh-
market tomato varieties
• > 7,500 additional KASP
assays for tomato have been
pre-designed
• Online search tool makes it
easy to ID assays relevant to
your research 16
SolCAP KASP tomato panels
• LGC Genomics> Luke Linz
> Mike New
> Mike Freeman
> Dusty Vyas17
Acknowledgements