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

4

Automated DNA

extractionAutomated SNP

genotyping• Seeds collected

from seven corn

varieties

• Samples crushed

using

Geno/Grinder

oKtopure & sbeadex

5

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

7

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

8

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

9

IntelliQube®

Fully automated PCR setup, amplification and analysis system

Array Tape Consumable Information

10

• 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

11

IntelliQube®

Fully automated PCR setup, amplification and analysis system

Components of a KASP reaction

12

Assay Mix Master Mix

Sample DNA

KASP reaction – PCR cycles 1 and 2

13

Round 2 - Complement of allele-specific tail sequence generated

Round 1 - Denatured template and annealing components

KASP reaction – PCR cycle 3 and beyond…

14

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