Complete High Throughput Pharmacogenomics Workflow: From Sample to Results

Michael Farrell1, Jeffrey Roeder1, Travis Butts1, Julie Baggs1, Luke Linz2, Mike Freeman2, Steven Nesbit3, Joan Gordon3
1 Omega Bio-tek, Inc., 2 Douglas Scientific, 3 Maine Molecular Quality Controls, Inc.

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Introduction

Rapid, high throughput, low cost and accurate screening of human DNA for the identification of single nucleotide polymorphisms (SNPs) is a useful tool for the development of therapeutics and diagnostics. Here, we demonstrated these metrics with an automated pharmacogenomics workflow consisting of a non-invasive buccal swab collection system and DNA extraction chemistry (Omega Bio-tek) on two open automated liquid handling platforms (Hamilton Microlab STAR and Qiagen BioSprint 96), followed by specific SNP identification via a high throughput genotyping instrument (Douglas Scientific). For demonstration purposes, this system was used to determine the SNP status of warfarin anticoagulant sensitivity mutations within the human VKORC1 and CYP2C9 genes (Ref 1). Genomic controls (Introl HD Multiplex WT, HET and MUT) were provided by Maine Molecular Quality Controls, Inc.

Workflow and Methods

Omega Bio-tek's E-Z 96® Spin-Out buccal swab collection device allows for rapid arraying of sample swabs (1) into 96-well plates for lysis configuration (2), followed by the raised configuration (3) for easy eluate spin-out and start of automated workflow.


1. Buccal swabs with breakpoints.


2. Sample lysis configuration.


3. Easy spin-out configuration

Sample Qiagen BioSprint 96 Hamilton® Microlab STAR
  Conc. (ng/µL) Total Yield (ng) Conc. (ng/µL) Total Yield (ng)
1 6.6 658 15.6 1559
2 17.0 1700 18.1 1808
3 7.0 697 9.8 982
4 8.2 825 17.4 1744
5 6.8 680 21.7 2166
6 7.9 791 5.5 548
7 13.6 1360 64.9 6487
8 7.0 702 13.2 1317
9 9.9 986 8.0 797
10 9.6 959 7.3 733
11 5.0 502 16.3 1634
12 6.5 646 30.0 3003
13 13.9 1388 33.7 3367
14 25.7 2575 26.1 2611
15 10.3 1031 15.3 1530
16 8.7 869 4.0 404
17 17.1 1711 9.9 987
Average 10.6 1063 20.8 2079
Figure 1. Extracted DNA quantification. Values are the average of duplicate extractions.


Sample CYP2C9*2 CYP2C9*3 VKORC1
  BS 96 STAR BS 96 STAR BS 96 STAR
1 WT WT WT WT HET HET
2 WT WT WT WT HET HET
3 WT WT WT WT MUT MUT
4 WT WT WT WT WT WT
5 WT WT WT WT WT WT
6 WT WT WT WT HET HET
7 WT WT WT WT WT WT
8 HET HET WT WT WT WT
9 WT WT WT WT HET HET
10 WT WT WT WT WT WT
11 WT WT WT WT WT WT
12 WT WT WT WT HET HET
13 WT WT WT WT WT WT
14 WT WT WT WT MUT MUT
15 WT WT WT WT MUT MUT
16 WT WT WT WT WT WT
17 WT WT WT WT MUT MUT
HET* HET* HET* HET* HET* HET* HET*
MUT* MUT* MUT* MUT* MUT* MUT* MUT*
WT* WT* WT* WT* WT* WT* WT*
Figure 2. Warfarin sensitivity SNP determination for all samples.

Omega Bio-tek's Mag-Bind® Blood & Tissue DNA HDQ 96 Kit is designed for the rapid and reliable isolation of high quality genomic DNA from swabs and other sample types. Mag-Bind® Particles HDQ provide quick magnetic response time. Utilizing paramagnetic particles allows use on multiple open liquid handling platforms and provides high quality DNA that is suitable for direct use in most downstream applications.

The IntelliQube® by Douglas Scientific combines liquid handling with real-time quantitative PCR (qPCR) analysis in miniaturized reaction volumes. The system utilizes Array Tape® in a unique and innovative 768-well format. Array Tape® is a thin and flexible polypropylene consumable that, in combination with miniaturized reaction volumes (1.6 µL), enables both outstanding PCR performance and profound reagent savings.

Results

The purified DNA from 17 replicate samples was quantified for DNA concentration and total DNA yield via PicoGreen® (Figure 1). DNA from these samples was then analyzed via BHQplus® probe-based qPCR for wild-type, mutant or heterozygous status for warfarin sensitivity at three known SNPs (Figures 2 and 3).

Figure 2. Genotype scatterplots from the IntelliQube®. WT = wild-type, MUT = mutant, HET = heterozygous.

Conclusions

These sample collection, extraction and analysis technologies offer a rapid, high throughput, low cost and accurate pharmacogenomics workflow solution for human DNA genotyping.

The Douglas Scientific IntelliQube®, Omega Bio-tek's Mag-Bind® Blood and Tissue DNA HDQ 96 Kit and Maine Molecular Quality Controls, Inc's products are for research use only.

References

1. Takeuchi, F., et al. (2009). PLoS Genet 5: e1000433. doi: 10.1371.

Workflow Reagents and Consumables Cost per Sample
Omega Bio-tek's E-Z 96® Spin-Out collection system $0.24
Omega Bio-tek's Mag-Bind® Blood and Tissue DNA HDQ 96 Kit $1.85
Disposables (Hamilton tips, etc.) $0.42
Quanta Biosciences Accustart Genotyping Toughmix $0.05
Biosearch BHQplus® SNP Assay (Primers and probes) $0.01
Control DNA $0.24
Total cost per sample: $2.81

info@omegabiotek.com

www.douglasscientific.com

www.mmqci.com