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Evaluation of DNA Whole Genome Amplification Technologies for Genotyping. Genomic Variation Research Group (GVRG). WGA Kits Tested. GE Healthcare illustra TM GenomiPhi TM V2 DNA Amplification Kit NuGen ® Ovation ® WGA System NuGEN ® Ovation ® WGA FFPE System Qiagen ® REPLI-g ® Mini
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Evaluation of DNA Whole Genome Amplification Technologies for Genotyping Genomic Variation Research Group (GVRG)
WGA Kits Tested • GE Healthcare illustraTM GenomiPhiTMV2 DNA Amplification Kit • NuGen® Ovation® WGA System • NuGEN® Ovation® WGA FFPE System • Qiagen® REPLI-g® Mini • Sigma-Aldrich® GenomePlex® Complete Whole Genome Amplification Kit (WGA2) • Sigma-Aldrich® GenomePlex® Single Cell Whole Genome Amplification Kit (WGA4)
NuGEN Ovation WGA System *The Ovation WGA FFPE System differs slightly. The first step is an enzymatic fragmentation of the dsDNA, followed by end-repair and a-tailing. Adaptors containing target hybridization sequences are then ligated to the end-repaired, A-tailed dsDNA to create SPIA templates.
Samples Tested • CEPH/Utah Pedigree 1463 Trio from HapMap Project • NA12891 (Father) • NA12892 (Mother) • NA12878 (Daughter) • FFPE Derived Sample • Newly extracted • Extracted 2 years ago • Fragmented Sample • NA12891 (average fragment length: 477 bp)
Quality Analysis of wgaDNA Product • Yield • Quantitation via PicoGreen assay and NanoDrop UV-Vis • Fragment Size • Agilent BioAnalyzer DNA 12000 Chip • Allelic Representation • Applied Biosystems Identifiler Assay
Genotyping Platforms Assessed • TaqMan SNP Assays • 18 assays duplicated with Infinium • TaqMan Copy Number Assays • Infinium Human Omni1Quad
Infinium Call Rates Across All Kits
Infinium Call Rates Genomic Control
Infinium Call Rates GenomiPhi V2
Infinium Call Rates Ovation
Infinium Call Rates Ovation FFPE
Infinium Call Rates Repli-G
Infinium Call Rates WGA2
Infinium Call Rates WGA4
Infinium Concordance • Every sample with a completion rate above 90% had concordance rate greater than 99.8% with genomic results • Mendelian Check • 100% Concordant
18 Duplicated Assays Between TaqMan and Infinium:A Closer Look
Comparing TaqMan Results To Infinium Results Across Duplicated Assays
Breakdown of Differences Between TaqMan and Infinium Results
Breakdown of Differences Between TaqMan and Infinium Results
Breakdown of Differences Between TaqMan and Infinium Results
Breakdown of Differences Between TaqMan and Infinium Results
Breakdown of Differences Between TaqMan and Infinium Results
What else could have been done… • aCGH Platform for CNVs • NGS Platforms
Acknowledgements • Corporate Sponsors • GE Healthcare Life Sciences • NuGen Technologies • Qiagen • Sigma-Aldrich • All generously donated the WGA kits for this study. • Additional Thanks • Alex McCary from SAIC-Frederick • Supriya Singh from Eurofins • Bruce Kingham for supplying the FFPE samples used in this study.
Current GVRG Members • Amy Hutchinson • Casey Dagnall • Charles Nicolet • Helaman Escobar • Sean Blake • Brian Sanderson • Bruce Kingham • Karen Jonscher • Looking to join? We’re always looking for new members…