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Evaluation of DNA Whole Genome Amplification Technologies for Genotyping

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

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  1. Evaluation of DNA Whole Genome Amplification Technologies for Genotyping Genomic Variation Research Group (GVRG)

  2. 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)

  3. GE Healthcare illustra GenomiPhi &Qiagen REPLI-g

  4. 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.

  5. Sigma-Aldrich GenomePlex Technology

  6. 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)

  7. Kit Information

  8. 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

  9. Product Yield

  10. Size of Product

  11. Quality of Allelic Representation

  12. Genotyping Platforms Assessed • TaqMan SNP Assays • 18 assays duplicated with Infinium • TaqMan Copy Number Assays • Infinium Human Omni1Quad

  13. TaqMan Call Rates by Sample

  14. TaqMan Call Rates by Kit

  15. TaqMan Concordance

  16. Copy Number Call Rate By Sample

  17. Copy Number Call Rate By Kit

  18. Copy Number Call Rate By Assay and Kit

  19. Copy Number Concordance

  20. Infinium Call Rates Across All Kits

  21. Infinium Call Rates Genomic Control

  22. Infinium Call Rates GenomiPhi V2

  23. Infinium Call Rates Ovation

  24. Infinium Call Rates Ovation FFPE

  25. Infinium Call Rates Repli-G

  26. Infinium Call Rates WGA2

  27. Infinium Call Rates WGA4

  28. Infinium Concordance • Every sample with a completion rate above 90% had concordance rate greater than 99.8% with genomic results • Mendelian Check • 100% Concordant

  29. 18 Duplicated Assays Between TaqMan and Infinium:A Closer Look

  30. Infinium Call Rates Across Duplicated Assays By Sample

  31. Infinium Call Rates Across Duplicated Assays By Kit

  32. Infinium Call Rates Across Duplicated Assays

  33. Infinium Concordance

  34. Comparing TaqMan Results To Infinium Results Across Duplicated Assays

  35. TaqMan to Infinium ComparisonAcross 18 Duplicated Assays

  36. Breakdown of Differences Between TaqMan and Infinium Results

  37. Breakdown of Differences Between TaqMan and Infinium Results

  38. Breakdown of Differences Between TaqMan and Infinium Results

  39. Breakdown of Differences Between TaqMan and Infinium Results

  40. Breakdown of Differences Between TaqMan and Infinium Results

  41. Final Thoughts…

  42. What else could have been done… • aCGH Platform for CNVs • NGS Platforms

  43. 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.

  44. 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…

  45. Questions?

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