Presentation on theme: "GVRG ABRF 2011 Research Study Evaluation of DNA Whole Genome Amplification (WGA) Technologies for Genotyping The evolution of genomic technologies is occurring."— Presentation transcript:
GVRG ABRF 2011 Research Study Evaluation of DNA Whole Genome Amplification (WGA) Technologies for Genotyping The evolution of genomic technologies is occurring rapidly and often requires large amounts of source DNA. There is also an expanded desire to analyze smaller numbers of cells for higher resolution studies as well as to take advantage of large numbers of archived samples (eg. FFPE, serum, etc.). To provide enough material for the newest genomic technologies, whole genome amplification (WGA) has reemerged as an important and necessary technique. With new WGA products on the market, the GVRG has completed a benchmarking study evaluation of 6 commercially available WGA kits using several QC and genotyping assays. Utilizing 6 samples, the WGA kits were tested following the manufacturer’s protocols. Quality metrics provided measurements on yield, fragment size, and concentration of material derived from each kit. Several widely adopted genotyping methods were then used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Amy Hutchinson 1, Casey Dagnall 1, Charles Nicolet 2, Helaman Escobar 3, Sean Blake 4, Brian Sanderson 5, Bruce Kingham 6, Karen Jonscher 7 Introduction Samples Amplification Kits Amplified Sample QC TaqMan & Copy Number Genotyping Illumina Infinium Genotyping Discussion 1 Core Genotyping Facility, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD; 2 Data Production Facility, USC Epigenome Center, Los Angeles, CA; 3 Eurofins MWG|Operon, Huntsville, AL; 4 DNA Core Facility, University of Mis souri, Columbia, MO; 5 Life Technologies, Austin, TX; 6 DNA Sequencing & Genotyping Center, University of Delaware, Newark, DE; 7 SBCF and TATC Proteomics, UC Denver, Denver CO 18 SNP Platform Comparison – TaqMan and Infinium Acknowledgements The ABRF Genomic Variation Research Group (GVRG) is very thankful for the support of its corporate sponsors whose generous help enabled the GVRG 2011 study. Specifically, the GVRG would like to thank GE Healthcare Life Sciences, NuGen Technologies, Qiagen and Sigma-Aldrich, who generously donated the whole genome amplification kits for this study. The GVRG also thanks Bruce Kingham for supplying the FFPE samples used in this study. The samples used for the experiment include Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both from the same individual (1 newly extracted and the other extracted 2 years ago), and 1 sample from the Coriell trio that was fragmented prior to amplification for a total of 6 samples tested. The WGA kits included 6 options from Sigma, NuGen, Qiagen and GE Life Sciences. Samples were amplified as blind duplicates (n = 12) at several GVRG member lab sites using 5 of the kits for a total of 60 products. Additionally, the Ovation FFPE kit provided reagents for the 6 samples only (no duplicates) adding another 6 WGA products for a grand total of 66 samples. All processing following the manufacturer’s provided protocol Following amplification, all samples were sent to a single laboratory for QC and genotyping. CEPH/Utah Pedigree 1463 Trio from HapMap Project NA12891 (Father) NA12892 (Mother) NA12878 (Daughter) All amplified and genomic control samples (66 wgaDNA + 6 gDNA) were subjected to several quality control (QC) measures: Quantification via NanoDrop (OD) and PicoGreen (Invitrogen Quant-iT™ dsDNA Reagent) Agilent BioAnalyzer analysis STR Fingerprinting via Applied BioSystem’s AmpFℓSTR® Identifiler® assay Table 1 – WGA Kit List and Comparison Figures 3 & 4 – Average fragment length data by sample and kit (left) and BioAnalyzer traces from amplified sample across kits (right) Figure 2 – Summary of allelic distribution by sample and kit from the Identifiler assay. FFPE Derived Sample Newly Extracted Extracted 2 years ago Fragmented Sample NA12891 (average fragment length: 477 bp) Whole genome amplification is a useful tool, and possibly a necessary step, for amplifying unique and rare samples in order to have enough input DNA material for use with current technology. This study has evaluated the practical use of several commercially available WGA kits in relation to ease of use, quality of amplification and maintenance of genetic integrity across various genotyping platforms in relation to regular, degraded, and FFPE DNA sources. There is excellent performance across the kits tested though there are several things to take into account when selecting a kit for use: Take into account the downstream application or platform (required input, fragment sizes, etc.) Consider QC measure to ensure the amplification products meet needs Identifiler assay is representative of LOH and no amplification in samples tested here FFPE samples can be accurately genotyped using a WGA sample Ease of kit automation if high-throughput is desired Figure 7 – Call Rates on Infinium platform of all samples run across all SNPs by kit Figure 1 – Fold increase of material yield by sample and kit TaqMan ® SNP Genotyping Assays (18 SNPs chosen from SNPs on Infinium chips) and TaqMan ® Gene Copy Number Assays (2 genes) Figure 5 – 5a)Concordance Rate by Kit and 5b) Discordances by Sample and Type for TaqMan SNP Assays Figure 6 – 6a) Call Rates by Gene and Kit, 6b) Concordance Rate By Kit, and 6c) Types of Discordances by Gene and Kit for TaqMan Copy Number Assays Infinium Human Omni1Quad Chips Concordance with genomic greater than 99.8% using 90% call rate cut-off Genotypes 100% consistent with Mendelian inheritance among trio Figure 8 – Call rates on Infinium platform of all samples by 18 duplicated SNPs by kit Figure 9 – 9a) Concordance rates of all samples on 18 duplicated SNPs by kit and 9b) Discordance type by kit Figure 10 – Call variance between TaqMan and Infinium across duplicated assays by kit Figure 11 – Breakdown of call variance between TaqMan and Infinium by kit and variance type Fig. 5aFig. 5b Fig. 6a Fig. 6bFig. 6c * 64pg was used as the input amount for the Sigma Single Cell kit (WGA4) For concordance checking, eighteen (18) TaqMan assays were selected and run on all WGA and genomic samples for SNPs also present on the Infinium Omni1 Quad BeadChip. Fig. 9aFig. 9b Kit Required Input Input Types Expected Yield Expected Product Length (in Kb) Process Time Hands-On Processing Time GE Life Sciences GenomiPhi 10 ngPurified gDNA, whole blood, mouth wash, buccal, dried blood cards 4 - 7 ugAverage: 10 Range: 2-100 2 hours20 min NuGen Ovation10 ngPurified gDNA3 - 6 ugAverage: 0.2 Range: 0.05-1.5 5 hours2 hours Sigma Complete (WGA2) 10 ngPurified gDNA10 ugAverage: 0.4 Range: 0.1-1 3.5 hours1 hour Qiagen REPLI-g10 ngPurified gDNA, whole blood, mouth wash, buccal, dried blood cards 10 ugAverage: 10 Range: 2-100 17 hours40 min Sigma Single Cell (WGA4) 1 cell or < 100 pg* Single cell or Purified gDNA Million- fold Average: 0.4 Range: 0.1-1 3.5 hours1 hour NuGen Ovation FFPE 100 ngPurified gDNA3.5 - 5 ugAverage: 0.2 Range: 0.05-1.5 8.5 hours4 hours
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