GVRG ABRF 2011 Research StudyEvaluation 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 Hutchinson1, Casey Dagnall1, Charles Nicolet2, Helaman Escobar3, Sean Blake4, Brian Sanderson5, Bruce Kingham6, Karen Jonscher7
Introduction
Samples
Amplification Kits
Amplified Sample QC
TaqMan & Copy Number Genotyping
Illumina Infinium Genotyping
Discussion
1Core Genotyping Facility, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD; 2Data Production Facility, USC Epigenome Center, Los Angeles, CA; 3Eurofins MWG|Operon, Huntsville, AL;4DNA Core Facility, University of Mis souri, Columbia, MO; 5Life Technologies, Austin, TX; 6DNA Sequencing & Genotyping Center, University of Delaware, Newark, DE; 7SBCF and TATC Proteomics, UC Denver, Denver CO
18 SNP Platform Comparison – TaqMan and Infinium
AcknowledgementsThe 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 ProjectNA12891 (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 SampleNewly ExtractedExtracted 2 years ago
• Fragmented SampleNA12891 (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. 5a Fig. 5b
Fig. 6a
Fig. 6b Fig. 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. 9a Fig. 9b
KitRequired
InputInput Types
ExpectedYield
Expected Product Length
(in Kb)
Process Time
Hands-On Processing
TimeGE Life Sciences GenomiPhi
10 ng Purified gDNA, whole blood, mouth wash,
buccal, dried blood cards
4 - 7 ug Average: 10Range: 2-100
2 hours 20 min
NuGen Ovation 10 ng Purified gDNA 3 - 6 ug Average: 0.2Range: 0.05-1.5
5 hours 2 hours
Sigma Complete(WGA2)
10 ng Purified gDNA 10 ug Average: 0.4Range: 0.1-1
3.5 hours 1 hour
Qiagen REPLI-g 10 ng Purified gDNA, whole blood, mouth wash,
buccal, dried blood cards
10 ug Average: 10Range: 2-100
17 hours 40 min
Sigma Single Cell(WGA4)
1 cell or< 100 pg*
Single cell orPurified gDNA
Million-fold
Average: 0.4Range: 0.1-1
3.5 hours 1 hour
NuGen Ovation FFPE
100 ng Purified gDNA 3.5 - 5 ug Average: 0.2Range: 0.05-1.5
8.5 hours 4 hours