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