The WGA is a promising solution to eliminate the practical problem in the limitation of the source of DNA needed for genome-wide scans. In order to fulfill the purpose, WGA must satisfy some basic requirements. First, the amplification process should be highly accurate to avoid undue errors. Second, amplification should not produce a bias in the distribution of the DNA products. Questions of amplification-induced error and template bias generated by the WGA process have been addressed elsewhere through small and large scale SNP detection methodologies (1
). Third, a high amplification factor is required so that WGA generates a useful amount of DNA from small starting samples. Finally, the WGA method should be applicable to a wide array of genomic platforms (24
Different methods of WGA have been used so far in different studies by different investigators. Three main methods have been used for WGA: (1
) Multiple Displacement Amplification (MDA) (22
) Primer extension pre-amplification (PEP) (28
), and (3
) Degenerate Oligonucleotide-primed PCR (DOP) (5
). Besides the methods of amplification, other critical issues include amount of DNA input (30
), amplified DNA yield (24
) and the level of bias (32
). Pinard et al compared the yield of WGA product using the different amplification methods from 25ng of gDNA as starting material: the MDA based REPLI-g method generated 2100 fold amplification, GenomiPhi 640 fold, PEP 120 fold and DOP 92 fold (24
). The sharp contrast between the yields derived from the two MDA based methods (REPLI-g and GenomiPhi) may be attributed to the use of KOH alkali denaturation prior to the amplification process which opens priming sites more efficiently than the thermal denaturation used in the GenomiPhi protocol (24
There is evidence that the level of error introduced during WGA reaction appears to be a function of amount of starting material. In this connection, Dean (22
) and Lovmar (33
) have evaluated the genotyping performance of MDA WGA using a range of gDNA inputs and both the authors focused attention in their evaluation of genotyping performance of WGA DNA derived from 3 ng of gDNA. Bergen et al carried out extensive investigation on the effect of gDNA mass (1,10, 25, 50, 100 and 200 ng) on WGA and genotyping performance (30
). They found that, for optimal performance in single-plex SNP genotyping using TaqMan platform, at least 10 ng of lymphoblastoid gDNA input in WGA reaction was required; but over 100 ng of lymphoblastoid gDNA input into WGA reaction was required to obtain optimal STR genotyping performance from WGA DNA. In their work, the WGA obtained from 25 ng of gDNA input showed 99.9% completion of genotyping with 2.3% discordance. Lasken and Egholm recommended 10 –100 ng of gDNA template in the MDA WGA reaction to avoid stochastic amplification (34
). In our lab, for single-plex SNP genotyping using fluorescent polarization method, we have seen up to 100% completion of genotyping with 25 ng of WGA-DNA sample per well in PCR reaction from the WGA stock obtained from 25 ng of gDNA input in 50 µL WGA reaction volume. shows the clustering of 84 genotype calls for rs1476413, using 25 ng of gDNA in left panel and 25 ng corresponding WGA-DNA (from stock of WGA obtained from 25 ng of g-DNA input in WGA reaction) on the right panel. SNP concordance was 100%. Among the g-DNA samples, five samples were not clustered tightly (undetermined or no call) but clearly three were heading towards GA genotype cluster and the other two were heading towards AA genotype cluster. However, in case of the corresponding WGA samples (right panel), the samples were nicely separated in three distinct genotype clusters. Sawcer et al used a total of 508 WGA samples for genotyping on the Illumina GoldenGate platform and found that the likelihood of successful genotyping from WGA DNA correlated with the starting concentration of genomic DNA used in the amplification reaction: a large proportion of samples (n=404) failed to produce genotype calls and the mean starting concentration was 5.9 ng/ul, whereas for the rest of samples (n=104) for which they had successful genotype calls, the concentration of the starting gDNA was 17.4 ng/ul (25
). The present study was not designed to find out optimal gDNA input into the WGA reaction. Rather we focused on the performance of WGA DNA derived from 25ng of gDNA as input in the WGA reaction. In the context of genome-wide genotyping, only 25 ng of good quality genomic DNA as starting material for subsequent WGA reaction may be considered a good alternative to standard requirement of 250 – 500 ng of gDNA for microarray-based high throughput genotyping.
Genotyping for rs1476413 using Fluorescent Polarization method for g-DNA samples (left) and corresponding WGA-DNA samples (right)
Arriola et al amplified genomic DNA at different starting amounts (0.5, 5, 10 and 50 ng) using the Phi29 based MDA method and found that the fold amplification was highest when the input DNA was low and this higher fold amplification was correlated to amplification bias in Comparative Genomic Hybridization (CGH) profiles (31
Paez et al, used the Phi 29 polymerase based amplification method, with or without alkali denaturation prior to amplification and tested the accuracy and genome-wide coverage of the derived WGA product through both direct sequencing of around 500,000 bp and high density oligonucleotide arrays interrogating 10K SNPs with mean inter-marker distance of 210 kb on the Affymetrix platform (32
). Their study showed better call rates with prior alkali denaturation. The call rate was 92.93% in genomic DNA and 92.06% in WGA samples with prior alkali denaturation. In the present study, we used 25 ng of gDNA as starting material and treated with KOH prior to WGA by the MDA method and used the Affymetrix Early Access Mendel Nsp 250K GeneChip containing 224,940 SNPs with mean and median inter-SNP distance of 11.19 kb and 4.815 kb respectively. We found that the overall call rate was 97.07% (95% CI 96.17–97.97) in genomic DNA samples and 97.77% (95% CI 97.26–98.28) in WGA samples.
In a small-scale genotyping study in which only 6 SNPs were genotyped in 172 samples, a concordance of 100% was found among gDNA and corresponding WGA DNA (35
). On the other hand, when genotyping was performed on a larger number of SNPs on the Illumina linkage panel (2320 SNPs) platform (36
) or using the Illumina GoldenGate method (345 SNPs) (7
), the call concordance was found to vary between 98.8% and 99.7%. One study explored the utility of MDA on 10K SNP arrays, reporting good coverage and high concordance rates but reduced call rates (32
). In our study, using 250K SNP chip, the overall concordance was 97.74% (95% CI 97.03–98.45) and when restricting the analysis to well performing SNPs (Com_gDNA and Com_WGA >90%), on an average, 99.11% (95%CI 98.80 – 99.42) of the SNPs were concordant and overall a SNP showed discordant call only in 0.92% (95%CI 0.90 – 0.94) of paired samples. Moreover, we used the early access chips where the SNP panel was not yet fully optimized for SNP performance. For practical purposes, in genome-wide analysis SNPs should be filtered by call rate (across the samples). Analyzing the small number of SNPs that caused discordant calls, we identified that there were very few regions with copy number loss and those were predominantly at the telomeric regions. We also looked at paired LOH regions for the WGA samples compared to the corresponding gDNA samples and found only 5 copy- neutral LOH regions (smallest region at 2q21.1 of 8540 bp and the largest one at 8q11.22 of 156577bp), none of which was located near telomeric regions. In a previous study, Paez et al. also found few chromosomal regions with loss of copy number in MDA-based WGA samples, but none of those regions were telomeric (32
). To our knowledge, this is one of the first studies to examine the SNP concordance of WGA product with healthy human germ line gDNA samples on very high-density oligonucleotide based SNP chips interrogating 224,940 SNPs. Although only in one pair of samples, we also tested the performance of MDA-based WGA product on a different platform – Illumina’s 610 Quad chip interrogating 592,532 SNPs, and noticed 99.998% concordance with the gDNA. Previous studies have not used such a high resolution microarray platform to address this issue. It may be noted that neither the Affymetrix nor the Illumina GoldenGate assay protocol uses further WGA step in sample processing, rather PCR amplification is used. On the other hand, Illumina’s Infinium chemistry uses WGA as a part of DNA sample processing before hybridization.
The present study was limited to the use of high quality intact gDNA as input into the WGA reaction. Considering the fragment size of the degraded DNA extracted from formalin-fixed paraffin embedded (FFPE) samples, MDA based WGA may not be suitable option for Affymetrix GeneChip. However, fragmentation-PCR-based method for WGA is an appropriate choice for the FFPE samples. In a very recent publication (Epub 2008 Jun 12), Mead et al have documented that degraded DNA amplified with MDA-based-WGA gave low call rates and concordance across all platforms at standard loading concentration; but the fragmentation-PCR-based method of WGA gave high call rate and concordance for degraded DNA (37
In summary, our results suggest that Phi29 MDA based WGA product provides a highly accurate and reasonably comprehensive representation of the unamplified human genome, suitable for high resolution genome-wide genotyping studies using oligonucleotide-based SNP genotyping arrays.