In this study we have evaluated the Φ29 polymerase MDA whole genome amplification method from MSI by assessing the compatibility of its product with the established TaqMan and Invader genotyping chemistries and with the highly multiplexed BeadArray genotyping platform. We have also evaluated Illumina's BeadArray genotyping platform for a large-scale experiment using gDNA.
At 95 SNPs, comparison of TaqMan genotypes generated from MDA product and gDNA templates revealed a very good concordance rate but a higher failure rate for MDA product compared to gDNA. This would need be estimated in a sample size larger than the current n
= 88 in order to be confirmed. This result is comparable to the smaller study by Tranah et al
], in which six SNPs were genotyped by TaqMan on 172 samples, resulting in 100% concordance of pre- and post-MDA DNA. In the present study, the MDA product genotypes were slightly more difficult to assign, owing to more dispersed clusters. This was not observed by Lovmar et al
. with fluorescent minisequencing on Amersham MDA products compared to gDNA [10
]. One marker in our study, which may be unusually prone to allelic bias, was impossible to score using MDA product but was acceptable when using gDNA as a template (INS
Compared to the yields indicated in Dean et al.
], our average yield from in-house amplification using the reagents in kit form were in the order of five- to six-fold higher. This was probably due to differences in the two protocols: for example, our protocol used an increased reaction volume compared to the protocol used in Dean et al.
]. Furthermore, the Dean et al.
] protocol omitted the denaturation step, which is now standard practice. One other potential explanation for this variation is possible differences between laboratories in the quantitation of DNA using PicoGreen, the application of which requires a standard reference data set. We cannot at present fully resolve the differences in yields between studies but we can conclude that very large amounts of DNA are synthesized during the Φ29 reaction and that this is an excellent template for genotyping. MDA product should, therefore, be quantified and its concentration on completion of the MDA reaction not assumed to be consistent. Genotype failure rate, concordance rates with gDNA and the nature of genotype clustering showed similar patterns to service-generated MDA. However, a larger number of SNP markers would need to be genotyped on the MDA product using purchased kit reagents in order to verify these figures for in-house amplifications.
In the evaluation of MDA product in conjunction with BeadArray technology, the high concordance rate between genotypes obtained from MDA product and gDNA templates is encouraging. A concordance rate of 99.86% has been reported by Barker et al
. using 2,320 SNPs and five samples [13
]. However, as our study used 86 samples, we were able to observe differences in genotype failure rate between the different templates, not noted in the previous study [11
]. As with the TaqMan evaluation, BeadArray had a higher genotype failure rate for MDA product compared to gDNA (0.2% for MDA versus 0.06% for gDNA). We did not find any evidence for allele drop-out with MDA compared to gDNA. BeadArray genotyping excluded more MDA samples than gDNA samples (10.5% for MDA versus 5.7% for gDNA) indicating that gDNA is a superior genotyping template for BeadArray technology. This 2-fold exclusion rate for MDA is consistent with the approximately 2 to 3-fold genotype failure rate of MDA typically observed with TaqMan and Invader, compared to gDNA (unpublished data).
The performance of MDA product is continuously being monitored in our laboratory. In a study blinded to all genotypers and database administrators, 288 family-based gDNA samples (prepared by the salting out method), were replaced with MDA product and left in continual use in our genotyping pipeline for 12 months. The change went undetected by all users. The failure rate for MDA was 3.34% for 15,921 genotypes, compared to 2.39% for 19,272 gDNA genotypes. Therefore, this improvement in the MDA performance for TaqMan is likely to be applicable to BeadArray, which improves the feasibility of mapping susceptibility loci in complex traits.
When using a highly multiplexed, highly automated genotyping platform, slight reductions in the quality of template material are likely to have a greater adverse effect on data than in scenarios in which markers are assessed individually and manual scoring is undertaken. Our results indicate that MDA is an adequate solution for the vast majority of SNP markers, even in this highly multiplexed allelic assay platform.
It is noted that 5.8% of markers that passed the Illumina acceptable scoring threshold were in fact showing high misinheritance rates in our family samples. This problem was at the same magnitude as TaqMan for individually genotyped markers. This highlights the importance of checking potential positive results with a second genotyping technology.
MDA should allow the continuation of genetic analysis on archived DNA in researchers' freezers worldwide, providing the very necessary increases in sample sizes so urgently required [1