Abstract
BackgroundThe use of pooled DNA on SNP microarrays (SNP-MaP) has been shown to be a cost effective and rapid manner to perform whole-genome association evaluations. While the accuracy of SNP-MaP was extensively evaluated on the early Affymetrix 10 k and 100 k platforms, there have not been as many similarly comprehensive studies on more recent platforms. In the present study, we used the data generated from the full Affymetrix 500 k SNP set together with the polynomial-based probe-specific correction (PPC) to derive allele frequency estimates. These estimates were compared to genotyping results of the same individuals on the same platform, as the basis to evaluate the reliability and accuracy of pooled genotyping on these high-throughput platforms. We subsequently extended this comparison to the new SNP6.0 platform capable of genotyping 1.8 million genetic variants.ResultsWe showed that pooled genotyping on the 500 k platform performed as well as those previously shown on the relatively lower throughput 10 k and 100 k array sets, with high levels of accuracy (correlation coefficient: 0.988) and low median error (0.036) in allele frequency estimates. Similar results were also obtained from the SNP6.0 array set. A novel pooling strategy of overlapping sub-pools was attempted and comparison of estimated allele frequencies showed this strategy to be as reliable as replicate pools. The importance of an appropriate reference genotyping data set for the application of the PPC algorithm was also evaluated; reference samples with similar ethnic background to the pooled samples were found to improve estimation of allele frequencies.ConclusionWe conclude that use of the PPC algorithm to estimate allele frequencies obtained from pooled genotyping on the high throughput 500 k and SNP6.0 platforms is highly accurate and reproducible especially when a suitable reference sample set is used to estimate the beta values for PPC.
Highlights
The use of pooled DNA on single nucleotide polymorphisms (SNPs) microarrays (SNP-MaP) has been shown to be a cost effective and rapid manner to perform whole-genome association evaluations
Pooled genotyping, yielded lower detection rates than individually genotyped samples of between 86% and 88%, likely due to the highly heterogeneous nature of the sample. These detection rates were comparable with that published from other studies working on the Affymetrix 100 k and 500 k array sets
On the SNP6.0 platform, detection rates for pooled genotyping were between 96% and 98%
Summary
The use of pooled DNA on SNP microarrays (SNP-MaP) has been shown to be a cost effective and rapid manner to perform whole-genome association evaluations. We used the data generated from the full Affymetrix 500 k SNP set together with the polynomial-based probe-specific correction (PPC) to derive allele frequency estimates These estimates were compared to genotyping results of the same individuals on the same platform, as the basis to evaluate the reliability and accuracy of pooled genotyping on these high-throughput platforms. Association of SNPs to a phenotype is usually identified by differences in allele frequencies of the variant between case and control samples While other factors such as population stratification, epistasis, pleiotropy and gene-environment interactions may play a part in the phenotypic expression of differently observed allele frequencies, casting a genome-wide net to "fish" for susceptibility genes allows researchers to perform an un-biased initial round of screening to obtain a list of leads for more focused analysis in follow-up studies. Known as the two-stage study design, has been shown to improve statistical power and reduce measurement errors [3]
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