Validation of pooled genotyping on the Affymetrix 500 k and SNP6.0 genotyping platforms using the polynomial-based probe-specific correction.

Background: 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. 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.

Results: We 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.

Conclusion: We 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.