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

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.

Pooling/bootstrap-based GWAS (pbGWAS) identifies new loci modifying the age of onset in PSEN1 p.Glu280Ala Alzheimer's disease

“…15,34,35 These results are even more consistent when the pool involves individuals of similar ethnic background (as it is the case of this study) 34 and remain still consistent throughout different platforms (in this study, we use a unique platform). 15,35 In order to evaluate both accuracy and reliability of allele frequencies estimated by DNA pooling in this particular study, we evaluated the correlation between gene frequencies estimated by DNA pooling with those determined by individual genotyping.…”

Single nucleotide polymorphisms to discriminate different classes of hybrid between wild Atlantic salmon and aquaculture escapees

“…The performance of these PPC correction coefficients in an independent set of samples has not yet been examined. In particular, in the sample used to generate the PPC correction, 2.3% of SNPs were missing one or more of the three possible genotypes, meaning that the coefficients for these SNPs were unlikely to be estimated correctly (Anantharaman & Chew, 2009 (Glover, Hansen, & Skaala, 2009;Karlsson et al, 2011). This is reflected in the relatively high heterozygosity that we observe within our aquaculture escapee F I G U R E 4 Results of NewHybrids analysis for 400 individuals produced by three generations of simulated hybridization between aquaculture escapees and wild fish.…”

“…The performance of these PPC correction coefficients in an independent set of samples has not yet been examined. In particular, in the sample used to generate the PPC correction, 2.3% of SNPs were missing one or more of the three possible genotypes, meaning that the coefficients for these SNPs were unlikely to be estimated correctly (Anantharaman & Chew, ). Further, the increased difficulty of estimating DNA concentration when it is partly degraded (Simbolo et al., ) may cause allelotyping accuracy to be lower for older scale samples because individuals contribute unequally to the pools.…”

“…We estimated allele frequency at each SNP in each pool by calculating the relative intensity of the B-allele probe signal (=B-allele intensity/ (A-allele intensity + B-allele intensity)), taking the median over all replicated pools, and applying a polynomial-based probe-specific (PPC) correction to account for differential hybridization efficiency of the two probes (Anantharaman & Chew, 2009;Brohede, Dunne, McKay, & Hannan, 2005). To obtain the PPC correction coefficients for each SNP, a second-order polynomial describing the relationship between relative B probe intensity and genotype call (AA, AB or BB) was fit over the 525 individually genotyped New Teno Mainstem samples, plus 86 samples from the Teno mainstem genotyped for a different study, F I G U R E 2 Multidimensional scaling analysis plot visualizing genomewide identity-by-state amongst Old Teno Mainstem, New Teno Mainstem and Teno Escapee samples.…”

Investigating highly replicated asthma genes as candidate genes for allergic rhinitis

“…More detailed phenotypic characteristics of the Singapore Chinese population have been described previously [5]. Genomic DNA was extracted from buccal cells obtained from a mouthwash of 0.9% saline solution following a standardized protocol [18]. Samples were quantified in triplicate on the Nanodrop (Thermo Fisher Scientific Inc, Wilmington, DE, USA).…”