Our results show that (1) pooling genomic samples is highly accurate; (2) unreliable SNPs most likely to give false-positives can be largely identified and removed prior to association analysis; and (3) a moving window of averaged test-statistics can be used to detect association signals. Additionally, we have described modifications as to how allelic frequencies are calculated from RAS values of pooled samples that remove systematic biases.

Pioneering work on pooling studies by other research groups has shown that the average Relative Allele Signal (RAS

_{ave}) can be effectively used to derive

*k*-correction factors by

*k *= RAS

_{AVE}/(1-(RAS

_{AVE}), and as such, can be used to accurately predict allelic frequencies [

8,

11,

12]. Pooling studies are intended to be screening approaches. RAS values are highly convenient since they are generated by the Affymetrix GDAS software on the 10K platform and fairly intuitive to understand. We suggest significant improvements to this innovative approach that will remove biases; allow for continued use of RAS values; and result in more accurate predictions. These improvements focus on lowering the number of false positives due to added variance or systematic biases, since the utility of pooling-based approaches will be based on how one can detect association signals given a high number of false positives.

First, RAS1 and RAS2 should not be averaged since they are separate probe sets with distinct variances. One may unnecessarily propagate unwanted variance by averaging. For example in Figure , it is clearly visible that RAS2 is highly predictable of the particular SNP allele whereas RAS1 is highly inaccurate. In this case, averaging RAS2 and RAS1 will produce a RAS_{AVE }value that is less accurate than RAS2 alone. We suggest instead that these values be treated as separate measures, each with their distinct variance. In the case of RAS values with a large variance, these values should not be used due to the increased chance of a false positive.

Second, we highly recommend that RAS values for each SNP be normalized prior to calculation of allelic frequencies. When these values are not normalized prior to calculating a predicted allelic frequency a significant bias is introduced since the RAS values, as produced by the Affymetrix GDAS software, generally are not 0.0 or 1.0 for homozygous BB and AA respective alleles. Indeed, on a training set of 1000 individuals we found that 34% of SNPs who were called AA had a RAS value less than 0.9 and 35% of SNPs called BB had a RAS value greater than 0.1. This bias can be seen in an example calculation using

*k*-correction factors derived from a typical RAS value directly obtained from the GDAS software. For example, the average RAS1 for a given SNP of an AA individual may be 0.9, the average RAS1 for a heterozygous individual may be 0.5, and the average RAS2 for a BB individual may be 0.1. When one uses the approach outlined by Butcher,

*et al*, the

*k*-correction factor is 1.0, whereby the RAS value of the average heterozygote is divided by one minus this value [

10]. In a pooled sample, the same SNP is expected to have a RAS value of 0.9 if it is completely homozygous for AA. However, using the

*k-correction *approach on non-normalized RAS values, one would predict an allelic frequency of 90%, whereas the actual frequency is 100%, a bias of 10%. These biases would be most pronounced as pools approach dominance by one allele type, as would often be the case for a SNP highly associated to a disease.

While RAS values are readily obtainable from the Affymetrix software for the 10K GeneChip

^{® }arrays, they are not provided for the 100K or 500K. This is partly due to the fact that RAS values are no longer used to make a SNP call. We have developed a simple Perl script which generates RAS values, still useful in pooling, for the 100K and 500K Affymetrix GeneChip

^{® }platform from CHP files. This tool is available on our website [

23]. While one may use these RAS values to find obvious differences in cases and controls, for many SNPs allelic frequencies are not linearly dependent on the RAS values; thus, one should calculate allelic frequencies when possible to reduce uneven biases between different SNPs.

Additionally, we are making public on the same site both normalized and non-normalized

*k-correction *factors derived from over 3,000 genotyped individuals for the 10K version 2.0 SNP genotyping platform. Other research groups have created central repositories for

*k-corrections *using non-normalized RAS values and we will work with these teams to contribute these values to this valuable centralized resource [

11].