A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

Pandya, Gagan A.; Holmes, Michael H.; Sunkara, Sirisha; Sparks, Andrew; Bai, Yun; Verratti, Kathleen; Saeed, Kelly; Venepally, Pratap; Jarrahi, Behnam; Fleischmann, Robert D.; Peterson, Scott N.

The next script, referred to as the alternate homology filter, is important particularly when resequencing DNAs of higher complexity. The query DNA sample may contain sequence capable of hybridizing with high efficiency to more than one probe pair at a locus on the array. The occurrence of such sequence, referred to as the 'alternate homology effect', is illustrated in Figure 1 . The ability of the GSEQ software to make a base call at any particular locus is dependent upon the relative signal strength of the best forward and reverse probe being above a certain threshold compared with the next best signal at that locus. When a locus contains two strongly hybridizing probe pairs, the GSEQ software may make a SNP call, a reference base call or a no-call ('N'), depending on the relative signal strengths of the probe pairs. In practice, we have found that the alternate sequence need not match the probe sequence over the entire 25-base length in order to cause a spurious base call. A whole-genome DNA sample will naturally contain a larger population of distinct 25-mer sequences than a sample composed of a small subset of the genome. For this reason, the alternate homology effect is significant in the context of whole-genome hybridization. This problem led us to develop an algorithm to identify and filter out base miscalls that resulted from this phenomenon.