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 basis of the Affymetrix GeneChip Õ resequencing by hybridization is shown in Supplementary Figure 1 . Each queryable base position in the reference sequence (nonrepetitive sequence) was represented by eight 25 nucleotide probes or 'features' that define a locus. For each locus, four probes were designed to query the forward strand and four probes represented the reverse complementary strand. The forward and reverse probes were identical at all base positions except the central (13th) position, where the reference base and each of the three alternative bases were represented. The next locus represented probes that were shifted by one base and placed a new base in the central query position. The tiling of loci along a genome sequence effectively allowed for base calls to be derived for each base represented on the array. High confidence base calls were made by virtue of two features within each locus hybridizing with greater efficiency than alternative features (e.g. an A call on the forward strand was accompanied by a T call on the reverse strand). The Affymetrix GSEQ software assigned a quality score to each base call that combined hybridization information from both the forward and reverse strands.