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 alternate homology filter identifies SNP calls that may have arisen as a result of this effect. For each SNP call in the analyzed results, the SNP 25-mer probe sequence is used to search for all perfect, ungapped alignments of at least 13 bases with the LVS genome sequence. The requirement of a minimum alignment length of 13 bases guarantees that the SNP base will be included in all alignments found. The program ExamineSNPs.pl examines the SNP alignments and calculates the binding energies, using the MUMmer package to obtain the sequence alignments (11) and the binding energy calculator from the ArrayOligoSelector package (12) for the binding energy calculations. The alignment representing the highest binding energy is selected and compared with the free energy of binding of the reference 25-mer to its reverse complement. If the difference between these two binding energies is 11.5 kcal/mol, the SNP call is assumed to be an artifact of the alternate sequence homology and it is removed from the list of high-confidence SNP calls. The set of SNP calls from the hybridization of a SCHU S4 sample was used to determine the threshold binding energy difference that identifies probable alternate homology artifacts. A delta binding energy threshold of 11.5 kcal/mol was chosen based on the effect of different threshold values on the false-negative and false-positive calls ( Figure 2 ). The next filter in our pipeline is a quality filter that simply eliminates SNP calls that have been assigned low quality scores by the GSEQ software. The quality score is based on the difference in signal intensity between the highest intensity probe pair and the next highest intensity The top pair represents a sample DNA sequence perfectly matching a reference probe. The next pair illustrates a sample DNA sequence partially matching a SNP probes and therefore capable of hybridizing with high efficiency to the SNP probe pair.