Using a Sanger-validated set of 32 BRCA gene regions from 16 patients, high-throughput colorspace (Life Technologies) sequencing performance was optimized by comparing various combinations of sequence aligners, re-aligners, de-duplicators, quality re-calibrators and genotype callers. Independently, six exomes were captured using the Agilent SureSelect v3 kit. The optimized pipeline was applied, and results were compared to microarray genotyping to characterize false positives and negatives. A further four exomes were pair-end sequenced on both the Life Technologies 5500x1 and Illumina HiSeq sequencers to check platform concordance. Variant metrics for each exome were compared to the literature.
In the clinic, individual exomes are manually triaged by a medical geneticist, and salient variants are confirmed by Sanger sequencing. For disease cohorts, software was developed to isolate variants possibly causing monogenic rare diseases, taking likely false positives into account.