Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for resequencing of hypertrophic cardiomyopathy-associated genes.

Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range PCR (LR-PCR), 40 GS FLX and 31 Genome Analyzer exon variants were identified using >or=30-fold read-coverage and >or=20% read-percentage selection criteria. Twenty-seven platform concordant variants were Sanger-confirmed. The discordant variants segregated into two categories: variants with read coverages >or=30 on one platform but <30-fold on the alternate platform and variants with read percentages >or=20% on one platform but <20% on the alternate platform. All variants with <30-fold coverage were Sanger-confirmed, suggesting that the coverage criterion of >or=30-fold is too stringent for variant discovery. The variants with <20% read percentage were identified as reference sequence based on Sanger sequencing. These variants were found in homopolymer tracts and short-read misalignments, specifically in genes with high identity. The results of the current study demonstrate the feasibility of combining LR-PCR with the Genome Analyzer or GS FLX for targeted resequencing of HCM-associated genes.