Overview of experimental design. Six genomic intervals, each encoding genes for K+/Na+ voltage-gated channel proteins, were amplified using DNA from four individuals and LR-PCR reactions to generate 260 kb of target sequence per sample. Amplicons from each individual were pooled in equimolar amounts and then sequenced using the three NGS platforms. The 260 kb examined in this study is representative of human sequences containing 38% repeats and 4% coding sequence compared with 47% and 1%, respectively, genome-wide. For each sample 88 kb was amplified using short range PCR (SR-PCR) reactions targeting the exons and evolutionarily conserved intronic regions. Each SR-PCR amplicon was individually sequenced in the forward and reverse directions using the ABI-3730xL platform (Additional data file 2). Data generated from the NGS platforms were analyzed to identify bases variants from the reference sequence (build 36) and the quality of the variant calls was assessed using platform specific methodologies. A comparative analysis of the sequence data from the NGS platforms and ABI Sanger was then performed to determine accuracy, and false positive and false negative rates.
Harismendy et al. Genome Biology 2009 10:R32 doi:10.1186/gb-2009-10-3-r32