SNP discovery performance of two second-generation sequencing platforms in the NOD2 gene region.

A potentially important application of second generation sequencing technologies is to identify disease-associated variation. For comparison of the performance in SNP detection, the Crohn's disease (CD)-associated NOD2 gene was subjected to targeted resequencing using two different second-generation sequencing technologies. Eleven CD patients were selected based on their haplotype background at the NOD2 locus. The 40-kb large NOD2 gene region was amplified using long-range PCR (LR-PCR), and sequenced with the Roche 454/FLX system, an Applied Biosystems SOLiD mate-pair library (2 x 25 bp), and a SOLiD fragment (50 bp) library. The entire NOD2 region was also sequenced using conventional Sanger technology. Four-hundred forty-two single nucleotide polymorphisms (SNPs) were discovered with the SOLiD mate-pair library, 454 with the fragment library, and 441 with the 454/FLX. For the homozygous SNPs, 98% were confirmed by Sanger for the mate-pair library, 100% for the fragment library and 99% for the 454/FLX. Ninety-six percent of the heterozygous SNPs detected with the SOLiD mate-pair library, 91% with the fragment library and 96% with the 454/FLX were confirmed by Sanger. In a simulation, the SNP detection performance fell rapidly when the achieved coverage was below 40 x. Due to uneven representation of the target region when using LR-PCR, oversequencing of other regions is necessary. Hum Mutat 31:875-885, 2010. (C) 2010 Wiley-Liss, Inc.