title: SNP discovery performance of two second-generation sequencing platforms in the NOD2 gene region.
abstract: 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.
authors.affiliation: Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany.
authors.forename: Espen
authors.initials: E
authors.lastname: Melum
authors.forename: Sandra
authors.initials: S
authors.lastname: May
authors.forename: Markus B
authors.initials: MB
authors.lastname: Schilhabel
authors.forename: Ingo
authors.initials: I
authors.lastname: Thomsen
authors.forename: Tom H
authors.initials: TH
authors.lastname: Karlsen
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Stefan
authors.initials: S
authors.lastname: Schreiber
authors.forename: Andre
authors.initials: A
authors.lastname: Franke
mesh_terms: D003424:Crohn Disease / Q000235:genetics; D020022:Genetic Predisposition to Disease; D006239:Haplotypes; D006801:Humans; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein