SNP Discovery Performance of Two Second-Generation Sequencing Platforms in the NOD2 Gene Region
SNP Discovery Performance of Two Second-Generation Sequencing Platforms in the NOD2 Gene Region
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DOI：
10.1002/humu.21276
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发表时间：
2010-07-01
期刊：
影响因子：
3.9
通讯作者：
Franke, Andre
中科院分区：
文献类型：
--
作者：
Melum, Espen;May, Sandra;Franke, Andre
第二代测序技术的一个潜在重要应用是识别与疾病相关的变异。为了比较 SNP 检测的性能，使用两种不同的第二代测序技术对克罗恩病 (CD) 相关的 NOD2 基因进行靶向重测序。根据 NOD2 基因座的单倍型背景选择了 11 名 CD 患者。使用长程 PCR (LR-PCR) 扩增 40 kb 大 NOD2 基因区域，并使用 Roche 454/FLX 系统、Applied Biosystems SOLiD 配对文库 (2 x 25 bp) 和 SOLiD 片段 (50 bp) 文库进行测序。整个 NOD2 区域也使用传统的 Sanger 技术进行了测序。 SOLiD 配对文库发现了 442 个单核苷酸多态性 (SNP)，片段文库发现了 454 个，454/FLX 发现了 441 个。对于纯合SNP，配对文库的Sanger 确认为98%，片段文库的纯合SNP 为100%，454/FLX 的纯合SNP 为99%。 Sanger 确认了 SOLiD 配对文库检测到的杂合 SNP 的 96%、片段文库检测到的 91% 以及 454/FLX 检测到的杂合 SNP 的 96%。在模拟中，当达到的覆盖率低于 40 倍时，SNP 检测性能迅速下降。由于使用 LR-PCR 时目标区域的代表性不均匀，因此有必要对其他区域进行过测序。 Hum Mutat 31:875-885, 2010。(C) 2010 Wiley-Liss, Inc.
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.