Department of Agronomy, Iowa State University, Ames, IA 50011, USA.
Plant J. 2010 Jun 1;62(5):898-909. doi: 10.1111/j.1365-313X.2010.04196.x. Epub 2010 Mar 4.
Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.
序列捕获技术是在哺乳动物基因组中开创的，可以对靶向基因组区域进行重测序。大多数捕获方案都需要阻断 DNA，而大量生产阻断 DNA 可能具有挑战性。本研究使用 NimbleGen 微阵列开发了一种无阻断剂、两阶段的捕获方案。第一阶段的捕获会耗尽文库中的重复序列，而第二阶段则富集目标基因座。该策略用于重测序约 2.2 Mb 染色体间隔区和一组分散在 2.3 Gb 玉米基因组中的 43 个基因的非重复部分。该方法实现了约 1800-3000 倍的富集和 80-98%的靶向碱基覆盖度。在目标基因中鉴定出了 2500 多个 SNP。即使存在捕获的同源序列，也获得了低假阳性 SNP 预测率。重要的是，有可能从非参考等位基因中恢复新序列。设计新的重复序列去除和目标捕获微阵列的能力使得这项技术在任何物种中都可以应用。
