Department of Computer Science, University of Montana, Missoula, MT, USA.
BMC Genomics. 2011 Jul 5;12:347. doi: 10.1186/1471-2164-12-347.
Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses.We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus) to capture (enrich for), and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison). Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits.
We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes.
This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.
通过提高对适应和物种形成遗传过程的理解，基因靶向和全基因组标记对于推进进化生物学、农业和生物多样性保护至关重要。不幸的是，对于具有大基因组的真核生物来说，获得基因组序列和开发基因组资源（如全基因组 SNP）仍然成本高昂。需要有一种方法来允许进行基因靶向的下一代测序，这种方法足够灵活，可以包括任何基因或多个基因，与转录组测序不同。这种方法将允许对许多个体进行测序，从而避免后续种群遗传分析中的确定偏差。
我们展示了一种最近的技术，即外显子捕获，在没有基因组资源的物种中用于全基因组、基因靶向标记发现的有用性。我们使用来自家养牛基因组序列（Bos taurus）的编码基因序列来捕获（富集），并随后对 B. taurus、B. indicus 和野牛（野生野牛）的数千个外显子进行测序。我们的捕获阵列有针对 2570 个基因中的 16131 个外显子的探针，包括 203 个具有已知功能的候选基因，这些候选基因与疾病和其他适应性特征有关。
我们成功地对 B. taurus 基因组序列的 29 条常染色体和 X 染色体上的外显子序列进行了测序和映射。外显子捕获和高通量测序在所有三个个体的所有参考染色体上均匀地鉴定出了数千个假定的 SNP，包括我们靶向候选基因中的数百个 SNP。
这项研究表明，外显子捕获可以针对许多个体和没有基因组资源的非模式物种进行 SNP 发现的定制。我们捕获的外显子集足够小，可以进行负担得起的下一代测序，并且使用家养牛基因组作为参考成功地捕获了来自不同物种的外显子。
