Expressed exome capture sequencing: A method for cost‐effective exome sequencing for all organisms
Expressed exome capture sequencing: A method for cost‐effective exome sequencing for all organisms
复制标题
DOI：
10.1111/1755-0998.12905
复制
发表时间：
2018-06
影响因子：
7.7
通讯作者：
Jonathan B. Puritz;K. Lotterhos
中科院分区：
文献类型：
--
作者：
Jonathan B. Puritz;K. Lotterhos
外显子组捕获是研究被选择基因座基因组的有效工具。然而，传统的方法需要带注释的基因组资源。在这里，我们提出了一种从表达的mRNA中创建cDNA探针的方法，然后将其用于丰富和捕获外显子区域的基因组DNA。这种方法被称为“EecSeq”，消除了昂贵的探针设计和合成的需要。我们使用受控暴露实验，在东方牡蛎Crassostrea virgiica中检测了EecSeq。4只成年牡蛎在36℃下热休克1小时，4只对照牡蛎在14℃下保存，每个处理为两个人准备单链mRNA文库并汇集。组合文库的一半用于探针合成，另一半用于测序以评估捕获效率。从所有个体中提取基因组DNA，通过捕获的探针进行浓缩，并直接测序。我们发现，EecSeq对所有已知外显子的平均捕获敏感度为86.8%，对mRNA文库中可检测到表达水平的外显子的敏感度超过99.4%。在所有的定位阅读中，超过47.9%映射到外显子，37.0%映射到表达靶标，这与以前发表的外显子捕获研究类似。EecSeq在外显子内显示了相对均匀的覆盖率(即，较小的“边缘效应”)，甚至覆盖了外显子GC内容。我们发现了5951个SNPs，最小平均覆盖率为80×，其中3508个SNPs出现在外显子区域。我们表明，与昂贵的传统方法相比，EecSeq提供了相当的(如果不是更好的)特异性和捕获效率。
Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called “EecSeq,” eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1 hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor “edge effects”) and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.