Efficient Mapping and Cloning of Mutations in Zebrafish by Low-Coverage Whole-Genome Sequencing
Efficient Mapping and Cloning of Mutations in Zebrafish by Low-Coverage Whole-Genome Sequencing
复制标题
DOI：
10.1534/genetics.111.136069
复制
发表时间：
2012-03-01
期刊：
影响因子：
3.3
通讯作者：
Harris, Matthew P.
中科院分区：
文献类型：
--
作者：
Bowen, Margot E.;Henke, Katrin;Harris, Matthew P.
斑马鱼突变体的产生和分析对于确定脊椎动物发育、生理和疾病的遗传调控起到了重要作用。然而，鉴定构成突变体表型基础的遗传变化仍然是突变体分析中的一个重大瓶颈。全基因组测序最近已成为一种在非脊椎动物模式生物中鉴定突变的快速且有效的方法。然而，由于斑马鱼基因组较大以及缺乏完全近交系等复杂因素，这种方法尚未应用于斑马鱼。在此我们提供一种利用这些新的平行测序技术高效定位和检测斑马鱼突变的方法。该方法利用一个广泛的参考单核苷酸多态性（SNP）数据库，通过对仅来自有限数量的突变F - 2代鱼的混合DNA进行低覆盖度的全基因组测序来确定同祖纯合区域。通过这种方法，我们对所测序的五种不同斑马鱼突变体中的每一种都进行了定位，并在其中两种中鉴定出可能的致无义突变，在其余的突变体中鉴定出候选突变。此外，我们提供证据表明，所鉴定出的其中一个突变，即bmp1a中的无义突变，是融合突变体表型的基础。
The generation and analysis of mutants in zebrafish has been instrumental in defining the genetic regulation of vertebrate development, physiology, and disease. However, identifying the genetic changes that underlie mutant phenotypes remains a significant bottleneck in the analysis of mutants. Whole-genome sequencing has recently emerged as a fast and efficient approach for identifying mutations in nonvertebrate model organisms. However, this approach has not been applied to zebrafish due to the complicating factors of having a large genome and lack of fully inbred lines. Here we provide a method for efficiently mapping and detecting mutations in zebrafish using these new parallel sequencing technologies. This method utilizes an extensive reference SNP database to define regions of homozygosity-by-descent by low coverage, whole-genome sequencing of pooled DNA from only a limited number of mutant F-2 fish. With this approach we mapped each of the five different zebrafish mutants we sequenced and identified likely causative nonsense mutations in two and candidate mutations in the remainder. Furthermore, we provide evidence that one of the identified mutations, a nonsense mutation in bmp1a, underlies the welded mutant phenotype.