Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing.
Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing.
复制标题
通过全基因组测序快速纯合性作图和鉴定斑马鱼 ENU 诱导的突变。
DOI：
10.1371/journal.pone.0034671
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Peers B
中科院分区：
文献类型：
--
作者：
Voz ML;Coppieters W;Manfroid I;Baudhuin A;Von Berg V;Charlier C;Meyer D;Driever W;Martial JA;Peers B
斑马鱼正向遗传学是通过大规模诱变研究脊椎动物发育的有力工具。尽管如此，分子损伤的鉴定仍然是费力的，并且涉及耗时的遗传作图。在这里，我们表明，整个斑马鱼基因组的高通量测序可以直接定位携带致病突变的间隔，同时查明分子病变。这种方法的可行性得到了验证，通过测序的m1045突变株，显示严重发育不全的外分泌胰腺。我们产生了13 Gb的序列，相当于一个八倍的基因组覆盖率，从50个突变体胚胎的游泳池从AB突变载体和WIK多态性菌株之间的地图杂交获得。携带因果突变的染色体区域基于其独特性质被定位，以在序列读段中显示高水平的纯合性，因为它仅来源于初始AB突变的等位基因。我们开发了一种算法，通过计算沿着所有染色体的纯合性得分来识别这样的区域。这突出了染色体5上的8 Mb窗口，在m1045突变体中得分接近1。在此间隔内的所有基因的序列分析揭示了snapc4基因中的无义突变。敲除实验证实了snapc4是导致胰腺外分泌发育不全的基因突变的断言。总之，这项研究构成了一个概念证明，全基因组测序是一个快速和有效的替代经典的位置克隆策略在斑马鱼。
Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.
登录
查看更多内容
影响因子：
10.5
作者：
Henry, RW;Mittal, V;Hernandez, N
通讯作者：
Hernandez, N
影响因子：
14.9
作者：
Hubbard, T;Barker, D;Clamp, M
通讯作者：
Clamp, M
影响因子：
14.9
作者：
Pruitt KD;Tatusova T;Maglott DR
通讯作者：
Maglott DR
影响因子：
3.3
作者：
Arnold, Carrie N.;Xia, Yu;Beutler, Bruce
通讯作者：
Beutler, Bruce
影响因子：
64.5
作者：
Koenig, Harald;Matter, Nathalie;Mueller, Ferenc
通讯作者：
Mueller, Ferenc