Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Genome Res. 2013 Apr;23(4):679-86. doi: 10.1101/gr.147322.112. Epub 2013 Jan 8.
Forward genetic screens have elucidated molecular pathways required for innumerable aspects of life; however, identifying the causal mutations from such screens has long been the bottleneck in the process, particularly in vertebrates. We have developed an RNA-seq-based approach that identifies both the region of the genome linked to a mutation and candidate lesions that may be causal for the phenotype of interest. We show that our method successfully identifies zebrafish mutations that cause nonsense or missense changes to codons, alter transcript splicing, or alter gene expression levels. Furthermore, we develop an easily accessible bioinformatics pipeline allowing for implementation of all steps of the method. Overall, we show that RNA-seq is a fast, reliable, and cost-effective method to map and identify mutations that will greatly facilitate the power of forward genetics in vertebrate models.
正向遗传学筛选已经阐明了生命无数方面所需的分子途径；然而，从这些筛选中识别出因果突变一直是该过程的瓶颈，特别是在脊椎动物中。我们开发了一种基于 RNA-seq 的方法，可以识别与突变相关的基因组区域和可能导致感兴趣表型的候选病变。我们表明，我们的方法成功地鉴定了导致密码子无义或错义变化、改变转录剪接或改变基因表达水平的斑马鱼突变。此外，我们开发了一个易于访问的生物信息学管道，允许实现该方法的所有步骤。总的来说，我们表明 RNA-seq 是一种快速、可靠且具有成本效益的方法，可以对突变进行定位和鉴定，这将极大地促进正向遗传学在脊椎动物模型中的应用。
