Forward genetic screens in Mus musculus have proved powerfully informative by revealing unsuspected mechanisms governing basic biological processes. This approach uses potent chemical mutagens, such as N-ethyl-N-nitrosourea (ENU), to randomly induce mutations in mice, which are then bred and phenotypically screened to identify lines that disrupt a specific biological process of interest. Although identifying a mutation using the rich resources of mouse genetics is straightforward, it is unfortunately neither fast nor cheap. Here we show that detecting newly induced causal variants in a forward genetic screen can be accelerated dramatically using a methodology that combines multiplex chromosome-specific exome capture, next-generation sequencing, rapid mapping, sequence annotation, and variation filtering. The key innovation of our method is multiplex capture and sequence that allows the simultaneous survey of both mutant, parental, and background strains in a single experiment. By comparing variants identified in mutant offspring with those found in dbSNP, the unmutagenized background strains, and parental lines, induced causative mutations can be distinguished immediately from preexisting variation or experimental artifact. Here we demonstrate this approach to find the causative mutations induced in four novel ENU lines identified from a recent ENU screen. In all four cases, after applying our method, we found six or fewer putative mutations (and sometimes only a single one). Determining the causative variant was then easily achieved through standard segregation approaches. We have developed this process into a community resource that will speed up individual labs' ability to identify the genetic lesion in mutant mouse lines; all of our reagents and software tools are open source and available to the broader scientific community.
Keywords: DNA sequencing; N-ethyl-N-nitrosourea (ENU) mutagenesis; genomics; next generation sequencing; targeted enrichment.