Mutation discovery in the mouse using genetically guided array capture and re-sequencing

Forward genetics (phenotype driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between phenotype and genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (~200 kb) from each of 5 non-complementing Kit mutants (one known allele and 4 unknown alleles) and have successfully identified and validated a non-synonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover mutations.