Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing

Abstract

BackgroundExome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.ResultsHere we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.ConclusionExon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

Genetic and Chemical Dissection of BloodBrain Barrier Development in Zebrafish

“…To clone this line, we simultaneously performed BSA and NGS experiments. Like other groups, we did not get a successful map position with BSA but were able to clone 22.6 with WES 134 . WES revealed a point mutation in the gpr124 allele which in turn was predicted to cause a premature stop codon in the protein sequence.…”

“…BSA has been efficacious at isolating genes mutated in forward genetic screens but not every mutant in a forward genetic screen can be successfully cloned with this strategy 50,134 . Positional cloning is quite time consuming and tedious if linked markers are not detected in the initial round of mapping.…”

“…After the gels were analyzed, another round of PCR was performed with follow-up markers on individual larvae. Combing both techniques has successfully identified linkage and subsequent alleles in Xenopus tropicalis mutants 134 . While this is an ideal strategy, positional cloning is not always guaranteed to work for every mutant.…”

“…While this is an ideal strategy, positional cloning is not always guaranteed to work for every mutant. For example, del Viso et al (2012) were able to perform this dual approach on the ruby mutant but not on the grinch mutant 134 . We were also unsuccessful at identifying a map position with BSA.…”

“…Positional cloning is commonly performed in the zebrafish with bulk segregant analysis PCR 129 . While this method has cloned zebrafish mutants, it is not fully guaranteed for every mutant identified in a genetic screen 134 . To increase our chances of cloning 22.6, we simultaneously performed exome sequencing on pools from 22.6 mutant and wild-type sibling genomic DNA.…”