MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature Sensitive Mutants via Molecular Inversion Probes

Temperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants of Caenorhabditis elegans and used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.