title: C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy.
abstract: Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York, United States of America. md2398@columbia.edu
authors.forename: Maria
authors.initials: M
authors.lastname: Doitsidou
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Poole
authors.forename: Sumeet
authors.initials: S
authors.lastname: Sarin
authors.forename: Henry
authors.initials: H
authors.lastname: Bigelow
authors.forename: Oliver
authors.initials: O
authors.lastname: Hobert
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D005260:Female; D056726:Genetic Association Studies / Q000379:methods; D049751:Genome, Helminth / Q000235:genetics*; D055106:Genome-Wide Association Study / Q000379:methods; D005838:Genotype; D018398:Homeodomain Proteins; D008297:Male; D009154:Mutation; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D034622:RNA Interference; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D012984:Software; D014157:Transcription Factors
chemical_list: D029742:Caenorhabditis elegans Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C095405:vab-3 protein, C elegans