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PLoS One. 2010 Nov 8;5(11):e15435. doi: 10.1371/journal.pone.0015435.

C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy.

Author information

  • 1Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York, United States of America. md2398@columbia.edu

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.

PMID:
21079745
[PubMed - indexed for MEDLINE]
PMCID:
PMC2975709
Free PMC Article

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