Computational protocol: Rapid and Efficient Identification of Caenorhabditis elegans Legacy Mutations Using Hawaiian SNP-Based Mapping and Whole-Genome Sequencing

Similar protocols

Protocol publication

[…] Identification of the molecular lesion responsible for each mutant was determined using a SNP-based mapping strategy combined with WGS (). Hermaphrodites from mutant strains were crossed with Hawaiian males (strain CB4856). Approximately 200 F2 progeny were singled to fresh 3.5-cm plates and shifted to 24° for 16 hr to reveal the mutant phenotype. The plates were returned to 15° and allowed to produce progeny for one to two generations. The animals from 20 to 50 mutant plates were then pooled and genomic DNA isolated and sheared by sonication. Libraries were prepared using TruSeq reagents and sequenced with a HiSequation 2500 (Illumina, San Diego, CA). Single-read 50-bp sequencing yielded a minimum of 22-fold genome coverage for each library. Variants were identified using a pipeline of BFAST for alignment (), SAMtools for variant calling (), and ANNOVAR for annotation () with C. elegans reference genome version WS220 ( Hawaiian SNP density was plotted against chromosome position using R (), and the mapping interval delimited by the absence of Hawaiian SNPs. Mutations within the mapping interval were filtered to remove variants derived from the Hawaiian and pre-mutagenesis strain backgrounds. Candidate genes were defined by homozygous (>85% variant call), nonsynonymous mutations. […]

Pipeline specifications

Software tools BFAST, SAMtools, ANNOVAR
Databases WormBase
Application WGS analysis
Organisms Caenorhabditis elegans