Genetic mapping by whole genomic DNA sequencing

FastQC was used to check the quality of raw sequence data [51]. Sequence reads were aligned to the ce10 C. elegans assembly using bwa with parameters: aln -n 0.04 -t 20, but only retaining single-hit alignments (‘‘bwa samse -n 1”) [52]. The resulting alignments were converted to BAM format, sorted and indexed using ‘‘samtools” [53]. Sequence variants were identified using GATK (version 3.0). Finally, the numbers of high quality (score > = 300) single nucleotide substitutions, absent from the parental strain, were counted in sequential windows of 1 Mb to identify regions of increased variant density. VCFTOOLs were used to filter out background SNPs [54]. For viable mutants, homozygous SNPs were plotted across chromosomes to estimate the location of mutated genes. For the sterile mutant, heterozygous SNPs were used for the analysis. SnpEff was employed to annotate the effects of SNP mutations [55]. Candidate genes were confirmed by RNAi or crossed with mutants obtained from the Caenorhabditis Genetics Center (CGC).

The goal of genetic mapping is to locate phenotype-causing SNPs in the genome. The rationale behind the mapping used for viable and sterile mutant was based on SNPs introduced randomly by EMS mutagenesis. The linkage between SNPs and mutated gene is distance-dependent. SNPs that are not linked to the phenotype-causing SNP/gene are diluted after backcrossing and phenotype selection. Only SNPs that are close to the phenotype-causing SNP/gene are enriched, which is revealed by SNP analysis. In case of viable mutants with recessive mutations, the mutants were homozygous for the phenotype-causing SNPs, so all corresponding sequencing reads displayed those SNPs. However, in case of the recessive sterile mutant, the progeny of heterozygous mutant were collected. Based on Mendelian genetics, these were ¼ wild-type, ½ heterozygous, and ¼ sterile. On average, half of the corresponding sequencing reads displayed the phenotype-causing SNP and the other half the wild-type sequence. In what we called “the heterozygous SNP frequency-based mapping”, or Het-Map for short, the location of phenotype-causing mutation was pinpointed based on the increased co-occurrence between the phenotype and “heterozygous” SNPs.

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