Identifying Suppressor Mutations in C. elegans

We first performed a conventional Sanger sequencing analysis for all suppressor alleles for cebp-1 and determined that the ju1367 allele affected cebp-1. We next sequenced mak-2, which was previously known to act in the same pathway as cebp-1 in neurons [22 (link)], and found ju1349 and ju1352 alleles to affect mak-2. All other suppressors were analysed by whole genome sequencing analysis and single-nucleotide polymorphism (SNP) mapping following established methods [51 (link)]. Briefly, genomic DNA was prepared using a Puregene Cell and Tissue Kit (Qiagen) according to the manufacturer’s instructions, and 20X coverage of sequences was obtained using a 90 bp paired-end Illumina HiSeq 2000 at Beijing Genomics Institute (BGI Americas). The raw sequences were mapped to the C. elegans reference genome (WS220/ce10) using Burrows-Wheeler Aligner (BWA) [52 (link)] in the Galaxy platform (http://usegalaxy.org) [53 (link)]. Following subtraction of the nucleotide variants in the original strains, we generated a list of candidate genes containing unique homozygous nucleotide variants that were predicated to alter the function of the gene. We then confirmed the causality of the candidate genes by testing the known null alleles on the suppression of nipi-3(0).