Mutagenesis was carried out using VC2010, a local subculture of the standard laboratory strain N2 (Brenner 1974 (link)) with either ethyl methanesulfonate (EMS) (Sulston and Hodgkin 1988 ), N-ethyl-N-nitrosourea (ENU) (De Stasio and Dorman 2001 (link)), trimethylpsoralen/UV (UV/TMP) (Flibotte et al. 2010 (link)), or with an EMS/ENU cocktail. F1 animals were screened in 1% nicotine (Moerman and Baillie 1979 (link)) to ensure mutagenesis and propagated through F10, with single F10 animals used to establish strains for DNA isolation and frozen stocks as shown in Figure 1. Genomic DNA from mutant strains and 40 wild isolates (Supplemental Table 10) was extracted as described earlier (Flibotte et al. 2010 (link)), and libraries prepared for sequencing using a modified Illumina protocol using only one addition of Agencourt AMPure XP beads per sample through Y-adaptor ligation. Multiplexed libraries were sequenced with Illumina GAII/HiSeq technology, and clusters passing default quality filters were demultiplexed using a custom perl script. Raw FASTQ files from each strain were aligned to build WS230 of the C. elegans genome (www.wormbase.org) using the alignment program phaster (P Green, pers. comm.) or BWA version 0.5.9 (Li and Durbin 2009 (link)) for comparison. All reported variants were generated using phaster (see Supplemental Material for details).