We detected SNPs according to a procedure outlined in Figure 2 and described in detail in the methods section. In short, alignments were scored for likely deviation from the reference sequence and then sequentially filtered for coverage, homozygosity, and a requirement that reads map to both strands and sequence **data** quality. Following this, SNPs were further filtered to remove all variants unlikely to be obtained by EMS treatment, which primarily induces G to A mutations (Greene et al., 2003) which also appear in sequence **data** as the reverse complement, C to T. Table 2 summarizes the SNP detection results and filtering steps. We obtained a total of 920,834 likely deviations from the reference sequence in initial SNP calling for the five samples using the SAMtools SNP calling package (Li and Durbin, 2009). Discarding low quality SNPs and SNPs of repetitive origin reduced the total by 59 percent to 376,533. Due to the low coverage and depth of sequencing **data** from the 12-2 sample an unsusually high number of sites initially scored as variants but derived from singleton reads were detected. These positions were efficiently removed by our procedure. Further filtering to retain only SNPs likely to be homozygous with a minimum Phred quality score of 20 retained 125,308 positions, removing 27% of the initial SNP calls. If we further require all reads overlapping a SNP site to contain the mutant allele and for reads to derive from both forward and reverse DNA strands, only 71,058 high-quality homozygous SNPs remain from the five lines. EMS-induced alleles are almost exclusively G:C to A:T transitions unlike background mutations which could be any of the eight possible sequence changes.