Specifically, significantly regulated genes were placed at the top or bottom of the list and ordered by descending or ascending fold changes, respectively. Less significantly regulated Wnt signaling pathway inhibitor genes were placed in the middle of the list and ordered by ascending or descending p values. GO terms with the top ten Normalized Enrichment Scores were Compound C selected and combined from both the upregulated and downregulated GO terms in each time point, compared to the 0 min treatment condition. 2 function in the gplots package in R selleck chem with default parameters. In the present study, prior to mutagenesis with ENU, we in troduced extra copies of human PIGA cDNA into the H129 2 haploid ESC line, but not into the HAP 1 haploid ESC line. As a result, X linked Piga mutants apparently dominated in the HAP 1 ESC population, whereas no Piga mutants, but instead various other autosomal mutants, appeared in the H129 2 ESC population. Pigg, Pign, and Pigq are known by their hypomorphic loss of function phenotypes. Consistently, a re cent ESC based mutagenesis study using the clustered regularly interspaced short palindromic repeats Cas system also screened for the resistance to toxin and failed to obtain Pigg, Pign, and Pigq mutants. This suggests that, apart from these three genes and Piga, the remaining 22 genes are essential for the maintenance of toxin sensitivity in our screening scheme. Because the present mutagenesis study identified 20 of the 22 essential genes, this corresponded to a degree of saturation of 91%. Whole exome sequencing for detection of mutations in haploid ESCs The degree of saturation in mutagenesis largely depends on the mutagen. We examined 10 independent H129 2 derived mutant ESC clones by whole exome sequencing and compared the results with those from ENU untreated parental ESC clones. Over 98% of the reads were success fully mapped to the NCBI37mm9 mouse reference gen ome with a mean coverage of 92. 4, and 84. 5% of the exome regions were analyzed at 30 fold depth. Given that each locus has in principle one allele, only mutations designated homozygous were taken into account by our WES analytical pipeline. To filter out potential false positive mutations, we adopted the following criteria the mutations are only positive when 90% of reads are called alteration in ENU treated mutant ESCs and 95% of the corresponding reads are called reference in ENU untreated control ESCs. These criteria were validated by the Sanger sequencing of 20 true positive mutations and 22 false positive mutations in mutant clone F 43. As a result, 19 of the 20 true positive mutations were con firmed, and 0 of the 22 false positive mutations were de tected.
One mutation thought to be true positive but not detected by Sanger sequencing was near the threshold line of our criteria. Using Fishers exact test, we obtained an extremely small P value, confirming the appropriateness of the above criteria for filtering WES data. Yoshida et al. previously reported a true positive rate of candidate mutations of only 53. 9% using WES data from patients with myelodysplastic syndrome.