Whole-genome sequencing data from next-generation DNA sequencers can provide comprehensive information about mutation spectrum, distribution etc.In this study, we focus on mutation accumulation in DNA repair deficient mutator strains of Escherichia coli that exhibit mutation rates 100 to 1,000 times higher than the wild type.The strains used in this study were YG6156, mutT;YG2250, mutM/mutY;AB1157/pYG782, a DinB-overproducing strain, and the parental wild-type AB1157.First, DNA from overnight cultures was sequenced and it was clarified that 40% of mutations (6/15) in mutT strain were A to C, 75% (18/24) of mutations in mutM/Ywere G to T.Second, we determined mutations generated in the genome of YG2250 with three-month sequential inoculation for 500-times-diluted overnight cultures in LB medium at 37CC.Genomic DNA was prepared from the four cultures at week 1, 4, 8 and 12.Total 88 independent mutations were identified in the four cultures.Of 88 mutations, 47 (53%) were base substitutions.Most of them (41/47) were identified at GC pairs and transversions accounted for 70% of the substitutions.Eighty percent of the base substitutions were identified in ORFs and the numbers of missense and nonsense mutations were almost equal.Besides the substitutions, 41 out of 88 were insertions (34) and deletions (7).The insertions were mainly one-base insertions whereas all the deletions were larger than one base.This new strategy generates more precise data without phenotypic selection which sometimes provides biased data in results.