AbstractSpontaneous mutations power evolution, whereas large-scale structural variations (SVs) remain poorly studied, primarily because of the lack of long-read sequencing techniques and powerful analytical tools. Here, we explore the SVs ofEscherichia coliby running 67 wild-type (WT) and 37 mismatch repair (MMR)–deficient (ΔmutS) mutation accumulation lines, each experiencing more than 4,000 cell divisions, by applying Nanopore long-read sequencing and Illumina PE150 sequencing and verifying the results by Sanger sequencing. In addition to precisely repeating previous mutation rates of base-pair substitutions and insertion and deletion (indel) mutation rates, we do find significant improvement in insertion and deletion detection using long-read sequencing. The long-read sequencing and corresponding software can particularly detect bacterial SVs in both simulated and real data sets with high accuracy. These lead to SV rates of 2.77 × 10−4(WT) and 5.26 × 10−4(MMR-deficient) per cell division per genome, which is comparable with previous reports. This study provides the SV rates ofE. coliby applying long-read sequencing and SV detection programs, revealing a broader and more accurate picture of spontaneous mutations in bacteria.