Investigation of spatial vulnerabilities of Bacterium Escherichia coli genome to spontaneous mutations by molecularly barcoded deep Sequencing

Investigation of spontaneous mutations by Next-Generation Sequencing technology has attracted extensive attention lately due to the fundamental roles of spontaneous mutations in evolution and pathological processes. However, these studies only focused on the mutations accumulated through many generations during long term --could be years of-- culturing, but not the freshly-generated spontaneous mutations that occurs at very low frequencies. In this study, we established a molecularly-barcoded deep sequencing strategy to detect low abundant spontaneous mutations in genomes of bacteria cell cultures. Genome-wide spontaneous mutations in 15 E. coli cell culture samples were defined with a high confidence (P < 0.01). We also developed a hotspot region-calling approach based on Run-Length Encoding algorithm to find the genomic regions that are vulnerable to the spontaneous mutations. The hotspots for the mutations appeared to be highly conserved across the bacteria samples. Further biological annotation of these regions indicated that most of the spontaneous mutations were located at the repeat domains or nonfunctional domains of the genomes, suggesting the existence of mechanisms that could somehow prevent the occurrence of mutations in crucial genic areas. This study provides a more faithful picture of mutation occurrence and spectra without the distortion of long term culturing. We conducted RNA-seq analysis for E.coli lines of E3C to study the relationship between mutation and gene expression.