Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes

Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although many ssDNA detection technologies exist, almost all methods have been developed for eukaryotic genomes. To apply these to bacterial genomes, which harbor a smaller amount of DNA than those of eukaryotes, more efficient, new methods are required. Therefore, we developed a novel strand-specific ssDNA sequencing method, called 4S-seq, for the bacterial genome. The 4S-seq method enriches ssDNA in the extracted genomic DNA by a dsDNA-specific nuclease and implements a strand-specific library using a biotin label with a customized tag. As a result, the 4S-seq is able to calculate the ssDNA content in each strand (Watson/Crick) at each position of the genome efficiently.