Bacterial DNA methylation occurs in diverse sequence contexts and plays important functional roles in cellular defense and gene regulation. An increasing number of studies have reported that bacterial DNA methylation has important roles affecting clinically relevant phenotypes such as virulence, host colonization, sporulation, biofilm formation, among others. Bacterial methylomes contain three primary forms of DNA methylation: N6-methyladenine (6 mA), N4-methylcytosine (4mC) and 5-methylcytosine (5mC). The widely used bisulfite sequencing for DNA methylation mapping in mammalian genomes are not effective at resolving bacterial methylomes. Single molecule real-time (SMRT) can effectively map 6mA and 4mC events, and have empowered the study of >4,000 bacterial methylomes in the past ten years. However, SMRT sequencing cannot effectively detect 5mC methylation.