DNA methylation occurs by the covalent addition of a methyl group (CH3) at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine (5-mC). DNA methylation is essential in regulating gene expression in nearly all biological processes, including development, growth, and differentiation. Alterations in DNA methylation have been demonstrated to cause a change in gene expression. For example, hypermethylation leads to gene silencing or decreased gene expression, while hypomethylation activates genes or increases gene expression. Aberrant DNA methylation is also associated with the pathogenesis of diseases such as cancer, autoimmune disorders, and schizophrenia. Thus, genome-wide analysis of DNA methylation could provide valuable information for discovering epigenetic markers used for disease diagnosis and potential targets used for therapeutics. Reduced representation bisulfite sequencing (RRBS) is the most used genome-wide DNA methylation analysis method. It enriches CpG-rich DNA fragments with restriction enzyme digestion and converts unmethylated cytosine to uracil, while 5-mC remains unmodified by the bisulfite treatment. Epigenetic differences are interpreted as genetic differences, which can then be detected by sequencing at single-base resolution and on a genome-wide scale. However, traditional RRBS is still not practical for many reasons. (1) It requires relatively large amounts of DNA as input material, which is difficult to prepare from limited biological samples such as tumor biopsy, early embryos, embryonic tissues, and circulating DNA. (2) DNA must be sheared first and then ligated to adaptors, followed by bisulfite conversion (post-ligation bisulfite conversion). This procedure causes most of the DNA fragments (contained in the adaptor-DNA fragment constructs) to be broken, resulting in the formation of mono-tagged templates that will be removed during library enrichment. Thus, incomplete coverage and bias occur when performing RRBS. (3) The method is also time-consuming, taking approximately 2 days.