DMN-seq enriches DNA hypomethylated regions for biomarker discovery using 5-methylcytosine glycosylase

Various methods have been developed for 5-methylcytosine (5mC) sequencing; however, effective ways to enrich hypomethylated DNA regions have been limited. Here, we describe the DEMETER-assisted 5-Methylcytosine Nicking sequencing (DMN-seq) utilizing 5mC-specific glycosylase DEMETER to nick DNA at 5mC sites, enabling 5mC detection at the single-base resolution. Leveraging this nicking activity to deplete hypermethylated sites, we adapt DMN-seq to preferentially enrich and investigate hypomethylated regions in colorectal cancer samples. When applied to cell-free DNA as low as 0.1 ng, DMN-seq significantly expands the scope of cancer biomarkers by capturing hypomethylated regions, with high sensitivity and reproducibility even in low-input clinical samples.