Deaminase-Assisted Sequencing for the Identification of 5-glyceryl-methylcytosine.

DNA epigenetic modifications play crucial roles in regulating gene expression and cellular function across diverse organisms. Among them, 5-glyceryl-methylcytosine (5gmC), a unique DNA modification first discovered in Chlamydomonas reinhardtii, represents a novel link between redox metabolism and epigenetic regulation. Accurate genome-wide detection of 5gmC is essential for investigating its biological functions, yet no streamlined method has been available. Here, we present deaminase-assisted sequencing (DEA-seq), a simple and robust approach for base-resolution mapping of 5gmC. DEA-seq employs a single DNA deaminase that efficiently converts unmodified cytosines (C) and 5-methylcytosine (5mC) into uracils or thymines, while leaving 5gmC intact. This selective resistance generates a clear sequence signature that enables precise identification of 5gmC sites across the genome. The method operates under mild reaction conditions and is compatible with low-input DNA, minimizing sample loss and improving detection sensitivity. Overall, DEA-seq provides an accessible, efficient, and highly accurate protocol for profiling 5gmC, offering clear advantages in workflow simplicity, DNA integrity, and analytical performance. Key features • A commercial deaminase mix (DEA) efficiently converts unmodified cytosines and 5-methylcytosines into uracils or thymines. • 5gmC specifically resists DEA-mediated deamination, enabling its identification at single-base resolution. • DEA-seq requires only minimal DNA input and supports high-sensitivity detection from nanogram-level samples.