ABSTRACT: 5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic mark that plays a key role in regulating various physiological and pathological processes. Although several methods have been developed to detect 5hmC, direct genome-wide mapping at single-base resolution remains highly desirable. Herein, we developed a double-stranded deamination sequencing (DDD-seq) method for genome-wide mapping of 5hmC at single-base resolution. This method utilizes the double-stranded DNA deaminase SsdAcat from Pseudomonas syringae, which efficiently deaminates cytosine (C), 5-methylcytosine (5mC), 5hmC, 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) in duplex DNA, but not glycosylated 5hmC (5gmC). In DDD-seq, C, 5mC, 5fC, and 5caC in dsDNA are deaminated by SsdAcat and read as T during sequencing, while 5gmC is resistant to deamination, allowing its identification by detecting the remaining C in sequence reads. The map of 5hmC generated by DDD-seq in mouse cerebellum tissue closely aligns with results obtained from the ACE-seq method. Additionally, applying DDD-seq to cerebellum tissue of mouse subjected to chronic sleep deprivation revealed significant global changes in 5hmC distribution on genomic DNA. In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA, DDD-seq eliminates this step, reducing risks associated with incomplete denaturation and simplifying sequencing library construction. Additionally, SsdAcat demonstrates superior thermostability and activity across a wide range of pH values and temperatures, making DDD-seq applicable in broader scenarios with more accessible conditions. Collectively, the DDD-seq method is straightforward, bisulfite-free, and eliminates the need for DNA denaturation step, making it a valuable tool for direct and quantitative detection of 5hmC in genomes at single-base resolution.
