DARESOME enables concurrent profiling of multiple DNA modifications with restriction enzymes in single cells and cell-free DNA.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.