5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）是最丰富的DNA修饰，对基因调控起重要作用。然而，由于当前方法无法同时测量这两种修饰，尤其是在数量有限的样本中，对这些不同的表观遗传标记的详细研究以了解它们的综合效应和动态相互转化受到了阻碍。我们提出了一种基于修饰敏感限制酶消化和顺序标签连接的DNA酶切分析联合检测多种表观组态（DARESOME）的技术，在全基因组范围内同时定量分析未修饰的胞嘧啶、5mC和5hmC在CCGG位点的分布。DARESOME揭示了5mC和5hmC在基因表达调控中的相互对立作用以及它们在老化过程中的相互转化。将DARESOME应用于单细胞中展示了显著的5hmC链偏好，反映了DNA的半保守复制特点。最后，我们展示了DARESOME能够对游离DNA进行整合基因组学、5mC和5hmC的综合分析，在液体活检中发现了多组学癌症特征。

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.