Direct Sequencing of 5‐Methylcytosine and 5‐Hydroxymethylcytosine at Single‐Base Resolution Unravels Their Distinct Roles in Alzheimer's Disease

Alzheimer's disease (AD) is associated with genetic risk factors and widespread epigenetic alterations. 5-Hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), constitutes up to 20% of 5mC in neuronal DNA and is implicated in aging and neurodegeneration. However, the precise roles of DNA modifications in AD remain unclear, partly due to the lack of accurate detection methods. Here, two orthogonal sequencing methods are introduced: CMD1-Deaminase sequencing (CD-seq) and CMD1-TET bisulfite sequencing (CT-seq), which enable direct, independent detection of 5mC. When combined with APOBEC-coupled epigenetic sequencing (ACE-seq) or TET-assisted bisulfite sequencing (TAB-seq) for 5hmC mapping, these techniques provide base-resolution, subtraction-free profiling of DNA modifications. Applying them to hippocampal tissue from AD model mice, a significant reduction in 5hmC levels is identified without corresponding changes in 5mC, suggesting that 5hmC functions as an independent epigenetic mark in AD pathogenesis. These findings underscore the importance of precise 5mC/5hmC discrimination and suggest that 5hmC and its regulatory pathways may serve as potential therapeutic targets for AD.