Direct sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution unravels their distinct roles in Alzheimer's disease [BS-Seq]
5-hydroxymethylcytosine (5hmC) constitutes up to 20% of 5-methylcytosine (5mC) in mammalian cells. As conventional bisulfite sequencing is unable to distinguish 5hmC from 5mC, several methods have been proposed to detect 5hmC and infer 5mC levels by subtracting 5hmC from whole-genome bisulfite sequencing data. This subtraction approach, however, can lead to the underestimation of 5mC levels at sites with high 5hmC abundance. Here, we present two orthogonal methods: CMD1-Deaminase sequencing and CMD1-TET bisulfite sequencing, which enable the direct and independent detection of 5mC. In combination with ACE-seq or TAB-seq, these techniques facilitate an accurate distinction between 5mC and 5hmC, eliminating the need for subtraction. Using these approaches, we generated a comprehensive landscape of 5mC and 5hmC in the hippocampus of the Alzheimer's disease (AD) model mice. Notably, while 5hmC levels were substantially reduced in the AD mice, 5mC levels remained largely unchanged, suggesting a critical role for 5hmC as an independent epigenetic mark in the pathogenesis of AD. Overall design: CD-seq?CT-seq?ACE-seq and TAB-seq in hippocampus of WT and Alzheimer's disease model mice.