Beyond Bisulfite Sequencing: Resolving 5-hmC with Nanopore Sequencing Unmasks the True Methylation Entropy Landscape

DNA methylation dynamically regulates cellular function and phenotype. At the tissue level, stochasticity in methylation patterns, as measured by methylation entropy, drives plasticity, development, cancer, and aging. Modulation of methylation patterns is facilitated by erasure of 5-methylcytosine (5mC) via the oxidized intermediate 5-hydroxymethylcytosine (5hmC). Bisulfite sequencing cannot distinguish the two modifications, labeling them both as 5-mC. We quantitatively analyze the effect of this historical conflation on the genome-wide distribution of methylation levels and methylation entropy. Using nanopore sequencing with direct 5mC and 5hmC calling, we compare True-mC to bisulfite-like analysis in two model systems, kidney cancer and the mouse medial prefrontal cortex. We show that bisulfite sequencing introduces systematic, tissue-specific shifts in methylation distribution that affect the mechanistic interpretation of the underlying biology. The distortion scaled with endogenous 5hmC content: substantial in brain, where over half of the entropy-associated gene ontology terms recovered under bisulfite-like analysis were absent under True-mC, and minimal in the low-5hmC kidney cancer sample, which delimits the regime in which bisulfite-derived entropy remains interpretable. Together, these findings establish that True-5mC-based methylation entropy redefines the physical mapping of certain epigenomes, demonstrating that in some contexts, what has previously been interpreted as stochastic maintenance failure is frequently the structured signature of distinct and mechanistically interpretable cytosine biochemistry.