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

Authors

Bertocchi, U., Katz, E., Jeffet, J., Grunwald, A., Gabay, N., Deek, J., Verma, S., Shwartz, A., Umschweif-Nevo, G., Lerer, B., Roichman, Y., Ebenstein, Y.

Abstract

DNA methylation dynamically regulates cellular function and phenotype. At the tissue level, stochastic variation in methylation patterns, measured as methylation entropy, drives plasticity, development, cancer, and aging. Demethylation is facilitated by erasure of 5-methylcytosine (5mC) via the oxidized intermediate 5-hydroxymethylcytosine (5hmC), but bisulfite sequencing cannot distinguish these modifications, classifying both as 5mC. Using nanopore sequencing with direct detection of 5mC and 5hmC, we quantified how this historical conflation affects genome-wide methylation levels and methylation entropy in kidney cancer and the mouse medial prefrontal cortex. Bisulfite-like analysis introduced systematic, tissue-specific shifts in methylation distributions, influencing biological interpretation. However, these effects were modest in the low-5hmC kidney cancer samples, where pathway-level results remained highly concordant. Our findings demonstrate that True-5mC-based methylation entropy redefines the physical mapping of epigenomes, demonstrating that, in some contexts, what was previously interpreted as stochastic maintenance failure is frequently the structured signature of distinct, mechanistically interpretable cytosine biochemistry.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 09 Jul 2026.

Stats

1-terrible, 9-excellent. How would you rate this preprint? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.