CpG methylation is a pivotal component of epigenetic landscapes that shape cell-specific transcriptional programs. Oxford Nanopore Technologies (ONT) long read sequencing has emerged as a powerful method to simultaneously profile 5-methylcytosine (5mC) as well as 5-hydroxymethylcytosine (5hmC), an intermediate of the active demethylation pathway. Current methods that profile 5mC and 5hmC are largely restricted to bulk tissue sequencing, often hiding cell-specific signals in heterogenous tissues.
To tackle this limitation, we developed a novel genome-wide unbiased deconvolution framework that clusters reads based on their 5mC and 5hmC patterns, annotates cell-type specific clusters, and infers cell type-specific methylome and hydroxymethylome. We evaluated the algorithm on in-silico mixtures of purified cells and highly heterogeneous samples. We further applied the algorithm on human pancreatic islets, and investigated cell specific changes of 5mC and 5hmC across age, disease and exposure to variable glucose concentrations. In sum, this ONT-based methodology offers an opportunity to assess cell-specific 5mC and 5hmC methylomes in complex tissue samples.

Co-authors: Laura Moutard, Merce Planas-Felix, Jakob Admard, Eva Bru-Tari, Javier Garci­a-Hurtado, Pere Santamaria, Pedro Herrera, Stephan Ossowski, Jorge Ferrer