Methylation pseudotime analysis for label-free profiling of the temporal chromatin landscape with long-read sequencing

Faithful epigenetic inheritance across cell divisions is essential to maintaining cell identity and involves numerous epigenetic modifications, whose roles in coordinating chromatin architecture are less understood. Technological approaches to temporally order epigenetic modifications throughout the cell cycle often face limitations in sequence resolution and rely on potentially damaging mitotic labeling or conversion steps. Herein, we present Methylation Pseudotime Analysis Through read-level Heterogeneity (MPATH), a label- and conversion-free method to infer post-replication DNA strand maturity from methylation patterns across single molecules. We showed that MPATH is generally applicable across human embryonic stem cell (hESC) lines in which CpG methylation heterogeneity is coupled to replication, but not applicable to breast cancer cells, in which CpG methylation heterogeneity may also be associated with other factors. We also analyzed differentiated human cell lines and human and mouse mesenchymal stem cells and found that while MPATH showed reduced ability to infer DNA strand maturity within these contexts, it still captured general trends in DNA methylation that were associated with chromatin maturation. When applied to long reads generated by NOMe-seq, MPATH revealed that nucleosome compaction and CpG methylation uniformity are coupled across replication-associated timescales. Finally, extension of MPATH to phased reads reveals allele-specific trends in pseudotime distribution associated with X chromosome inactivation. Our findings suggest that when coupled with multimodal sequencing strategies, MPATH could provide valuable insights into chromatin restoration dynamics.