DNA methylation patterns are primarily studied at the bulk level, where information is averaged over a cellular population. While some studies have examined methylation patterns within single molecules, the short-read nature of the data used has restricted these analyses to short patterns. However, the ability of nanopore sequencing to generate megabase-scale reads provides the opportunity to study single-molecule patterns up to four orders of magnitude longer than those previously considered. Here, we analyse methylation within single nanopore reads with a mean length of 24.6 kb. Using the correlation in methylation state between neighbouring sites, we quantify heterogeneity within single reads and find that the most heterogeneous single-molecule methylation patterns are observed in heterochromatin and partially methylated domains. Moreover, by analysing single-molecule patterns in more detail, we observe a 185 bp periodicity in DNA methylation that sheds light on the nature of the patterns observed within single molecules.
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