Genome-wide single-molecule analysis of long-read DNA methylation reveals heterogeneous patterns at heterochromatin that reflect nucleosome organisation

High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulklevel patterns of DNA methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome. Author summary DNA methylation is an epigenetic DNA modification that is often associated with the repression of gene expression. Correct patterning of DNA methylation is crucial in development and for normal cellular function. Aberrant patterns of DNA methylation are also observed in diseases such as cancer. Here, we examine DNA methylation patterns within long DNA molecules to identify how methylation heterogeneity varies throughout the human genome within single molecules. We find that single molecule DNA methylation heterogeneity varies widely, with some genomic regions, particularly those away from genes, having very heterogeneous patterns. In contrast, we find that the regions of the genome around active genes have more homogeneous, uniform methylation states. When we examined the nature of heterogeneous methylation patterns, we find they have an underlying periodicity. We also find that this periodicity is reflective of the single molecule placement of nucleosomes, proteins that are important in the packaging of DNA. Overall, our study indicates that DNA methylation heterogeneity shows wide variation across the human genome and that DNA packaging could play a role in shaping DNA methylation patterns at the level of single molecules.