Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m 6 A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m 6 A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.