Figure 1. Global distribution of CpG and non-CpG methylation in human cell types.

(A) Schematic of RRBS data visualization and a selected 36 bp read. Blue lines indicate covered cytosines (CpN), black lines MspI restriction sites (middle). One selected RRBS read in this region is shown (bottom). Red circles indicate CpGs, light red boxes CpTs, dark red boxes CpAs and yellow boxes CpCs. Filled circles and boxes indicate dinucleotides with detectable levels of methylation. The percent below indicate the methylation levels by averaging the methylation state of a given cytosine over all reads that cover its position. (B) Venn diagrams show the theoretical RRBS coverage compared to the whole genome for CpGs (top) and non-CpGs (bottom) based on a 40–260 bp size selection. (C) Enrichment of cytosine dinucleotide frequency for RRBS relative to the whole genome. (D) Venn diagrams show the overlap of methylated CpGs (top) as well as methylated non-CpGs (bottom) exhibiting above threshold (≥10% and ≥5%) methylation in the whole methylome (WM) data by Lister et al. 2009 and our RRBS data for the same cell line and passage. Only those dinucleotides were considered that were covered in both data sets simultaneously by at least 5 reads. Numbers below the venn diagrams indicate overlap of both dinucleotide sets. (E) Pie chart of sequence context distribution of methylated cytosines in the human ESC line H1 (passage 25) and human fibroblasts 18 (passage 7). (F) Boxplots of the methylation levels as assessed by RRBS across six biological replicates of hESC line H1. Boxplots are based on all cytosine dinucleotides that show any evidence for methylation in H1 (median methylation ≥0.1% over all six replicates). Boxes are 25^th and 75^th quartiles, whiskers indicate most extreme data point less than 1.5 interquartile range from box and black bar represents the median. n indicates the number of dinucleotides covered in all and methylated in at least one of the six samples. (G) Distribution of methylated (≥10%) cytosine dinucleotides in human ES cells (ES, n = 30), iPS cells (n = 12), embryoid bodies (EB, n = 10) and 10 somatic cell types (n = 18). Percentages are methylated cytosine dinucleotides divided by corresponding total number of each cytosine dinucleotide with ≥5x coverage. (H) Barplot showing the average reduction in the number of methylated cytosine dinucleotides in EBs (n = 10) and somatic cells (n = 18) relative to pluripotent cells (n = 42). (I) Distribution of distinct CpG (left) and CpA (right) methylation levels for all CpA and CpG dinucleotides averaged over all hES samples (n = 30). The medians of the CpA methylation level distribution are fitted by the exponential distribution (yellow circle). Boxplots are defined as in (F).