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Methylation of cytosine at the 5 position of the pyrimidine ring is the most prevalent and significant epigenetic modifications in mammalian DNA. The CpG methylation level shows a bimodal distribution but the bimodality can be overestimated due to the heterogeneity of per-base depth. Here, we developed an algorithm to eliminate the effect of per-base depth inhomogeneity on the bimodality and obtained a random CpG methylation distribution. By quantifying the deviation of the observed methylation distribution and the random one using the information formula, we find that in tetranucleotides 5’-N 5 CGN 3 -3’ (N 5 , N 3  = A, C, G or T), GCGN 3  and CCGN 3  show less apparent deviation than ACGN 3  and TCGN 3 , indicating that GCGN 3  and CCGN 3  are less variant in their level of methylation. The methylation variation of N 5 CGN 3  are conserved among different cells, tissues and species, implying common features in the mechanisms of methylation and demethylation, presumably mediated by DNMTs and TETs in mammalians, respectively. Sequence dependence of DNA methylation variation also relates to gene regulatory and promotes the reexamination of the role of DNA sequence in fundamental biological processes.

The sequence preference of DNA methylation variation in mammalians