Abstract
Motivation: The two major epigenetic modifications of cytosines, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), coexist with each other in a range of mammalian cell populations. Increasing evidence points to important roles of 5-hmC in demethylation of 5-mC and epigenomic regulation in development. Recently developed experimental methods allow direct single-base profiling of either 5-hmC or 5-mC. Meaningful analyses seem to require combining these experiments with bisulfite sequencing, but doing so naively produces inconsistent estimates of 5-mC or 5-hmC levels.Results: We present a method to jointly model read counts from bisulfite sequencing, oxidative bisulfite sequencing and Tet-Assisted Bisulfite sequencing, providing simultaneous estimates of 5-hmC and 5-mC levels that are consistent across experiment types.Availability: http://smithlab.usc.edu/software/mlmlContact: andrewds@usc.eduSupplementary information: Supplementary material is available at Bioinformatics online.
Highlights
DNA methylation is an important epigenetic mark in mammals
Combining Tet-Assisted Bisulfite sequencing (TAB-seq) and oxBS-seq could lead to estimates of 5-mC and 5-hmC levels exceeding 100%
In one dataset based on oxBS-seq technology, 17% of CpG sites captured by reduced representation bisulfite sequencing (RRBS) and oxRRBS experiments exhibited overshoot (Booth et al, 2012)
Summary
DNA methylation is an important epigenetic mark in mammals. In addition to the extensively studied 5-methylcytosine (5-mC) modification, its oxidation product, 5-hydroxymethylcytosine (5-hmC), has been observed at substantial levels in both somatic and embryonic stem cells (Kriaucionis and Heintz, 2009; Tahiliani et al, 2009). Any two of BS-seq, TAB-seq or oxBS-seq can be combined to profile both the 5-mC and 5-hmC methylomes of a cell population, and especially when studying 5-hmC, proper interpretation of results depends on having some estimate of the 5-mC level. Naive manipulation of read count frequencies from independent sequencing experiments often produces two kinds of ‘overshoot’ problems in estimating 5-mC and 5-hmC levels.
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