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Investigating 5-methylcytosine (5mC) has led to many hypotheses regarding molecular mechanism underlying human diseases and disorders. Many of these studies, however, utilize bisulfite conversion alone, which cannot distinguish 5mC from its recently discovered oxidative product, 5-hydroxymethylcytosine (5hmC). Furthermore, previous array-based technologies do not have the necessary probes to adequately investigate both modifications simultaneously. In this manuscript, we used technical replicates of DNA from human brain, human blood, and human saliva, in combination with oxidative bisulfite conversion and Illumina’s Infinium MethylationEPIC array, to analyze 5mC and 5hmC at more than 650 000 and 450 000 relevant loci, respectively, in the human genome. We show the presence of loci with detectable 5mC and 5hmC to be equally distributed across chromosomes and genomic features, while also being present in genomic regions with transcriptional regulatory properties. We also describe 2528 5hmC sites common across tissue types that show a strong association with immune-related functions. Lastly, in human brain, we show that 5hmC accounts for one-third of the total signal from bisulfite-converted data. As such, not only do our results confirm the efficacy and sensitivity of pairing oxidative bisulfite conversion and the EPIC array to detect 5mC and 5hmC in all three tissue types, but they also highlight the importance of dissociating 5hmC from 5mC in future studies related to cytosine modifications.

Variations in 5-methylcytosine and 5-hydroxymethylcytosine among human brain, blood, and saliva using oxBS and the Infinium MethylationEPIC array