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Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays

ABSTRACT: Aim: Tandem bisulfite (BS) and oxidative bisulfite (oxBS) conversion on DNA followed by hybridization to Infinium HumanMethylation BeadChips allows nucleotide resolution of 5-hydroxymethylcytosine genome-wide. Here, the authors compared data quality acquired from BS-treated and oxBS-treated samples. Materials & methods: Raw BeadArray data from 417 pairs of samples across 12 independent datasets were included in the study. Probe call rates were compared between paired BS and oxBS treatments controlling for technical variables. Results: oxBS-treated samples had a significantly lower call-rate. Among technical variables, DNA-specific extraction kits performed better with higher call rates after oxBS conversion. Conclusion: The authors emphasize the importance of quality control during oxBS conversion to minimize information loss and recommend using a DNA-specific extraction kit for DNA extraction and an oxBSQC package for data preprocessing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182919 | GEO | 2022/01/14

REPOSITORIES: GEO

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Project description:5-hydroxymethylcytosine (5hmC), converted from 5-methylcytosine (5mC) by Tet enzymes, has recently drawn attention as the ‘sixth base’ of DNA since it is considered to be an intermediate of the demethylation pathway. Nonetheless, it remains to be addressed how 5hmC is linked to the development of human imprinting disorders. In this regard, conventional bisulfite (BS) treatment is unable to differentiate 5hmC from 5mC. It is thus hypothesized that BS conversion-derived ‘hypermethylation’ at imprinting control regions (ICRs), which may cause human imprinting disorders, would in fact be attributable to excessively increased levels of 5hmC as well as 5mC. To test this hypothesis, we applied the newly developed oxidative BS (oxBS) treatment to detect 5hmC in blood samples from Kagami-Ogata syndrome (KOS14) patients caused by perturbed expression of clustered imprinted genes on 14q32.2 resulting from the hypermethylation of ICRs at this locus, IG-DMR and MEG3-DMR. oxBS with pyrosequencing and cloning-based sequencing revealed that there were few amounts of 5hmC at the hypermethylated IG-DMR in blood samples from KOS14 patients. oxBS with genome-wide methylation array analysis demonstrated that global levels of 5hmC were very low with similar distribution patterns in blood samples from KOS14 patients and normal controls. We also confirmed the presence of a large amount of 5hmC in the brain sample from a normal control. 5hmC is not a major component in abnormally hypermethylated ICRs or at a global level, at least in blood from KOS14 patients. As the brain sample contained a large amount of 5hmC, the neural tissues of KOS14 patients are promising candidates for analysis in elucidating the role of 5hmC in the neurodevelopmental context. We generated illumina 450k DNA methylation data for BS or oxBS converted samples of three KOS14 blood samples, one control pooled blood sample and one control brain sample with two technical replicates for each sample.