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

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. Bisulphite and oxidative bissulfite converted DNA from 22 pairs of clear cell renal cell carcinoma samples, 18 pairs of human fetal oligodendrocyte replicate samples, and 16 pairs of whole blood samples from glioma patients treated with or without Dexamethasone, were hybridised to the Illumina Infinium HumanMethylationEPIC Beadchip v1.0_B4