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. Lay abstract DNA hydroxymethylation (5-hydroxymethylcytosine [5hmC]) is a chemical modification of the cytosines in the DNA that affects gene transcription. 5hmC has been used as a biomarker for early cancer detection and survival prediction in recent years. 5hmC is measured using tandem bisulfite (BS) and oxidative bisulfite (oxBS) conversion of DNA followed by quantification through DNA methylation microarrays. This study observed a consistent loss of high-quality data in oxBS-treated samples compared with BS-treated samples. The authors offer a bioinformatic tool to evaluate potential quality issues in the process and some technical advice to reduce false signals in the data. Thus, they emphasize the importance of preserving DNA integrity when using tandem BS- and oxBS-treated DNA to measure 5-methylcytosine and 5hmC.

Automated summary

This study compared the data quality of BS-treated and oxBS-treated samples and observed a consistent loss of high-quality data in oxBS-treated samples. The authors offer a bioinformatic tool to evaluate potential quality issues in the process and provide some technical advice to reduce false signals in the data. Therefore, they emphasize the importance of preserving DNA integrity when using tandem BS- and oxBS-treated DNA to measure 5-methylcytosine and 5hmC.