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.

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.

Keywords::

• bisulfite conversion
• DNA hydroxymethylation
• DNA methylation
• epigenetics
• Infinium HumanMethylation450K BeadChip
• Infinium MethylationEPIC BeadChip
• oxidative bisulfite conversion

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2217/epi-2021-0490

Z Zhang, MK Lee and LA Salas designed the study. Z Zhang acquired data and performed data analyses of the paper. Z Zhang, MK Lee, L Perreard, BC Christensen and LA Salas participated in the interpretation of data for the work. Z Zhang and MK Lee were responsible for the initial draft of the work. Z Zhang, MK Lee, L Perreard, BC Christensen, KT Kelsey and LA Salas participated in the final drafting and critical revision for important intellectual content. Z Zhang, MK Lee, L Perreard, BC Christensen, KT Kelsey and LA Salas read and approved the final manuscript.

Financial & competing interests disclosure

This study is funded by United States Congressionally Directed Medical Research Programs Funding (number: W81XWH-20-1-0778), United States National Institute of General Medical Sciences (number: P20GM104416/8299) and National Institutes of Health (number: R01CA216265, R01CA253976, R01CA207360). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

For local human samples, the authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Data sharing statement

The datasets analyzed during the current study are publicly available on the Gene Expression Omnibus data repository: https://www.ncbi.nlm.nih.gov/geo/ (accession numbers: GSE182919, GSE152561, GSE63179, GSE71328, GSE71719, GSE73895, GSE104723, GSE105109, GSE116402, GSE133062). For publicly available data, the original data sources are referenced in the manuscript methods.

Additional information

Funding

This study is funded by United States Congressionally Directed Medical Research Programs Funding (number: W81XWH-20-1-0778), United States National Institute of General Medical Sciences (number: P20GM104416/8299) and National Institutes of Health (number: R01CA216265, R01CA253976, R01CA207360). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.