Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol

Borre, Marie De; Branco, Miguel R.

doi:10.1007/978-1-0716-0876-0_26

Cited by 11 publications

(4 citation statements)

References 28 publications

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“…Crucially, with the deepening of research, it was found that bisulfite sequencing, previously considered as the “gold standard” for detecting DNA methylation, could not distinguish DNA methylation (5mC) from DNA hydroxymethylation (5hmC) [ 168 ]. Chemical oxBS-Seq can accurately detect not only DNA methylation and eliminate the influence of DNA hydroxymethylation but also DNA hydroxymethylation, by simultaneously combining double libraries with single base resolution [ 169 ]. The technical principle of DNA hydroxymethylation can be summarized as follows: oxBS-Seq oxidizes 5hmC to 5fC, which can be converted into U by bisulfite to achieve accurate detection of 5mC; simultaneously, the accurate detection of 5hmC can be achieved by comparing with the conventional bisulfite results.…”

Section: Methods For Studying the Epigenetic Modificationmentioning

confidence: 99%

Targeting the chromatin structural changes of antitumor immunity

Li,

Lun,

Gong

et al. 2024

Journal of Pharmaceutical Analysis

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Section: Methods For Studying the Epigenetic Modificationmentioning

confidence: 99%

Targeting the chromatin structural changes of antitumor immunity

Li,

Lun,

Gong

et al. 2024

Journal of Pharmaceutical Analysis

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“…The 5fC is susceptible to BS treatment, and it is deaminated into uracil (equivalent to an unmethylated cytosine), while 5mC is preserved as a cytosine upon BS treatment. Thus, oxBS conversion enables the specific measurement of nucleotide-level 5mC [ 24 , 25 ]. Subsequent methylation analysis for all samples was conducted on an Illumina (San Diego, CA, USA) iScan platform using the Infinium Methylation EPIC BeadChip v1, following standard protocols provided by Illumina.…”

Section: Methodsmentioning

confidence: 99%

Smoking-Induced DNA Hydroxymethylation Signature Is Less Pronounced than True DNA Methylation: The Population-Based KORA Fit Cohort

Lai,

Matías-García,

Kretschmer

et al. 2024

Biomolecules

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Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite (BS) and oxidative bisulphite (oxBS) treatment, measured with the Illumina Infinium MethylationEPIC BeadChip. An epigenome-wide association study (EWAS) of 5mC+5hmC methylation revealed a total of 38,575 differentially methylated positions (DMPs) and 2023 differentially methylated regions (DMRs) associated with current smoking, along with 82 DMPs and 76 DMRs associated with former smoking (FDR-adjusted p < 0.05). Additionally, a focused examination of 5mC identified 33 DMPs linked to current smoking and 1 DMP associated with former smoking (FDR-adjusted p < 0.05). In the 5hmC category, eight DMPs related to current smoking and two DMPs tied to former smoking were identified, each meeting a suggestive threshold (p < 1 × 10−5). The substantial number of recognized DMPs, including 5mC+5hmC (7069/38,575, 2/82), 5mC (0/33, 1/1), and 5hmC (2/8, 0/2), have not been previously reported. Our findings corroborated previously established methylation positions and revealed novel candidates linked to tobacco smoking. Moreover, the identification of hydroxymethylated CpG sites with suggestive links provides avenues for future research.

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“…To analyze DNA methylation/demethylation in 3 pools of mPDCs treated with vehicle or AMA for 10 days, DNA was extracted from cell lysates using silica columns following manufacturer’s instructions (DNeasy Blood and Tissue kit, Qiagen), followed by RNASE H and Proteinase K treatment and an additional purification using solid phase reversible immobilisation (AMPure XP, Beckman Coulter). DNA was next converted using the oxidative bisulfite or normal bisulfite method as described previously ( De Borre and Branco, 2021 ), and hybridized to Illumina mouse EPIC Beadchip arrays (Life & Brain, Bonn, Germany). The resultant.idat files were analyzed using the minfi package.…”

Section: Methodsmentioning

confidence: 99%