title: oxBS-450K: a method for analysing hydroxymethylation using 450K BeadChips.
abstract: DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2)>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
authors.affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK.
authors.forename: Sabrina K
authors.initials: SK
authors.lastname: Stewart
authors.affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK.
authors.forename: Tiffany J
authors.initials: TJ
authors.lastname: Morris
authors.affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Guilhamon
authors.affiliation: Scottish Centre for Regenerative Medicine, Edinburgh EH16 4UU, UK.
authors.forename: Harry
authors.initials: H
authors.lastname: Bulstrode
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Bachman
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
authors.affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK. Electronic address: s.beck@ucl.ac.uk.
authors.forename: Stephan
authors.initials: S
authors.lastname: Beck
mesh_terms: D019175:DNA Methylation; D057890:Epigenomics / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods
keywords: 450K BeadChip; Bisulfite conversion; DNA methylation; Epigenetics; Hydroxymethylation; Oxidation