title: Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine.
abstract: To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Booth
authors.forename: Tobias W B
authors.initials: TW
authors.lastname: Ost
authors.forename: Dario
authors.initials: D
authors.lastname: Beraldi
authors.forename: Neil M
authors.initials: NM
authors.lastname: Bell
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Branco
authors.forename: Wolf
authors.initials: W
authors.lastname: Reik
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry* / Q000031:analogs & derivatives; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D019175:DNA Methylation; D010084:Oxidation-Reduction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine