Cytosine methylation has an important role in the regulation of mammalian gene expression. Additional forms of cytosine modification, including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), are intermediates of 5mC that occur during DNA demethylation, and are hypothesized to have additional functional roles in cellular development.Recent efforts in method development for quantifying cytosine modifications and mapping their genomic location, preferentially at single-base resolution, have focused on improving accuracy, increasing throughput, lowering sample input and reducing costs.One major class of DNA methylation assays relies heavily on bisulphite treatment and sequencing approaches that provide single-base resolution. Whole-genome bisulphite sequencing (WGBS) and reduced representation bisulphite sequencing (RRBS) are widely used to generate genome-wide maps of DNA methylation. Application of low input methods such as tagmentation-based WGBS (T-WGBS) and post-bisulphite adaptor tagging (PBAT) allows for the detection of 5mC using input DNA from hundreds to thousands of cells.Various targeted methylation-sequencing approaches, such as liquid hybridization and parallel amplification, have been developed to characterize DNA methylation at selected regions. These approaches are more cost effective for analysing large numbers of samples than non-targeted methods.Improvements in several techniques, including restriction enzyme-based single-cell methylation assay (RSMA), limiting dilution bisulphite (pyro) sequencing and single-cell RRBS (scRRBS) and single-cell BS-seq (scBS-seq), have enabled 5mC detection in single cells.Array-based methods are widely used in many studies of large cohorts. A major improvement is the dramatic increase in features per array and, hence, genome coverage.The combination of chromatin immunoprecipitation (ChIP) assay and bisulphite sequencing (BS-seq) in sequential order allows for simultaneous detection of DNA methylation and other epigenetic marks. Profiling of fluorescence-labelled DNA fragments using nanofluidic devices has helped to correlate multiple chromatin marks with DNA methylation. In addition, the incidence of DNA methylation and nucleosome occupancy can be mapped with nucleosome occupancy and methylome sequencing (NOMe-seq).A major challenge in the quantification of 5mC oxidation derivatives is their rarity in mammalian genomes. The use of specific antibodies and chemical or enzymatic modifications has enabled the enrichment of 5mC oxidation derivatives and the determination of their relative abundance in the genome, albeit with limited resolution. Chemical modifications coupled with BS-seq can be used to identify 5mC oxidation derivatives at single-base resolution, as in the oxidative bisulphite sequencing (oxBS-seq) and Tet-assisted bisulphite sequencing (TAB-seq) approaches for 5hmC quantification, the 5fC chemical modification-assisted bisulphite sequencing (fCAB-seq) and redBS-seq approaches for 5fC quantification, and the chemical modification-assisted bisulphite sequencing (CAB-seq) approach for 5caC quantification.Third-generation DNA sequencing technologies, including single-molecule, real-time (SMRT) sequencing and nanopore sequencing, are very appealing for direct reading of 5mC and other DNA modifications on the same DNA molecule, with the potential advantages of speed, read length and the lack of chemical treatment. Nevertheless, the throughput and accuracy of these technologies are still not sufficient for routine use.
