Advances in the profiling of DNA modifications: cytosine methylation and beyond

Journal name:
Nature Reviews Genetics
Year published:
Published online


Chemical modifications of DNA have been recognized as key epigenetic mechanisms for maintenance of the cellular state and memory. Such DNA modifications include canonical 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Recent advances in detection and quantification of DNA modifications have enabled epigenetic variation to be connected to phenotypic consequences on an unprecedented scale. These methods may use chemical or enzymatic DNA treatment, may be targeted or non-targeted and may utilize array-based hybridization or sequencing. Key considerations in the choice of assay are cost, minimum sample input requirements, accuracy and throughput. This Review discusses the principles behind recently developed techniques, compares their respective strengths and limitations and provides general guidelines for selecting appropriate methods for specific experimental contexts.

At a glance


  1. Methods for quantifying 5-methylcytosine (5mC) and its oxidized derivatives.
    Figure 1: Methods for quantifying 5-methylcytosine (5mC) and its oxidized derivatives.

    The procedures may involve fragmentation, treatment, library preparation, amplification and analysis. Sonication, restriction enzyme (RE) digestion or Tn5 (transposase) may be used to fragment the genomic DNA (gDNA). Treatment of gDNA may involve enzymatic or chemical modifications, or selection by IP (immunoprecipitation), fragment size or probe. Bisulphite conversion is the primary treatment of gDNA that also results in fragmentation of the gDNA. Sequencing-based methods are usually performed with library preparation for sequencing, which can be done with 'standard' adaptors (standard adaptors used in sequencing library construction) or 'M-adaptors' (methylated adaptors used prior to bisulphite treatment). Other library preparation variants are also used. Amplification or limited amplification is necessary for methods using bisulphite treatment and sequencing-by-synthesis technologies and for low input. Finally, analysis comprises of sequencing, arrays scanning or quantitative PCR. Aba-seq, DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing; βGT, β-glucosyltransferase; BSPP, bisulphite padlock probe; CAB-RRBS, chemical modification-assisted bisulphite-representation bisulphite sequencing; CAB-seq, chemical modification-assisted bisulphite sequencing; CHARM, comprehensive high-throughput arrays for relative methylation; CMS, cytosine-5-methylsulphonate; DIP-seq, DNA immunoprecipitation and shotgun sequencing; fCAB-RRBS, Fc antigen binding- representation bisulphite sequencing; fCAB-seq, 5fC chemical modification-assisted bisulphite sequencing; fC-Seal, 5-formylcytosine selective chemical labelling; GLIB, glucosylation, periodate oxidation and biotinylation; JBP1, J-binding protein 1; LHC-BS, liquid hybridization capture-based bisulphite sequencing; MDB, methyl-DNA binding domain; MeKL-ChIP, methylated DNA kinase pretreated ligation-mediated PCR amplification chromatin immunoprecipitation; MethylCap-seq, methylation DNA capture sequencing; MRE-seq, methylation restriction enzyme sequencing; mTACL, methylation target capture and ligation; oxBS-seq, oxidative bisulphite sequencing; oxRRBS, oxidative RRBS; PBAT, post-bisulphite adaptor tagging; redBS-seq, reduced bisulphite sequencing; red-RRBS, reduced representation bisulphite sequencing; RRHP, reduced representation 5hmC profiling; RSMA, methylation-sensitive restriction enzyme-based assay; scRRBS, single cell reduced representation bisulphite sequencing; TAB-seq, TET-assisted bisulphite sequencing; T-WGBS, transposase-based library construction; WGBS/BS-seq, whole-genome bisulphite sequencing.

  2. Simultaneous detection of 5mC and other epigenetic modifications.
    Figure 2: Simultaneous detection of 5mC and other epigenetic modifications.

    a |Chromatin-immunoprecipitation bisulphite sequencing (ChIP-BS-seq; also known as BisChIP-seq and ChIP-BMS) assays identify the interaction between DNA methylation and histone marks. Chromatin is immunoprecipitated with a specific antibody or binding protein to enrich for the histone mark of interest before performing BS-seq of enriched DNA. Nucleosome occupancy and methylome sequencing (NOMe-seq) determines the position of nucleosome and 5-methylcytosine (5mC) on the same DNA. GpC methyltransferase treatment is used to map the nucleosome position. Only accessible DNA is methylated at GpC positions. Standard BS-seq is used to map the position of 5mC. b | For single chromatin molecule analysis at the nanoscale (SCAN), the histone marks of interest or 5mC are bound with fluorescent-labelled proteins or antibodies. The labelled DNA passes through the nanofluidic channel and is sorted according to their fluorescent labels. MBD, methyl-CpG-binding domain.

  3. Assays for mapping 5-methylcytosine (5mC) oxidation derivatives at single-base resolution.
    Figure 3: Assays for mapping 5-methylcytosine (5mC) oxidation derivatives at single-base resolution.

    5-hydroxymethylcytosine (5hmC) in single-base resolution is characterized by the two methods, oxidative bisulphite sequencing (oxBS-seq) and TET-assisted bisulphite sequencing (TAB-seq). In oxBS-seq, 5hmC is oxidized to 5-formylcytosine (5fC) by potassium perruthenate (KRuO4). After bisulphite treatment and amplification, it appears as thymidine. 5hmC can be identified by subtracting thymidine from oxBS-seq from cytosine by traditional bisulphite sequencing (BS-seq). For TAB-seq, 5hmC in DNA is glucosylated to 5-glucosylmethylcytosine (5gmC) by β-glucosyltransferase (βGT). DNA is subsequently treated with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) to convert all modified cytosines except 5gmC to 5-carboxyctosine (5caC). After BS-seq of TET1 treated DNA, only 5gmC from original 5hmC appears as cytosine. To identify 5caC, DNA is treated with 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDC) to protect deamination of 5caC during bisulphite treatment. The protected 5caC is read out as cytosine in chemical modification-assisted bisulphite sequencing (CAB-seq) instead of thymidine in regular BS-seq. Similar to 5caC, 5fC chemically modification-assisted bisulphite sequencing (fCAB-seq) identifies 5fC by treating DNA with O-ethylhydroxylamine (EtONH2) to modify 5fC and protect it from deamination during bisulphite treatment. 5fC can be reduced by sodium borohydride (NaBH4) into 5hmC by the reduced bisulphite sequencing (redBS-seq) method. Both protected 5fC and 5hmC from original 5fC are read out as cytosine in fCAB-seq and redBS-seq. The signals of 5caC and 5fC are identified by subtracting cytosine from CAB-seq and fCAB-seq and/or redBS-seq, respectively, from thymidine in BS-seq. UDG-Glc, uridine diphosphate glucose.


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Author information

  1. These authors contributed equally to this work.

    • Nongluk Plongthongkum &
    • Dinh H. Diep


  1. Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92037, USA.

    • Nongluk Plongthongkum,
    • Dinh H. Diep &
    • Kun Zhang

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  • Nongluk Plongthongkum

    Nongluk Plongthongkum is a graduate student in the Bioengineering Graduate Program at the University of California, San Diego, USA. She is currently working on developing techniques for targeted DNA methylation assays and applying them to the study of genome–methylome interactions.

  • Dinh H. Diep

    Dinh H. Diep is a graduate student in Bioinformatics and Systems Biology at the University of California, San Diego, USA. She is currently working on the development and application of targeted assays to study the epigenetics of stem cells. She is a pre-doctoral fellow in the California Institute for Regenerative Medicine (CIRM) Interdisciplinary Stem Cell training program.

  • Kun Zhang

    Kun Zhang is an associate professor of Bioengineering at the University of California, San Diego, USA. His research focus is on the development of genomic assays, including targeted genome sequencing and methylation sequencing, as well as single-cell genome sequencing and transcriptome sequencing. His research group also applies these assays to the study of stem cells and brain. The Zhang laboratory's homepage.

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