Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

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

Key Points

  • 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.

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Methods for quantifying 5-methylcytosine (5mC) and its oxidized derivatives.
Figure 2: Simultaneous detection of 5mC and other epigenetic modifications.
Figure 3: Assays for mapping 5-methylcytosine (5mC) oxidation derivatives at single-base resolution.

Similar content being viewed by others

References

  1. Arand, J. et al. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases. PLoS Genet. 8, e1002750 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hon, G. C. et al. Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nature Genet. 45, 1198–1206 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Lister, R. et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lister, R. et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Ziller, M. J. et al. Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS Genet 7, e1002389 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jeong, M. et al. Large conserved domains of low DNA methylation maintained by Dnmt3a. Nature Genet. 46, 17–23 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Stadler, M. B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Xie, W. et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134–1148 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Jones, P. A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nature Rev. Genet. 13, 484–492 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nature Rev. Genet. 14, 204–220 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Xie, M. et al. DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape. Nature Genet. 45, 836–841 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Yu, M. et al. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nature Protoc. 7, 2159–2170 (2012).

    Article  CAS  Google Scholar 

  15. Pastor, W. A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Szulwach, K. E. et al. 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nature Neurosci. 14, 1607–1616 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Iurlaro, M. et al. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Laird, P. W., Principles and challenges of genome-wide DNA methylation analysis. Nature Rev. Genet. 11, 191–203 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Shen, L. & Zhang, Y. 5-hydroxymethylcytosine: generation, fate, and genomic distribution. Curr. Opin. Cell Biol. 25, 289–296 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Song, C.-X., Yi, C. & He, C. Mapping recently identified nucleotide variants in the genome and transcriptome. Nature Biotech. 30, 1107–1116 (2012).

    Article  CAS  Google Scholar 

  23. Bock, C. Analysing and interpreting DNA methylation data. Nature Rev. Genet. 13, 705–719 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Krueger, F. et al. DNA methylome analysis using short bisulfite sequencing data. Nature Methods 9, 145–151 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Grunau, C., Clark, S. J. & Rosenthal, A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, e65 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ogino, S. et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J. Mol. Diagn. 8, 209–217 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Nestor, C. E. et al. Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res. 22, 467–477 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Boyle, P. et al. Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling. Genome Biol. 13, R92 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wang, J. et al. Double restriction-enzyme digestion improves the coverage and accuracy of genome-wide CpG methylation profiling by reduced representation bisulfite sequencing. BMC Genomics 14, 11 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schillebeeckx, M. et al. Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Nucleic Acids Res. 41, e116 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Guo, H. et al. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 23, 2126–2135 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ruiz, S. et al. Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. Proc. Natl Acad. Sci. USA 109, 16196–16201 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Okuizumi, H. et al. Restriction landmark genome scanning. Methods Mol. Biol. 791, 101–112 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Maunakea, A. K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Taiwo, O. et al. Methylome analysis using MeDIP-seq with low DNA concentrations. Nature Protoc. 7, 617–636 (2012).

    Article  CAS  Google Scholar 

  36. Clark, C. et al. A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the infinium HumanMethylation450 BeadChip® for methylome profiling. PLoS ONE 7, e50233 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Aberg, K. A. et al. MBD-seq as a cost-effective approach for methylome-wide association studies: demonstration in 1500 case–control samples. Epigenomics 4, 605–621 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Lan, X. et al. High resolution detection and analysis of CpG dinucleotides methylation using MBD-seq technology. PLoS ONE 6, e22226 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Brinkman, A. B. et al. Whole-genome DNA methylation profiling using MethylCap-seq. Methods 52, 232–236 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Butcher, L. M. & Beck, S. AutoMeDIP-seq: a high-throughput, whole genome, DNA methylation assay. Methods 52, 223–231 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bock, C. et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotech. 28, 1106–1114 (2010).

    Article  CAS  Google Scholar 

  42. Matarese, F., Carrillo-de Santa Pau, E. & Stunnenberg, H. G. 5-hydroxymethylcytosine: a new kid on the epigenetic block? Mol. Syst. Biol. 7, 562 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Komori, H. K. et al. Application of microdroplet PCR for large-scale targeted bisulfite sequencing. Genome Res. 21, 1738–1745 (2011). This paper demonstrates a fully automated method for quantification of DNA methylation on 2100 genes.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nautiyal, S. et al. High-throughput method for analyzing methylation of CpGs in targeted genomic regions. Proc. Natl Acad. Sci. USA 107, 12587–12592 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Varley, K. E. & Mitra, R. D. Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome Res. 20, 1279–1287 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Diep, D. et al. Library-free methylation sequencing with bisulfite padlock probes. Nature Methods 9, 270–272 (2012). This paper describes a method for high-throughput padlock probes that sequence methylated DNA.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature Biotech. 27, 353–360 (2009).

    Article  CAS  Google Scholar 

  48. Lee, E.-J. et al. Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing. Nucleic Acids Res. 39, e127 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang, J. et al. High resolution profiling of human exon methylation by liquid hybridization capture-based bisulfite sequencing. BMC Genomics 12, 597 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ivanov, M. et al. In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes. Nucleic Acids Res. 41, e72 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Yamaguchi, S. et al. Tet1 controls meiosis by regulating meiotic gene expression. Nature 492, 443–447 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kobayashi, H. et al. High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome Res. 23, 616–627 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Shirane, K. et al. Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet. 9, e1003439 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kobayashi, H. & Kono, T. DNA methylation analysis of germ cells by using bisulfite-based sequencing methods. Methods Mol. Biol. 825, 223–235 (2012).

    Article  CAS  PubMed  Google Scholar 

  55. Adey, A. & Shendure, J. Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Res. 22, 1139–1143 (2012). This study shows that whole-genome bisulphite sequencing can be performed on ~1–10 ng of genomic DNA.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wang, Q. et al. Tagmentation-based whole-genome bisulfite sequencing. Nature Protoc. 8, 2022–2032 (2013).

    Article  CAS  Google Scholar 

  57. Miura, F. et al. Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 40, e136 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kantlehner, M. et al. A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res. 39, e44 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lorthongpanich, C. et al. Single-cell DNA-methylation analysis reveals epigenetic chimerism in preimplantation embryos. Science 341, 1110–1112 (2013). The first paper to use single-cell DNA methylation analysis to address an important biological problem.

    Article  CAS  PubMed  Google Scholar 

  60. El Hajj, N. et al. Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics 6, 1176–1188 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Guo, H. et al. The DNA methylation landscape of human early embryos. Nature 511, 606–610 (2014).

    Article  CAS  PubMed  Google Scholar 

  62. Smallwood, S.A. et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nature Methods 11, 817–820 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Irizarry, R. A. et al. Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18, 780–790 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Bibikova, M. et al. High density DNA methylation array with single CpG site resolution. Genomics 98, 288–295 (2011).

    Article  CAS  PubMed  Google Scholar 

  65. Yalcin, A. et al. MeDIP coupled with a promoter tiling array as a platform to investigate global DNA methylation patterns in AML cells. Leukemia Res. 37, 102–111 (2013).

    Article  CAS  Google Scholar 

  66. Gilson, E. & Horard, B. Comprehensive DNA methylation profiling of human repetitive DNA elements using an MeDIP-on-RepArray assay. Methods Mol. Biol. 859, 267–291 (2012).

    Article  CAS  PubMed  Google Scholar 

  67. Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126, 1189–1201 (2006).

    Article  CAS  PubMed  Google Scholar 

  68. Oliver, V. F. et al. A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples. Epigenetics Chromatin 6, 1–11 (2013).

    Article  CAS  Google Scholar 

  69. Dumenil, T. D. et al. Genome-wide DNA methylation analysis of formalin-fixed paraffin embedded colorectal cancer tissue. Genes Chromosomes Cancer 53, 537–548 (2014).

    Article  CAS  PubMed  Google Scholar 

  70. Brinkman, A. B. et al. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 22, 1128–1138 (2012). Together with reference 69, these papers describe DNA precipitation followed by bisulphite treatment to create a map of DNA methylation patterns associated with chromatin modifications.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Statham, A. L. et al. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA. Genome Res. 22, 1120–1127 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Li, Y. & Tollefsbol, T. O. Combined chromatin immunoprecipitation and bisulfite methylation sequencing analysis. Methods Mol. Biol. 791, 239–251 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Cipriany, B. R. et al. Single molecule epigenetic analysis in a nanofluidic channel. Anal. Chem. 82, 2480–2487 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Cipriany, B. R. et al. Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel. Proc. Natl Acad. Sci. USA 109, 8477–8482 (2012). This study discusses a nanofluidic device for sorting single methylated DNA molecules.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Murphy, P. J. et al. Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells. Proc. Natl Acad. Sci. USA 110, 7772–7777 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. You, J. S. et al. OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes. Proc. Natl Acad. Sci. USA 108, 14497–14502 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Kelly, T. K. et al. Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res. 22, 2497–2506 (2012). This study describes NOMe-seq, a method using M. Cvi PI treatment and bisulphite conversion to produce a genome-wide base resolution map of nucleosome positioning and DNA methylation on the same DNA molecules.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).

    Article  CAS  PubMed  Google Scholar 

  80. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Stroud, H. et al. 5-hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 12, R54 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Thomson, J. P. et al. Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res. 41, e206 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Inoue, A. et al. Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development. Cell Res. 21, 1670–1676 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Ko, M. et al. Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature 497, 122–126 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  87. Szwagierczak, A. et al. Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  88. Song, C. X. et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nature Biotech. 29, 68–72 (2011).

    Article  CAS  Google Scholar 

  89. Baskin, J. M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Pastor, W. A. et al. The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine. Nature Protocols 7, 1909–1917 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Robertson, A. B. et al. Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nature Protocols 7, 340–350 (2012).

    Article  CAS  PubMed  Google Scholar 

  92. Michaeli, Y. et al. Optical detection of epigenetic marks: sensitive quantification and direct imaging of individual hydroxymethylcytosine bases. Chem. Commun. (Camb.) 49, 8599–8601 (2013).

    Article  CAS  Google Scholar 

  93. Song, C.-X. et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691 (2013). This study describes the first genome-wide base resolution map of 5fC in mESCs.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Hu, J. et al. Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes. Chemistry 19, 5836–5840 (2013).

    Article  CAS  PubMed  Google Scholar 

  95. Kinney, S. M. et al. Tissue-specific distribution and dynamic changes of 5-Hydroxymethylcytosine in mammalian genomes. J. Biol. Chem. 286, 24685–24693 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Sun, Z. et al. High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep. 3, 567–576 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Shankaranarayanan, P. et al. Single-tube linear DNA amplification (LinDA) for robust ChIP–seq. Nature Methods 8, 565–567 (2011).

    Article  CAS  PubMed  Google Scholar 

  98. Booth, M. J. et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012).

    Article  CAS  PubMed  Google Scholar 

  99. Booth, M. J. et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protoc. 8, 1841–1851 (2013).

    Article  CAS  Google Scholar 

  100. Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Lu, X. et al. Chemical modification-assisted bisulfite sequencing (CAB-seq) for 5-Carboxylcytosine detection in DNA. J. Am. Chem. Soc. 135, 9315–9317 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Booth, M. J. et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chem. 6, 435–440 (2014). Together with reference 98 this paper describes a method for producing a whole-genome base resolution map of 5hmC (in mice and humans).

    Article  CAS  Google Scholar 

  103. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  PubMed  Google Scholar 

  104. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature Methods 7, 461–465 (2010). Together with reference 96 this paper describes a method for producing a whole-genome base resolution map of 5hmC.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Clark, T. et al. Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation. BMC Biol. 11, 4 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Song, C.-X. et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nature Methods 9, 75–77 (2012).

    Article  CAS  Google Scholar 

  107. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotech. 26, 1146–1153 (2008).

    Article  CAS  Google Scholar 

  108. Purnell, R. & Schmidt, J. Measurements of DNA immobilized in the alpha-hemolysin nanopore. Methods Mol. Biol. 870, 39–53 (2012).

    Article  CAS  PubMed  Google Scholar 

  109. Stoddart, D. et al. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl Acad. Sci. USA 106, 7702–7707 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Butler, T. Z. et al. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl Acad. Sci. USA 105, 20647–20652 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Manrao, E. A. et al. Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS ONE 6, e25723 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Wanunu, M. et al. Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules. J. Am. Chem. Soc. 133, 486–492 (2011).

    Article  CAS  PubMed  Google Scholar 

  113. Laszlo, A. H. et al. Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc. Natl Acad. Sci. USA 110, 18904–18909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Ross, M. G. et al. Characterizing and measuring bias in sequence data. Genome Biol. 14, R51 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge members of the Zhang laboratory for their proofreading of the manuscript. We apologize to those authors whose works were not covered here due to space constraints. This work is funded by the US National Institutes of Health grants R01GM097253 and R01AG042187. D.H.D. is supported by a UCSD-CIRM pre-doctoral fellowship.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kun Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

PowerPoint slides

Glossary

Partially methylated domains

(PMDs). Large contiguous regions of the genome (mean length ~ 153 kb) that display an intermediate level of DNA methylation (average <70%).

Ten-eleven translocation

(TET). DNA-binding enzymes that have been found to be methylcytosine dioxygenases in mammals and include TET1, TET2 and TET3. They were named for the genetic variant found in the TET1 gene sequence in acute myeloid and lymphocytic leukaemia.

Short interspersed nuclear elements

(SINEs). A subtype of transposable elements reverse-transcribed from RNA molecules. They do not encode a functional reverse transcriptase protein and cover a substantial portion of primate genomes, including all Alu sequences.

Long terminal repeats

(LTRs). Stretches of DNA sequences that are identical and repeat in hundreds to thousands of copies. LTRs were founds at the end of retrotransposons and act as insertion sites for viruses to insert their genetic material into the host genome.

Type I errors

The errors that result when there are false positives or when falsely rejecting the null hypothesis.

Type II errors

The errors that result when there are false negatives or incorrect failure to reject the null hypothesis

Laser-capture microdissection (LCM)

A method for isolating specific cells or specific areas from cell, tissue or organism samples using laser cutting under microscopic visualization.

Ligation capture

A method for capturing restriction enzyme-digested DNA molecules via the annealing of an oligonucleotide containing complementary sequences to adaptor oligonucleotides to the DNA molecules and to the adaptor oligonucleotides. The adaptors and DNA molecules are then ligated together, allowing for PCR amplification of only the ligated products.

Bisulphite padlock probe (BSPP) capture

A method for capturing the target CpG sites of bisulphite treated genomic DNA with bisulphite padlock probes (BSPP). The two capturing arms of the BSPPs are designed to flank the region of interest. After annealing padlock probes to target regions, polymerization is preformed to fill the gap and two ends of the padlock probe are joined together to form circularized DNA. The captured regions are amplified with barcoded adaptor primers and sequenced.

Liquid hybridization

A method for capturing fragmented DNA molecules via the annealing of biotinylated oligonucleotides to the DNA molecules. The binding of biotin to streptavidin beads allows for washing and removal of uncaptured DNA molecules, and subsequent elution of the captured DNA molecules.

Microdroplet PCR

Massively parallel PCR amplification of target sequences in microdroplets. The process involves the preparation of template and PCR mix in picoliter volume and primer droplets, combination of individual template and primer pair droplet, pooling the fused droplets for thermal cycling, and releasing of PCR amplicons for purification and sequencing.

Barcoded primers

Unique DNA sequences that are incorporated into adaptor sequences for tagging of different samples before sample pooling and shotgun sequencing.

Pyrosequencing

A sequencing-by-synthesis method based on the detection of phyrophosphate released upon nucleotide incorporation.

Shotgun library construction

The generation of a sequencing library involving random fragmentation of DNA and addition of adaptor sequences to both ends of DNA fragments before sequencing.

Transposase-based library construction

A procedure to generate a sequencing library using the transposase Tn5 to insert common transposon sequences to DNA. DNA segments are then amplified by annealing of primers to the transposon sequences.

Tn5 transposase

A member of the RNase superfamily of proteins that harbours retroviral integrases to catalyse random insertion of transposon DNA into target DNA.

Binning

A computational technique frequently used to reduce noise by grouping sequencing reads mapped to contiguous genomic segments.

Nucleosome

A basic unit of DNA packaging in eukaryotes that consists of section of DNA (~ 166 bp) wrapping around a histone core. Nucleosome structure helps to condense DNA into smaller volume. Nucleosomes are subunits of chromatin.

GpC methyltransferase (M.CviPI)

An enzyme from Chlorella virus that methylates all cytosines within the double-stranded dinucleotide recognition sequence 5′... GC...3′.

CCCTC-binding factor

(CTCF). A chromatin binding factor with highly conserved zinc finger domains that control binding to consensus DNA target sequences. CTCF regulates transcription by binding to chromatin insulators and preventing interaction between the promoter and enhancers or silencers.

Third-generation sequencing

A new progression of sequencing technology that aims to improve throughput and reduce sequencing cost and time. The main goals of third-generation sequencing are to eliminate the DNA amplification step before sequencing and to enable real-time signal monitoring.

β-glucosyltransferase

(βGT). An enzyme that transfers the glucose residue of uridine diphosphosphate glucose (UDP-Glc) specifically to the hydroxyl group of 5-hydroxymethylcytosine to generate β-glucosyl-5hmC (5gmC).

Click chemistry

A nonspecific chemical reaction that combines small modular units and is used to generate or label a substance. Azide alkyne Huisgen cycloaddition, in which an azide and alkyne interact to form a 1,2,3-triazole (with 5-membered ring) is the most popular click chemistry reaction. Click chemistry has been used for selectively labelling biomolecules.

Glucosylation

The process of transferring a glucose residue from a nucleotide sugar derivative, such as from uridine diphosphate glucose (UDP-Glc) to a target molecule.

J-binding proteins

Proteins that specifically bind to the base J (β-D-glucopyranosyloxymethyluracil), a modified form of uracil found in the DNA of a number of organisms, such as human pathogen Trypanosoma and the kinetoplastids. Base J is formed by hydroxylation of thymidine and subsequent glycosylation by glycosyltransferase enzyme.

Isoschizomer

Restriction enzymes that have the same recognition sequences and cleave at the same positions.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Plongthongkum, N., Diep, D. & Zhang, K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet 15, 647–661 (2014). https://doi.org/10.1038/nrg3772

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg3772

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing