Science China Life Sciences

, Volume 59, Issue 3, pp 219–226 | Cite as

Single-base resolution analysis of DNA epigenome via high-throughput sequencing

  • Jinying PengEmail author
  • Bo Xia
  • Chengqi YiEmail author
Open Access
Review SPECIAL TOPIC: From epigenetic to epigenomic regulation


Epigenetic changes caused by DNA methylation and histone modifications play important roles in the regulation of various cellular processes and development. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC) and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the epigenetic regulation. Analysis of DNA modification patterns relies increasingly on sequencing-based profiling methods. A number of different approaches have been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing (BS-seq), TAB-seq (TET-assisted bisulfite sequencing), oxBS-seq (oxidative bisulfite sequencing) and etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this review will be to discuss the chemical methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.


epigenetics DNA methylation bisulfite sequencing (BS-Seq) TAB-seq oxBS-seq fC-CET 


  1. Bender, J. (2004). DNA methylation and epigenetics. Annu Rev Plant Biol 55, 41–68.CrossRefPubMedGoogle Scholar
  2. Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev 16, 6–21.CrossRefPubMedGoogle Scholar
  3. Booth, M.J., Branco, M.R., Ficz, G., Oxley, D., Krueger, F., Reik, W., and Balasubramanian, S. (2012). Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937.CrossRefPubMedGoogle Scholar
  4. Booth, M.J., Marsico, G., Bachman, M., Beraldi, D., and Balasubramanian, S. (2014). Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem 6, 435–440.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Booth, M.J., Ost, T.W., Beraldi, D., Bell, N.M., Branco, M.R., Reik, W., and Balasubramanian, S. (2013). Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8, 1841–1851.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Booth, M.J., Raiber, E.A., and Balasubramanian, S. (2015). Chemical methods for decoding cytosine modifications in DNA. Chem Rev 115, 2240–2254.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chan, S.W., Henderson, I.R., and Jacobsen, S.E. (2005). Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6, 351–360.CrossRefPubMedGoogle Scholar
  8. Cokus, S.J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C.D., Pradhan, S., Nelson, S.F., Pellegrini, M., and Jacobsen, S.E. (2008). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Doerfler, W. (1983). DNA methylation and gene activity. Annu Rev Biochem 52, 93–124.CrossRefPubMedGoogle Scholar
  10. Flusberg, B.A., Webster, D.R., Lee, J.H., Travers, K.J., Olivares, E.C., Clark, T.A., Korlach, J., and Turner, S.W. (2010). Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7, 461–465.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Goll, M.G., and Bestor, T.H. (2005). Eukaryotic cytosine methyltransferases. Annu Rev Biochem 74, 481–514.CrossRefPubMedGoogle Scholar
  12. Guo, F., Li, X., Liang, D., Li, T., Zhu, P., Guo, H., Wu, X., Wen, L., Gu, T.P., Hu, B., Walsh, C.P., Li, J., Tang, F., and Xu, G.L. (2014). Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15, 447–458.CrossRefPubMedGoogle Scholar
  13. Guo, H., Zhu, P., Wu, X., Li, X., Wen, L., and Tang, F. (2013). Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res 23, 2126–2135.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hayatsu, H., Wataya, Y., Kai, K., and Iida, S. (1970). Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9, 2858–2865.CrossRefPubMedGoogle Scholar
  15. He, Y.F., Li, B.Z., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y., Chen, Z., Li, L., Sun Y., Li, X., Dai, Q., Song, C.X., Zhang, K., He, C., and Xu, G.L. (2011). Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Ito, S., Shen, L., Dai, Q., Wu, S.C., Collins, L.B., Swenberg, J.A., He, C., and Zhang, Y. (2011). Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Iurlaro, M., Ficz, G., Oxley, D., Raiber, E.A., Bachman, M., Booth, M.J., Andrews, S., Balasubramanian, S., and Reik, W. (2013). A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol 14, R119.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Jablonka, E., and Lamb, M.J. (2002). The changing concept of epigenetics. Ann N YAcad Sci 981, 82–96.CrossRefGoogle Scholar
  19. Johnson., T.B., and Coghill, R.D. (1925). Research on pyrimidines. C111. The discovery of 5-methyl-cytosine in tuberculinic acid, the nucleic acid of the Tubercle bacillus. J Am Chem Soc 47, 2844.CrossRefGoogle Scholar
  20. Jones, P.A. (2012). Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13, 484–492.CrossRefPubMedGoogle Scholar
  21. Kellinger, M.W., Song, C.X., Chong, J., Lu, X.Y., He, C., and Wang, D. (2012). 5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nat Struct Mol Biol 19, 831–833.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kriaucionis, S., and Heintz, N. (2009). The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lister, R., and Ecker, J.R. (2009). Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res 19, 959–966.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lister, R., Mukamel, E.A., Nery, J.R., Urich, M., Puddifoot, C.A., Johnson, N.D., Lucero, J., Huang, Y., Dwork, A.J., Schultz, M.D., Yu, M., Tonti-Filippini, J., Heyn, H., Hu, S., Wu, J.C., Rao, A., Esteller, M., He, C., Haghighi, F.G., Sejnowski, T.J., Behrens, M.M., and Ecker, J.R. (2013). Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lister, R., O’Malley, R.C., Tonti-Filippini, J., Gregory, B.D., Berry, C.C., Millar, A.H., and Ecker, J.R. (2008). Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti-Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M., Edsall, L., Antosiewicz-Bourget, J., Stewart, R., Ruotti, V., Millar, A.H., Thomson, J.A., Ren, B., and Ecker, J.R. (2009a). Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lu, X., Song, C.X., Szulwach, K., Wang, Z., Weidenbacher, P., Jin, P., and He, C. (2013). Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc 135, 9315–9317.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pastor, W.A., Pape, U.J., Huang, Y., Henderson, H.R., Lister, R., Ko, M., McLoughlin, E.M., Brudno, Y., Mahapatra, S., Kapranov, P., Tahiliani M., Daley, G.Q., Liu, X.S., Ecker, J.R., Milos, P.M., Agarwal, S., and Rao, A. (2011). Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Pfaffeneder, T., Hackner, B., Truss, M., Munzel, M., Muller, M., Deiml, C.A., Hagemeier, C., and Carell, T. (2011). The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem Int Ed Engl 50, 7008–7012.CrossRefPubMedGoogle Scholar
  30. Raiber, E.A., Beraldi, D., Ficz, G., Burgess, H.E., Branco, M.R., Murat, P., Oxley, D., Booth, M.J., Reik, W., and Balasubramanian, S. (2012). Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase. Genome Biol 13, R69.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Roberts, R.J., and Cheng, X. (1998). Base flipping. Annu Rev Biochem 67, 181–198.CrossRefPubMedGoogle Scholar
  32. Robertson, A.B., Dahl, J.A., Vagbo, C.B., Tripathi, P., Krokan, H.E., and Klungland, A. (2011). A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res 39, e55.CrossRefGoogle Scholar
  33. Shapiro, R., and Weisgras, J.M. (1970). Bisulfite-catalyzed transamination of cytosine and cytidine. Biochem Biophys Res Commun 40, 839–843.CrossRefPubMedGoogle Scholar
  34. Shen, L., Wu, H., Diep, D., Yamaguchi, S., D’Alessio, A.C., Fung, H.L., Zhang, K., and Zhang, Y. (2013). Genome-wide analysis reveals TETand TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Smallwood, S.A., Lee, H.J., Angermueller, C., Krueger, F., Saadeh, H., Peat, J., Andrews, S.R., Stegle, O., Reik, W., and Kelsey, G. (2014). Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 11, 817–820.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Song, C.X., Szulwach, K.E., Dai, Q., Fu, Y., Mao, S.Q., Lin, L., Street, C., Li, Y., Poidevin, M., Wu, H., Gao, J., Liu, P., Li, L., Xu, G.L., Jin, P., and He, C. (2013). Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Song, C.X., Szulwach, K.E., Fu, Y., Dai, Q., Yi, C., Li, X., Li, Y., Chen, C.H., Zhang, W., Jian, X., Wang, J., Zhang, L., Looney, T.J., Zhang, B., Godley, L.A., Hicks, L.M., Lahn, B.T., Jin, P., and He, C. (2011). Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29, 68–72.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Song, C.X., Yi, C., and He, C. (2012). Mapping recently identified nucleotide variants in the genome and transcriptome. Nat Biotechnol 30, 1107–1116.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Spruijt, C.G., Gnerlich, F., Smits, A.H., Pfaffeneder, T., Jansen, P.W., Bauer, C., Munzel, M., Wagner, M., Muller, M., Khan, F., Eberl, H.C., Mensinga, A., Brinkman, A.B., Lephikov, K., Müller, U., Walter, J., Boelens, R., Van Ingen, H., Leonhardt, H., Carell, T., and Vermeulen, M. (2013). Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159.CrossRefPubMedGoogle Scholar
  40. Sun, Z., Terragni, J., Borgaro, J.G., Liu, Y., Yu, L., Guan, S., Wang, H., Sun, D., Cheng, X., Zhu, Z., Pradhan, S., and Zheng, Y. (2013). Highresolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep 3, 567–576.CrossRefPubMedPubMedCentralGoogle Scholar
  41. Tahiliani, M., Koh, K.P., Shen, Y., Pastor, W.A., Bandukwala, H., Brudno, Y., Agarwal, S., Iyer, L.M., Liu, D.R., Aravind, L., and Rao, A. (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Waddington, C.H. (1939). Preliminary Notes on the development of the wings in normal and mutant strains of Drosophila. Proc Natl Acad Sci USA 25, 299–307.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Wang, L., Zhang, J., Duan, J., Gao, X., Zhu, W., Lu, X., Yang, L., Li, G., Ci, W., Li, W., Zhou, Q., Aluru, N., Tang, F., He, C., Huang, X., and Liu, J. (2014). Programming and inheritance of parental DNA methylomes in mammals. Cell 157, 979–991.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Wescoe, Z.L., Schreiber, J., and Akeson, M. (2014). Nanopores discriminate among five C5-cytosine variants in DNA. J Am Chem Soc 136, 16582–16587.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wu, H., D’Alessio, A.C., Ito, S., Wang, Z., Cui, K., Zhao, K., Sun, Y.E., and Zhang, Y. (2011). Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev 25, 679–684.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Wu, H., Wu, X., Shen, L., and Zhang, Y. (2014). Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol 32, 1231–1240.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wu, S.C., and Zhang, Y. (2010). Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11, 607–620.CrossRefPubMedPubMedCentralGoogle Scholar
  48. Xia, B., Han, D., Lu, X., Sun, Z., Zhou, A., Yin, Q., Zeng, H., Liu, M., Jiang, X., Xie, W., He, C., and Yi, C. (2015). Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods 12, 1047–1050.CrossRefPubMedGoogle Scholar
  49. Yildirim, O., Li, R., Hung, J.H., Chen, P.B., Dong, X., Ee, L.S., Weng, Z., Rando, O.J., and Fazzio, T.G. (2011). Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells. Cell 147, 1498–1510.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Yu, M., Hon, G.C., Szulwach, K.E., Song, C.X., Zhang, L., Kim, A., Li, X., Dai, Q., Shen, Y., Park, B., Min, J.H., Jin, P., Ren, B., and He, C. (2012). Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380.CrossRefPubMedPubMedCentralGoogle Scholar
  51. Zhang, L., Szulwach, K.E., Hon, G.C., Song, C.X., Park, B., Yu, M., Lu, X., Dai, Q., Wang, X., Street, C.R., Tan, H., Min, J.H., Ren, B., Jin, P., and He, C. (2013). Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nat Commun 4, 1517.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhu, J.K. (2009). Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet 43, 143–166.CrossRefPubMedPubMedCentralGoogle Scholar
  53. Ziller, M.J., Gu, H., Muller, F., Donaghey, J., Tsai, L.T., Kohlbacher, O., De Jager, P.L., Rosen, E.D., Bennett, D.A., Bernstein, B.E., Gnirke, A., and Meissner, A. (2013). Charting a dynamic DNA methylation landscape of the human genome. Nature 500, 477–481.CrossRefPubMedPubMedCentralGoogle Scholar

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© The Author(s) 2016

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits use, duplication, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, and Peking-Tsinghua Center for Life SciencesPeking UniversityBeijingChina
  2. 2.Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular EngineeringPeking UniversityBeijingChina

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