Skip to main content
SpringerLink
Log in

Sequencing the Cancer Methylome

  • Protocol
  • First Online:
Cancer Epigenetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1238))

Abstract

DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14(3):204–220. doi:10.1038/nrg3354

    Article  CAS  PubMed  Google Scholar 

  2. Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99(3):247–257

    Article  CAS  PubMed  Google Scholar 

  3. Robertson KD (2005) DNA methylation and human disease. Nat Rev Genet 6(8):597–610. doi:10.1038/nrg1655

    Article  CAS  PubMed  Google Scholar 

  4. Ito S, D’Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466(7310):1129–1133. doi:10.1038/nature09303

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324(5929):929–930. doi:10.1126/science.1169786

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Tahiliani M, Koh KP, Shen YH, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324(5929):930–935. doi:10.1126/science.1170116

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, Dai Q, Song CX, Zhang K, He C, Xu GL (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333(6047):1303–1307. doi:10.1126/science.1210944

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333(6047):1300–1303. doi:10.1126/science.1210597

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Monk M, Boubelik M, Lehnert S (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99(3):371–382

    CAS  PubMed  Google Scholar 

  10. Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, Laiho A, Tahiliani M, Sommer CA, Mostoslavsky G, Lahesmaa R, Orkin SH, Rodig SJ, Daley GQ, Rao A (2011) Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8(2):200–213. doi:10.1016/j.stem.2011.01.008

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, Iqbal K, Shi YG, Deng Z, Szabo PE, Pfeifer GP, Li J, Xu GL (2011) The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477(7366):606–610. doi:10.1038/nature10443

    Article  CAS  PubMed  Google Scholar 

  12. Pastor WA, Aravind L, Rao A (2013) TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat Rev Mol Cell Biol 14(6):341–356. doi:10.1038/nrm3589

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Wu H, Zhang Y (2011) Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev 25(23):2436–2452. doi:10.1101/gad.179184.111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Lian CG, Xu Y, Ceol C, Wu F, Larson A, Dresser K, Xu W, Tan L, Hu Y, Zhan Q, Lee CW, Hu D, Lian BQ, Kleffel S, Yang Y, Neiswender J, Khorasani AJ, Fang R, Lezcano C, Duncan LM, Scolyer RA, Thompson JF, Kakavand H, Houvras Y, Zon LI, Mihm MC Jr, Kaiser UB, Schatton T, Woda BA, Murphy GF, Shi YG (2012) Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 150(6):1135–1146. doi:10.1016/j.cell.2012.07.033

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Callinan PA, Feinberg AP (2006) The emerging science of epigenomics. Hum Mol Genet 15(Suppl 1):R95–R101. doi:10.1093/hmg/ddl095

    Article  CAS  PubMed  Google Scholar 

  16. Laird PW (2010) Principles and challenges of genome-wide DNA methylation analysis. Nat Rev Genet 11(3):191–203

    Article  CAS  PubMed  Google Scholar 

  17. Lister R, Ecker JR (2009) Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res 19(6):959–966. doi:10.1101/gr.083451.108

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Zilberman D, Henikoff S (2007) Genome-wide analysis of DNA methylation patterns. Development 134(22):3959–3965. doi:10.1242/dev.001131

    Article  CAS  PubMed  Google Scholar 

  19. Yan PS, Chen CM, Shi H, Rahmatpanah F, Wei SH, Caldwell CW, Huang TH (2001) Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res 61(23):8375–8380

    CAS  PubMed  Google Scholar 

  20. Khulan B, Thompson RF, Ye K, Fazzari MJ, Suzuki M, Stasiek E, Figueroa ME, Glass JL, Chen Q, Montagna C, Hatchwell E, Selzer RR, Richmond TA, Green RD, Melnick A, Greally JM (2006) Comparative isoschizomer profiling of cytosine methylation: the HELP assay. Genome Res 16(8):1046–1055. doi:10.1101/gr.5273806

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Estecio MR, Yan PS, Ibrahim AE, Tellez CS, Shen L, Huang TH, Issa JP (2007) High-throughput methylation profiling by MCA coupled to CpG island microarray. Genome Res 17(10):1529–1536. doi:10.1101/gr.6417007

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37(8):853–862. http://www.nature.com/ng/journal/v37/n8/suppinfo/ng1598_S1.html

  23. Rauch T, Li H, Wu X, Pfeifer GP (2006) MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells. Cancer Res 66(16):7939–7947. doi:10.1158/0008-5472.can-06-1888

    Article  CAS  PubMed  Google Scholar 

  24. Sandoval J, Heyn H, Moran S, Serra-Musach J, Pujana MA, Bibikova M, Esteller M (2011) Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome. Epigenetics 6(6):692–702

    Article  CAS  PubMed  Google Scholar 

  25. Michels KB, Binder AM, Dedeurwaerder S, Epstein CB, Greally JM, Gut I, Houseman EA, Izzi B, Kelsey KT, Meissner A, Milosavljevic A, Siegmund KD, Bock C, Irizarry RA (2013) Recommendations for the design and analysis of epigenome-wide association studies. Nat Methods 10(10):949–955. doi:10.1038/nmeth.2632. http://www.nature.com/nmeth/journal/v10/n10/abs/nmeth.2632.html#supplementary-information

  26. Suzuki M, Jing Q, Lia D, Pascual M, McLellan A, Greally J (2010) Optimized design and data analysis of tag-based cytosine methylation assays. Genome Biol 11(4):R36

    Article  PubMed Central  PubMed  Google Scholar 

  27. Challen GA, Sun D, Jeong M, Luo M, Jelinek J, Berg JS, Bock C, Vasanthakumar A, Gu H, Xi Y, Liang S, Lu Y, Darlington GJ, Meissner A, Issa J-PJ, Godley LA, Li W, Goodell MA (2012) Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 44(1):23–31. http://www.nature.com/ng/journal/v44/n1/abs/ng.1009.html#supplementary-information

  28. Harris RA, Wang T, Coarfa C, Nagarajan RP, Hong C, Downey SL, Johnson BE, Fouse SD, Delaney A, Zhao Y, Olshen A, Ballinger T, Zhou X, Forsberg KJ, Gu J, Echipare L, O’Geen H, Lister R, Pelizzola M, Xi Y, Epstein CB, Bernstein BE, Hawkins RD, Ren B, Chung WY, Gu H, Bock C, Gnirke A, Zhang MQ, Haussler D, Ecker JR, Li W, Farnham PJ, Waterland RA, Meissner A, Marra MA, Hirst M, Milosavljevic A, Costello JF (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nat Biotechnol 28(10):1097–1105. doi:10.1038/nbt.1682

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Choi JH, Li Y, Guo J, Pei L, Rauch TA, Kramer RS, Macmil SL, Wiley GB, Bennett LB, Schnabel JL, Taylor KH, Kim S, Xu D, Sreekumar A, Pfeifer GP, Roe BA, Caldwell CW, Bhalla KN, Shi H (2010) Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing. PLoS One 5(9):e13020. doi:10.1371/journal.pone.0013020

    Article  PubMed Central  PubMed  Google Scholar 

  30. Irizarry RA, Ladd-Acosta C, Carvalho B, Wu H, Brandenburg SA, Jeddeloh JA, Wen B, Feinberg AP (2008) Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res 18(5):780–790. doi:10.1101/gr.7301508

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Bhattacharyya S, Yu Y, Suzuki M, Campbell N, Mazdo J, Vasanthakumar A, Bhagat TD, Nischal S, Christopeit M, Parekh S, Steidl U, Godley L, Maitra A, Greally JM, Verma A (2013) Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. Nucleic Acids Res 41(16):e157. doi:10.1093/nar/gkt601

    Article  PubMed Central  PubMed  Google Scholar 

  32. Borgel J, Guibert S, Weber M (2012) Methylated DNA immunoprecipitation (MeDIP) from low amounts of cells. Methods Mol Biol 925:149–158. doi:10.1007/978-1-62703-011-3_9

    Article  CAS  PubMed  Google Scholar 

  33. Mohn F, Weber M, Schubeler D, Roloff TC (2009) Methylated DNA immunoprecipitation (MeDIP). Methods Mol Biol 507:55–64. doi:10.1007/978-1-59745-522-0_5

    Article  CAS  PubMed  Google Scholar 

  34. Nair SS, Coolen MW, Stirzaker C, Song JZ, Statham AL, Strbenac D, Robinson MD, Clark SJ (2011) Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias. Epigenetics 6(1):34–44. doi:10.4161/epi.6.1.13313

    Article  CAS  PubMed  Google Scholar 

  35. Taiwo O, Wilson GA, Morris T, Seisenberger S, Reik W, Pearce D, Beck S, Butcher LM (2012) Methylome analysis using MeDIP-seq with low DNA concentrations. Nat Protoc 7(4):617–636. doi:10.1038/nprot.2012.012

    Article  CAS  PubMed  Google Scholar 

  36. Vucic EA, Wilson IM, Campbell JM, Lam WL (2009) Methylation analysis by DNA immunoprecipitation (MeDIP). Methods Mol Biol 556:141–153. doi:10.1007/978-1-60327-192-9_10

    Article  CAS  PubMed  Google Scholar 

  37. Feber A, Wilson GA, Zhang L, Presneau N, Idowu B, Down TA, Rakyan VK, Noon LA, Lloyd AC, Stupka E, Schiza V, Teschendorff AE, Schroth GP, Flanagan A, Beck S (2011) Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors. Genome Res 21(4):515–524. doi:10.1101/gr.109678.110

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Rauch TA, Pfeifer GP (2009) The MIRA method for DNA methylation analysis. Methods Mol Biol 507:65–75. doi:10.1007/978-1-59745-522-0_6

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Jin B, Ernst J, Tiedemann RL, Xu H, Sureshchandra S, Kellis M, Dalton S, Liu C, Choi JH, Robertson KD (2012) Linking DNA methyltransferases to epigenetic marks and nucleosome structure genome-wide in human tumor cells. Cell Rep 2(5):1411–1424. doi:10.1016/j.celrep.2012.10.017

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473(7347):398–402. doi:10.1038/nature10008

    Article  CAS  PubMed  Google Scholar 

  41. Wu H, D’Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, 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(7):679–684. doi:10.1101/gad.2036011

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Song CX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen CH, Zhang W, Jian X, Wang J, Zhang L, Looney TJ, Zhang B, Godley LA, Hicks LM, Lahn BT, Jin P, He C (2011) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29(1):68–72. doi:10.1038/nbt.1732

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Robertson AB, Dahl JA, Ougland R, Klungland A (2012) Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads. Nat Protoc 7(2):340–350. doi:10.1038/nprot.2011.443

    Article  CAS  PubMed  Google Scholar 

  44. Thomson JP, Hunter JM, Nestor CE, Dunican DS, Terranova R, Moggs JG, Meehan RR (2013) Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res 41(22):e206. doi:10.1093/nar/gkt1080

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Laurent L, Wong E, Li G, Huynh T, Tsirigos A, Ong CT, Low HM, Sung KWK, Rigoutsos I, Loring J, Wei CL (2010) Dynamic changes in the human methylome during differentiation. Genome Res 20(3):320–331. doi:10.1101/gr.101907.109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, Nery JR, Lee L, Ye Z, Ngo QM, Edsall L, Antosiewicz-Bourget J, Stewart R, Ruotti V, Millar AH, Thomson JA, Ren B, Ecker JR (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462(7271):315–322. doi:10.1038/nature08514

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Li YR, Zhu JD, Tian G, Li N, Li QB, Ye MZ, Zheng HC, Yu JA, Wu HL, Sun JH, Zhang HY, Chen QA, Luo RB, Chen MF, He YH, Jin X, Zhang QH, Yu C, Zhou GY, Sun JF, Huang YB, Zheng HS, Cao HZ, Zhou XY, Guo SC, Hu XD, Li X, Kristiansen K, Bolund L, Xu JJ, Wang W, Yang HM, Wang JA, Li RQ, Beck S, Wang J, Zhang XQ (2010) The DNA methylome of human peripheral blood mononuclear cells. PLoS Biol 8(11). ARTN e1000533. doi:10.1371/journal.pbio.1000533

  48. Hodges E, Molaro A, Dos Santos CO, Thekkat P, Song Q, Uren PJ, Park J, Butler J, Rafii S, McCombie WR, Smith AD, Hannon GJ (2011) Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment. Mol Cell 44(1):17–28. doi:10.1016/j.molcel.2011.08.026

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Berman BP, Weisenberger DJ, Aman JF, Hinoue T, Ramjan Z, Liu YP, Noushmehr H, Lange CPE, van Dijk CM, Tollenaar RAEM, Van den Berg D, Laird PW (2012) Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet 44(1):40–U62. doi:10.1038/Ng.969

    Article  CAS  Google Scholar 

  50. Hon GC, Hawkins RD, Caballero OL, Lo C, Lister R, Pelizzola M, Valsesia A, Ye Z, Kuan S, Edsall LE, Camargo AA, Stevenson BJ, Ecker JR, Bafna V, Strausberg RL, Simpson AJ, Ren B (2012) Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res 22(2):246–258. doi:10.1101/gr.125872.111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Hansen KD, Timp W, Bravo HC, Sabunciyan S, Langmead B, McDonald OG, Wen B, Wu H, Liu Y, Diep D, Briem E, Zhang K, Irizarry RA, Feinberg AP (2011) Increased methylation variation in epigenetic domains across cancer types. Nat Genet 43(8):768–U777. doi:10.1038/Ng.865

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. Berman BP, Weisenberger DJ, Aman JF, Hinoue T, Ramjan Z, Liu Y, Noushmehr H, Lange CPE, van Dijk CM, Tollenaar RAEM, Van Den Berg D, Laird PW (2012) Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet 44(1):40–46. http://www.nature.com/ng/journal/v44/n1/abs/ng.969.html#supplementary-information

  53. Hansen KD, Timp W, Bravo HC, Sabunciyan S, Langmead B, McDonald OG, Wen B, Wu H, Liu Y, Diep D, Briem E, Zhang K, Irizarry RA, Feinberg AP (2011) Increased methylation variation in epigenetic domains across cancer types. Nat Genet 43(8):768–775. http://www.nature.com/ng/journal/v43/n8/abs/ng.865.html#supplementary-information

  54. Lee EJ, Luo J, Wilson JM, Shi H (2013) Analyzing the cancer methylome through targeted bisulfite sequencing. Cancer Lett 340(2):171–178. doi:10.1016/j.canlet.2012.10.040

    Article  CAS  PubMed  Google Scholar 

  55. Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J, Egli D, Maherali N, Park IH, Yu JY, Daley GQ, Eggan K, Hochedlinger K, Thomson J, Wang W, Gao Y, Zhang K (2009) Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 27(4):353–360. doi:10.1038/Nbt.1530

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  56. Ball MP, Li JB, Gao Y, Lee JH, LeProust EM, Park IH, Xie B, Daley GQ, Church GM (2009) Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells (vol 27, pg 361, 2009). Nat Biotechnol 27(5):485. doi:10.1038/Nbt0509-485b

    Article  CAS  Google Scholar 

  57. Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, Gao Y, Church GM, Shendure J (2007) Multiplex amplification of large sets of human exons. Nat Methods 4(11):931–936. doi:10.1038/Nmeth1110

    Article  CAS  PubMed  Google Scholar 

  58. Hodges E, Smith AD, Kendall J, Xuan ZY, Ravi K, Rooks M, Zhang MQ, Ye K, Bhattacharjee A, Brizuela L, McCombie WR, Wigler M, Hannon GJ, Hicks JB (2009) High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing. Genome Res 19(9):1593–1605. doi:10.1101/gr.095190.109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  59. Lee EJ, Pei L, Srivastava G, Joshi T, Kushwaha G, Choi JH, Robertson KD, Wang X, Colbourne JK, Zhang L, Schroth GP, Xu D, Zhang K, Shi H (2011) Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing. Nucleic Acids Res 39(19):e127. doi:10.1093/nar/gkr598, gkr598 [pii]

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  60. Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27(2):182–189. doi:10.1038/nbt.1523

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  61. Harrow J, Denoeud F, Frankish A, Reymond A, Chen C-K, Chrast J, Lagarde J, Gilbert J, Storey R, Swarbreck D, Rossier C, Ucla C, Hubbard T, Antonarakis S, Guigo R (2006) GENCODE: producing a reference annotation for ENCODE. Genome Biol 7(Suppl 1):S4

    Article  PubMed Central  PubMed  Google Scholar 

  62. Varley KE, Mitra RD (2010) Bisulfite patch PCR enables multiplexed sequencing of promoter methylation across cancer samples. Genome Res 20(9):1279–1287. doi:10.1101/gr.101212.109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  63. Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK, Samuels ML, Hutchison JB, Larson JW, Topol EJ, Weiner MP, Harismendy O, Olson J, Link DR, Frazer KA (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27(11):1025–U1094. doi:10.1038/Nbt.1583

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  64. Komori HK, LaMere SA, Torkamani A, Hart GT, Kotsopoulos S, Warner J, Samuels ML, Olson J, Head SR, Ordoukhanian P, Lee PL, Link DR, Salomon DR (2011) Application of microdroplet PCR for large-scale targeted bisulfite sequencing. Genome Res 21(10):1738–1745. doi:10.1101/gr.116863.110

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  65. Meissner A, Gnirke A, Bell GW, Ramsahoye B, Lander ES, Jaenisch R (2005) Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res 33(18):5868–5877. doi:10.1093/Nar/Gki901

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  66. Gu H, Bock C, Mikkelsen TS, Jager N, Smith ZD, Tomazou E, Gnirke A, Lander ES, Meissner A (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat Methods 7(2):133–U169. doi:10.1038/Nmeth.1414

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  67. Boyle P, Clement K, Gu HC, Smith ZD, Ziller M, Fostel JL, Holmes L, Meldrim J, Kelley F, Gnirke A, Meissner A (2012) Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling. Genome Biol 13(10). Artn R92. doi:10.1186/Gb-2012-13-10-R92

  68. Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C, Hartig R, Schonfeld P, Roessner A, Gali-Muhtasib H (2005) 5-Aza-cytidine is a potent inhibitor of DNA methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53-dependent mechanisms. J Pharmacol Exp Ther 312(2):525–536. doi:10.1124/jpet.104.074195

    Article  CAS  PubMed  Google Scholar 

  69. Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR, Rao A (2010) The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5(1):e8888. doi:10.1371/journal.pone.0008888

    Article  PubMed Central  PubMed  Google Scholar 

  70. Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W, Balasubramanian S (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336(6083):934–937. doi:10.1126/science.1220671

    Article  CAS  PubMed  Google Scholar 

  71. Booth MJ, Ost TWB, Beraldi D, Bell NM, Branco MR, Reik W, Balasubramanian S (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8(10):1841–1851. doi:10.1038/nprot.2013.115

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  72. Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li XK, Dai Q, Shen Y, Park B, Min JH, Jin P, Ren B, He C (2012) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149(6):1368–1380. doi:10.1016/j.cell.2012.04.027

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  73. Brinkman AB, Gu H, Bartels SJJ, Zhang Y, Matarese F, Simmer F, Marks H, Bock C, Gnirke A, Meissner A, Stunnenberg HG (2012) Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res 22(6):1128–1138. doi:10.1101/gr.133728.111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  74. Statham AL, Robinson MD, Song JZ, Coolen MW, Stirzaker C, Clark SJ (2012) Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA. Genome Res 22(6):1120–1127. doi:10.1101/gr.132076.111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  75. Huang Y, Pastor WA, Zepeda-Martínez JA, Rao A (2012) The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7(10):1897–1908

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  76. Pondugula S, Kladde MP (2008) Single-molecule analysis of chromatin: changing the view of genomes one molecule at a time. J Cell Biochem 105(2):330–337. doi:10.1002/jcb.21849

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  77. Kelly TK, Miranda TB, Liang G, Berman BP, Lin JC, Tanay A, Jones PA (2010) H2A.Z maintenance during mitosis reveals nucleosome shifting on mitotically silenced genes. Mol Cell 39(6):901–911. doi:10.1016/j.molcel.2010.08.026, S1097-2765(10)00636-2 [pii]

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  78. Lin JC, Jeong S, Liang G, Takai D, Fatemi M, Tsai YC, Egger G, Gal-Yam EN, Jones PA (2007) Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island. Cancer Cell 12(5):432–444. doi:10.1016/j.ccr.2007.10.014, S1535-6108(07)00299-1. [pii]

    Article  CAS  PubMed  Google Scholar 

  79. Kelly TK, Liu Y, Lay FD, Liang G, Berman BP, Jones PA (2012) Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res 22(12):2497–2506. doi:10.1101/gr.143008.112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  80. Nabilsi NH, Deleyrolle LP, Darst RP, Riva A, Reynolds BA, Kladde MP (2014) Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma. Genome Res 24(2):329–339. doi:10.1101/gr.161737.113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  81. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25(14):1754–1760. doi:10.1093/bioinformatics/btp324

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  82. Langmead B, Trapnell C, Pop M, Salzberg S (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25

    Article  PubMed Central  PubMed  Google Scholar 

  83. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24(5):713–714. doi:10.1093/bioinformatics/btn025

    Article  CAS  PubMed  Google Scholar 

  84. Down TA, Rakyan VK, Turner DJ, Flicek P, Li H, Kulesha E, Graf S, Johnson N, Herrero J, Tomazou EM, Thorne NP, Backdahl L, Herberth M, Howe KL, Jackson DK, Miretti MM, Marioni JC, Birney E, Hubbard TJP, Durbin R, Tavare S, Beck S (2008) A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis. Nat Biotechnol 26(7):779–785. http://www.nature.com/nbt/journal/v26/n7/suppinfo/nbt1414_S1.html

  85. Lienhard M, Grimm C, Morkel M, Herwig R, Chavez L (2013) MEDIPS: genome-wide differential coverage analysis of sequencing data derived from DNA enrichment experiments. Bioinformatics. doi:10.1093/bioinformatics/btt650

    PubMed Central  PubMed  Google Scholar 

  86. Lan X, Adams C, Landers M, Dudas M, Krissinger D, Marnellos G, Bonneville R, Xu M, Wang J, Huang THM, Meredith G, Jin VX (2011) High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology. PLoS One 6(7):e22226. doi:10.1371/journal.pone.0022226

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  87. Wilson G, Dhami P, Feber A, Cortazar D, Suzuki Y, Schulz R, Schar P, Beck S (2012) Resources for methylome analysis suitable for gene knockout studies of potential epigenome modifiers. Gigascience 1(1):3

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  88. Huang J, Renault V, Sengenès J, Touleimat N, Michel S, Lathrop M, Tost J (2012) MeQA: a pipeline for MeDIP-seq data quality assessment and analysis. Bioinformatics 28(4):587–588. doi:10.1093/bioinformatics/btr699

    Article  CAS  PubMed  Google Scholar 

  89. He J, Sun X, Shao X, Liang L, Xie H (2013) DMEAS: DNA methylation entropy analysis software. Bioinformatics 29(16):2044–2045. doi:10.1093/bioinformatics/btt332

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  90. Hansen K, Langmead B, Irizarry R (2012) BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions. Genome Biol 13(10):R83

    Article  PubMed Central  PubMed  Google Scholar 

  91. Akalin A AMP for aligning ERRBS and RRBS reads. http://code.google.com/p/amp-errbs/

  92. Akalin A, Kormaksson M, Li S, Garrett-Bakelman F, Figueroa M, Melnick A, Mason C (2012) methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol 13(10):R87

    Article  PubMed Central  PubMed  Google Scholar 

  93. Choi J-H BSpipe: a comprehensive pipeline for BS-seq. http://sourceforge.net/projects/bspipe/

  94. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D (2002) The human genome browser at UCSC. Genome Res 12(6):996–1006. doi:10.1101/gr.229102

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  95. Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14(2):178–192. doi:10.1093/bib/bbs017

    Article  PubMed Central  PubMed  Google Scholar 

  96. Nicol JW, Helt GA, Blanchard SG, Raja A, Loraine AE (2009) The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25(20):2730–2731. doi:10.1093/bioinformatics/btp472

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  97. Mi H, Lazareva-Ulitsky B, Loo R, Kejariwal A, Vandergriff J, Rabkin S, Guo N, Muruganujan A, Doremieux O, Campbell MJ, Kitano H, Thomas PD (2005) The PANTHER database of protein families, subfamilies, functions and pathways. Nucleic Acids Res 33(suppl 1):D284–D288. doi:10.1093/nar/gki078

    CAS  PubMed Central  PubMed  Google Scholar 

  98. Huang DW, Sherman BT, Lempicki RA (2008) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57. http://www.nature.com/nprot/journal/v4/n1/suppinfo/nprot.2008.211_S1.html

  99. Ingenuity Pathway Analysis. http://www.ingenuity.com

  100. Ghoshal K, Datta J, Majumder S, Bai S, Kutay H, Motiwala T, Jacob ST (2005) 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25(11):4727–4741. doi:10.1128/MCB.25.11.4727-4741.2005

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  101. Li F, Papworth M, Minczuk M, Rohde C, Zhang Y, Ragozin S, Jeltsch A (2007) Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes. Nucleic Acids Res 35(1):100–112. doi:10.1093/nar/gkl1035

    Article  PubMed Central  PubMed  Google Scholar 

  102. Siddique AN, Nunna S, Rajavelu A, Zhang Y, Jurkowska RZ, Reinhardt R, Rots MG, Ragozin S, Jurkowski TP, Jeltsch A (2013) Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity. J Mol Biol 425(3):479–491. doi:10.1016/j.jmb.2012.11.038

    Article  CAS  PubMed  Google Scholar 

  103. Chen H, Kazemier HG, de Groote ML, Ruiters MH, Xu GL, Rots MG (2014) Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter. Nucleic Acids Res 42(3):1563–1574. doi:10.1093/nar/gkt1019

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  104. Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE, Costello JF, Wilkinson MF, Joung JK (2013) Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol. doi:10.1038/nbt.2726

    Google Scholar 

  105. Gifford CA, Ziller MJ, Gu H, Trapnell C, Donaghey J, Tsankov A, Shalek AK, Kelley DR, Shishkin AA, Issner R, Zhang X, Coyne M, Fostel JL, Holmes L, Meldrim J, Guttman M, Epstein C, Park H, Kohlbacher O, Rinn J, Gnirke A, Lander ES, Bernstein BE, Meissner A (2013) Transcriptional and epigenetic dynamics during specification of human embryonic stem cells. Cell 153(5):1149–1163. doi:10.1016/j.cell.2013.04.037

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  106. Sharma MD, Huang L, Choi JH, Lee EJ, Wilson JM, Lemos H, Pan F, Blazar BR, Pardoll DM, Mellor AL, Shi H, Munn DH (2013) An inherently bifunctional subset of Foxp3+ T helper cells is controlled by the transcription factor eos. Immunity 38(5):998–1012. doi:10.1016/j.immuni.2013.01.013

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgement

This work was supported in part by National Institute of Health Grants CA134304, DA025779. Huidong Shi is a Georgia Cancer Coalition Distinguished Cancer Scientist.

Author information

Authors and Affiliations

  1. GRU Cancer Center, Georgia Regents University, Augusta, GA, 30912, USA

    Austin Y. Shull, Satish K. Noonepalle, Eun-Joon Lee, Jeong-Hyeon Choi & Huidong Shi

  2. Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, GA, 30912, USA

    Austin Y. Shull, Satish K. Noonepalle & Huidong Shi

  3. Department of Biostatistics and Epidemiology, Georgia Regents University, Augusta, GA, 30912, USA

    Jeong-Hyeon Choi

  4. Georgia Regents University Cancer Center, CN2138, 1120 15th Street, Augusta, GA, 30907, USA

    Huidong Shi

Authors
  1. Austin Y. Shull
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satish K. Noonepalle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eun-Joon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeong-Hyeon Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huidong Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huidong Shi .

Editor information

Editors and Affiliations

  1. National Institutes of Health, Rockville, Maryland, USA

    Mukesh Verma

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Shull, A.Y., Noonepalle, S.K., Lee, EJ., Choi, JH., Shi, H. (2015). Sequencing the Cancer Methylome. In: Verma, M. (eds) Cancer Epigenetics. Methods in Molecular Biology, vol 1238. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1804-1_33

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1804-1_33

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1803-4

  • Online ISBN: 978-1-4939-1804-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation