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Generating Multiple Base-Resolution DNA Methylomes Using Reduced Representation Bisulfite Sequencing

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Oral Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1537))

Abstract

Reduced representation bisulfite sequencing (RRBS) is an effective technique for profiling genome-wide DNA methylation patterns in eukaryotes. RRBS couples size selection, bisulfite conversion, and second-generation sequencing to enrich for CpG-dense regions of the genome. The progressive improvement of second-generation sequencing technologies and reduction in cost provided an opportunity to examine the DNA methylation patterns of multiple genomes. Here, we describe a protocol for sequencing multiple RRBS libraries in a single sequencing reaction to generate base-resolution methylomes. Furthermore, we provide a brief guideline for base-calling and data analysis of multiplexed RRBS libraries. These strategies will be useful to perform large-scale, genome-wide DNA methylation analysis.

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References

  1. Elango N, Yi SV (2008) DNA methylation and structural and functional bimodality of vertebrate promoters. Mol Biol Evol 25:1602–1608

    Article  CAS  PubMed  Google Scholar 

  2. Bock C, Beerman I, Lien WH, Smith ZD, Gu H, Boyle P, Gnirke A, Fuchs E, Rossi DJ, Meissner A (2012) DNA methylation dynamics during in vivo differentiation of blood and skin stem cells. Mol Cell 47:633–647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Carrel L, Willard HF (2005) X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature 434:400–404

    Article  CAS  PubMed  Google Scholar 

  4. Rollins RA, Haghighi F, Edwards JR, Das R, Zhang MQ, Ju J, Bestor TH (2006) Large-scale structure of genomic methylation patterns. Genome Res 16:157–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9:465–476

    Article  CAS  PubMed  Google Scholar 

  6. Igarashi J, Muroi S, Kawashima H, Wang X, Shinojima Y, Kitamura E, Oinuma T, Nemoto N, Song F, Ghosh S, Held WA, Nagase H (2008) Quantitative analysis of human tissue-specific differences in methylation. Biochem Biophys Res Commun 376:658–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chatterjee A, Morison IM (2011) Monozygotic twins: genes are not the destiny? Bioinformation 7:369–370

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chatterjee A, Eccles MR (2015) DNA methylation and epigenomics: new technologies and emerging concepts. Genome Biol 16:103

    Article  PubMed  PubMed Central  Google Scholar 

  9. Chatterjee A (2012) Conference scene: epigenetic regulation: from mechanism to intervention. Epigenomics 4:487–490

    Article  CAS  PubMed  Google Scholar 

  10. Foy JP, Pickering CR, Papadimitrakopoulou VA, Jelinek J, Lin SH, William WN Jr, Frederick MJ, Wang J, Lang W, Feng L, Zhang L, Kim ES, Fan YH, Hong WK, El-Naggar AK, Lee JJ, Myers JN, Issa JP, Lippman SM, Mao L, Saintigny P (2015) New DNA methylation markers and global DNA hypomethylation are associated with oral cancer development. Cancer Prev Res (Phila) 8:1027–1035

    Article  CAS  PubMed Central  Google Scholar 

  11. Blackledge NP, Klose R (2011) CpG island chromatin: a platform for gene regulation. Epigenetics 6:147–152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhou H, Hu H, Lai M (2010) Non-coding RNAs and their epigenetic regulatory mechanisms. Biol Cell 102:645–655

    Article  CAS  PubMed  Google Scholar 

  13. Ziller MJ, Gu H, Müller F, Donaghey J, Tsai LT, Kohlbacher O, De Jager PL, Rosen ED, Bennett DA, Bernstein BE, Gnirke A, Meissner A (2013) Charting a dynamic DNA methylation landscape of the human genome. Nature 500:477–481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xi Y, Bock C, Müller F, Sun D, Meissner A, Li W (2012) RRBSMAP: a fast, accurate and user-friendly alignment tool for reduced representation bisulfite sequencing. Bioinformatics 28:430–432

    Article  CAS  PubMed  Google Scholar 

  15. Chatterjee A, Ozaki Y, Stockwell PA, Horsfield JA, Morison IM, Nakagawa S (2013) Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing. Epigenetics 8:979–989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chatterjee A, Stockwell PA, Rodger EJ, Morison IM (2012) Comparison of alignment software for genome-wide bisulphite sequence data. Nucleic Acids Res 40:e79

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, Gnirke A, Jaenisch R, Lander ES (2008) Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454:766–770

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Smith ZD, Gu H, Bock C, Gnirke A, Meissner A (2009) High-throughput bisulfite sequencing in mammalian genomes. Methods 48:226–232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Baranzini SE, Mudge J, van Velkinburgh JC, Khankhanian P, Khrebtukova I, Miller NA, Zhang L, Farmer AD, Bell CJ, Kim RW, May GD, Woodward JE, Caillier SJ, McElroy JP, Gomez R, Pando MJ, Clendenen LE, Ganusova EE, Schilkey FD, Ramaraj T, Khan OA, Huntley JJ, Luo S, Kwok PY, Wu TD, Schroth GP, Oksenberg JR, Hauser SL, Kingsmore SF (2010) Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis. Nature 464:1351–1356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, Smith ZD, Ziller M, Croft GF, Amoroso MW, Oakley DH, Gnirke A, Eggan K, Meissner A (2011) Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144:439–452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gertz J, Varley KE, Reddy TE, Bowling KM, Pauli F, Parker SL, Kucera KS, Willard HF, Myers RM (2011) Analysis of DNA methylation in a three-generation family reveals widespread genetic influence on epigenetic regulation. PLoS Genet 7:e1002228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gu H, Bock C, Mikkelsen TS, Jäger 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 7:133–136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Smallwood SA, Tomizawa S, Krueger F, Ruf N, Carli N, Segonds-Pichon A, Sato S, Hata K, Andrews SR, Kelsey G (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43:811–814

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Steine EJ, Ehrich M, Bell GW, Raj A, Reddy S, van Oudenaarden A, Jaenisch R, Linhart HG (2011) Genes methylated by DNA methyltransferase 3b are similar in mouse intestine and human colon cancer. J Clin Invest 121:1748–1752

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hartung T, Zhang L, Kanwar R, Khrebtukova I, Reinhardt M, Wang C, Therneau TM, Banck MS, Schroth GP, Beutler AS (2012) Diametrically opposite methylome-transcriptome relationships in high- and low-CpG promoter genes in postmitotic neural rat tissue. Epigenetics 7:421–428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chatterjee A, Stockwell PA, Horsfield JA, Morison IM, Nakagawa S (2014) Base-resolution DNA methylation landscape of zebrafish brain and liver. Genomics Data 2:342–344

    Article  PubMed  PubMed Central  Google Scholar 

  27. Chatterjee A, Rodger EJ, Stockwell PA, Weeks RJ, Morison IM (2012) Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries. J Biomed Biotechnol 2012:741542

    Article  PubMed  PubMed Central  Google Scholar 

  28. Cox MP, Peterson DA, Biggs PJ (2010) SolexaQA: at-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485

    Article  PubMed  PubMed Central  Google Scholar 

  29. Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sun S, Noviski A, Yu X (2013) MethyQA: a pipeline for bisulfite-treated methylation sequencing quality assessment. BMC Bioinformatics 14:259

    Article  PubMed  PubMed Central  Google Scholar 

  31. Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27:1571–1572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Xi Y, Li W (2009) BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics 10:232

    Article  PubMed  PubMed Central  Google Scholar 

  33. Chen PY, Cokus SJ, Pellegrini M (2010) BS Seeker: precise mapping for bisulfite sequencing. BMC Bioinformatics 11:203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lim JQ, Tennakoon C, Li G, Wong E, Ruan Y, Wei CL, Sung WK (2012) BatMeth: improved mapper for bisulfite sequencing reads on DNA methylation. Genome Biol 13:R82

    Article  PubMed  PubMed Central  Google Scholar 

  35. Campagna D, Telatin A, Forcato C, Vitulo N, Valle G (2013) PASS-bis: a bisulfite aligner suitable for whole methylome analysis of illumina and SOLiD reads. Bioinformatics 29:268–270

    Article  CAS  PubMed  Google Scholar 

  36. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rodger EJ, Chatterjee A, Morison IM (2014) 5-hydroxymethylcytosine: a potential therapeutic target in cancer. Epigenomics 6:503–514

    Article  CAS  PubMed  Google Scholar 

  38. Smallwood SA, Kelsey G (2012) Genome-wide analysis of DNA methylation in low cell numbers by reduced representation bisulfite sequencing. Methods Mol Biol 925:187–197

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  40. Hebestreit K, Dugas M, Klein HU (2013) Detection of significantly differentially methylated regions in targeted bisulfite sequencing data. Bioinformatics 29:1647–1653

    Article  CAS  PubMed  Google Scholar 

  41. Stockwell PA, Chatterjee A, Rodger EJ, Morison IM (2014) DMAP: differential methylation analysis package for RRBS and WGBS data. Bioinformatics 30:1814–1822

    Article  CAS  PubMed  Google Scholar 

  42. Chatterjee A, Stockwell PA, Rodger EJ, Duncan EJ, Parry MF, Weeks RJ, Morison IM (2015) Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation. Sci Rep 5:17328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gu H, Smith ZD, Bock C, Boyle P, Gnirke A, Meissner A (2011) Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat Protoc 6:468–481

    Article  CAS  PubMed  Google Scholar 

  44. Akalin A, Garrett-Bakelman FE, Kormaksson M, Busuttil J, Zhang L, Khrebtukova I, Milne TA, Huang Y, Biswas D, Hess JL, Allis CD, Roeder RG, Valk PJ, Löwenberg B, Delwel R, Fernandez HF, Paietta E, Tallman MS, Schroth GP, Mason CE, Melnick A, Figueroa ME (2012) Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia. PLoS Genet 8:e1002781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We gratefully acknowledge the help and support of Dr. Robert Day, Dr. Rebecca Laurie, and Les McNoe of the Otago Genomics and Bioinformatics Facility (OGBF), Dunedin, New Zealand, during the development of this method. This work was supported by Gravida, National Centre for Growth and Development (formerly NRCGD) and the Health Research Council (HRC), New Zealand. A.C. would like to gratefully acknowledge the New Zealand Institute for Cancer Research Trust (NZICRT) for their support.

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Authors and Affiliations

  1. Department of Pathology, Dunedin School of Medicine, University of Otago, 270 Great King Street, P.O. Box 56, Dunedin, 9054, New Zealand

    Aniruddha Chatterjee, Euan J. Rodger, Gwenn Le Mée & Ian M. Morison

  2. Gravida: National Centre for Growth and Development, University of Auckland, 85 Park Road, Grafton, Auckland, New Zealand

    Aniruddha Chatterjee & Ian M. Morison

  3. Department of Biochemistry, University of Otago, 710 Cumberland Street, Dunedin, 9054, New Zealand

    Peter A. Stockwell

Authors
  1. Aniruddha Chatterjee
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  2. Euan J. Rodger
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  3. Peter A. Stockwell
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  4. Gwenn Le Mée
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  5. Ian M. Morison
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Corresponding authors

Correspondence to Aniruddha Chatterjee or Ian M. Morison .

Editor information

Editors and Affiliations

  1. University of Otago Fac. Dentistry, Dunedin, New Zealand

    Gregory J. Seymour

  2. Department of Oral Sciences, University of Otago Faculty of Dentistry, Dunedin, New Zealand

    Mary P. Cullinan

  3. Sir John Walsh Research Institute, University of Otago Faculty of Dentistry, Dunedin, New Zealand

    Nicholas C.K. Heng

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Chatterjee, A., Rodger, E.J., Stockwell, P.A., Le Mée, G., Morison, I.M. (2017). Generating Multiple Base-Resolution DNA Methylomes Using Reduced Representation Bisulfite Sequencing. In: Seymour, G., Cullinan, M., Heng, N. (eds) Oral Biology. Methods in Molecular Biology, vol 1537. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6685-1_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6685-1_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6683-7

  • Online ISBN: 978-1-4939-6685-1

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