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Whole-Genome Bisulfite Sequencing for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution

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Epigenome Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1767))

Abstract

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and disease-associated investigations. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered as the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here we provide two detailed protocols based on commercial kits for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. If only DNA methylation is of interest, sequencing libraries can be constructed from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For samples with significant levels of hydroxymethylation such as stem cells or brain tissue, we describe the protocol of oxidative bisulfite sequencing (OxBs-seq), which in its current version uses a post-bisulfite adaptor tagging (PBAT) approach. Two methylomes need to be generated: a classic methylome following bisulfite conversion and analyzing both methylated and hydroxymethylated cytosines and a methylome analyzing only methylated cytosines, respectively. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocols have been successfully applied to different human samples and yield robust and reproducible results.

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References

  1. How Kit A, Nielsen HM, Tost J (2012) DNA methylation based biomarkers: practical considerations and applications. Biochimie 94:2314–2337

    Article  CAS  Google Scholar 

  2. Baylin SB, Jones PA (2011) A decade of exploring the cancer epigenome – biological and translational implications. Nat Rev Cancer 11:726–734

    Article  CAS  Google Scholar 

  3. Lardenoije R, Iatrou A, Kenis G et al (2015) The epigenetics of aging and neurodegeneration. Prog Neurobiol 131:21–64

    Article  CAS  Google Scholar 

  4. Nielsen HM, Tost J (2012) Epigenetic changes in inflammatory and autoimmune diseases. Subcell Biochem 61:455–478

    Article  Google Scholar 

  5. Harb H, Renz H (2015) Update on epigenetics in allergic disease. J Allergy Clin Immunol 135:15–24

    Article  CAS  Google Scholar 

  6. Zhang Z, Zhang R (2015) Epigenetics in autoimmune diseases: pathogenesis and prospects for therapy. Autoimmun Rev 14:854–863

    Article  CAS  Google Scholar 

  7. Shorter KR, Miller BH (2015) Epigenetic mechanisms in schizophrenia. Prog Biophys Mol Biol 118:1–7

    Article  CAS  Google Scholar 

  8. Abdolmaleky HM, Zhou JR, Thiagalingam S (2015) An update on the epigenetics of psychotic diseases and autism. Epigenomics 7:427–449

    Article  CAS  Google Scholar 

  9. Ronn T, Ling C (2015) DNA methylation as a diagnostic and therapeutic target in the battle against type 2 diabetes. Epigenomics 7:451–460

    Article  Google Scholar 

  10. Urich MA, Nery JR, Lister R et al (2015) MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10:475–483

    Article  CAS  Google Scholar 

  11. Kobayashi H, Sakurai T, Imai M et al (2012) Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet 8:e1002440

    Article  CAS  Google Scholar 

  12. Adusumalli S, Mohd Omar MF, Soong R et al (2015) Methodological aspects of whole-genome bisulfite sequencing analysis. Brief Bioinform 16:369–379

    Article  CAS  Google Scholar 

  13. Lister R, Pelizzola M, Dowen RH et al (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315–322

    Article  CAS  Google Scholar 

  14. Lister R, O'Malley RC, Tonti-Filippini J et al (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133:523–536

    Article  CAS  Google Scholar 

  15. Lister R, Pelizzola M, Kida YS et al (2011) Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471:68–73

    Article  CAS  Google Scholar 

  16. Li Y, Zhu J, Tian G et al (2010) The DNA methylome of human peripheral blood mononuclear cells. PLoS Biol 8:e1000533

    Article  Google Scholar 

  17. Chalhoub B, Denoeud F, Liu S et al (2014) Plant genetics. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953

    Article  CAS  Google Scholar 

  18. Lyko F, Foret S, Kucharski R et al (2010) The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol 8:e1000506

    Article  Google Scholar 

  19. Guo JU, Su Y, Shin JH et al (2014) Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat Neurosci 17:215–222

    Article  CAS  Google Scholar 

  20. Adey A, Shendure J (2012) Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Res 22:1139–1143

    Article  CAS  Google Scholar 

  21. Miura F, Enomoto Y, Dairiki R et al (2012) Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res 40:e136

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935

    Article  CAS  Google Scholar 

  24. Tan L, Shi YG (2012) Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139:1895–1902

    Article  CAS  Google Scholar 

  25. Ludwig AK, Zhang P, Cardoso MC (2016) Modifiers and readers of DNA modifications and their impact on genome structure, expression, and stability in disease. Front Genet 7:115

    Article  Google Scholar 

  26. Wu SC, Zhang Y (2010) Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11:607–620

    Article  CAS  Google Scholar 

  27. Ito S, Shen L, Dai Q et al (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333:1300–1303

    Article  CAS  Google Scholar 

  28. Nestor CE, Ottaviano R, Reddington J et al (2012) Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res 22:467–477

    Article  CAS  Google Scholar 

  29. Langemeijer SM, Aslanyan MG, Jansen JH (2009) TET proteins in malignant hematopoiesis. Cell Cycle 8:4044–4048

    Article  CAS  Google Scholar 

  30. Pastor WA, Pape UJ, Huang Y et al (2011) Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473:394–397

    Article  CAS  Google Scholar 

  31. Ficz G, Branco MR, Seisenberger S et al (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473:398–402

    Article  CAS  Google Scholar 

  32. Jin SG, Wu X, Li AX et al (2011) Genomic mapping of 5-hydroxymethylcytosine in the human brain. Nucleic Acids Res 39:5015–5024

    Article  CAS  Google Scholar 

  33. Ono R, Taki T, Taketani T et al (2002) LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23). Cancer Res 62:4075–4080

    CAS  PubMed  Google Scholar 

  34. Mercher T, Quivoron C, Couronne L et al (2012) TET2, a tumor suppressor in hematological disorders. Biochim Biophys Acta 1825:173–177

    CAS  PubMed  Google Scholar 

  35. Putiri EL, Tiedemann RL, Thompson JJ et al (2014) Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells. Genome Biol 15:R81

    Article  Google Scholar 

  36. Jin SG, Jiang Y, Qiu R et al (2011) 5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations. Cancer Res 71:7360–7365

    Article  CAS  Google Scholar 

  37. Booth MJ, Branco MR, Ficz G et al (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336:934–937

    Article  CAS  Google Scholar 

  38. Yu M, Hon GC, Szulwach KE et al (2012) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149:1368–1380

    Article  CAS  Google Scholar 

  39. Skvortsova K, Zotenko E, Luu PL et al (2017) Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA. Epigenetics Chromatin 10:16

    Article  Google Scholar 

  40. Song CX, Szulwach KE, Fu Y et al (2011) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29:68–72

    Article  CAS  Google Scholar 

  41. Daviaud C, Renault V, Mauger F et al (2018) Whole-genome bisulfite sequencing using the Ovation® Ultralow Methyl-Seq protocol. Methods Mol Biol 1708:83–104

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079

    Article  Google Scholar 

  44. Akalin A, Kormaksson M, Li S et al (2012) methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol 13:R87

    Article  Google Scholar 

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Acknowledgments

The protocol for hydroxymethylation analysis has been set up in the laboratory of Jörg Tost in the framework of the ANR-BMBF-funded project “Epigenomics of Parkinson’s Disease” (EpiPD, ANR-13-EPIG-0003-05). Further work is supported by grants from the ANR (ANR-13-CESA-0011-05), Aviesan/INSERM (EPlGl2014-18 and EPIG2014-01), INCa (PRT-K14-049), a Sirius research award (UCB Pharma S.A.), a Passerelle research award (Pfizer), iCARE (MSD Avenir), and the institutional budget of the CNRGH.

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Author notes

  1. Magali Kernaleguen and Christian Daviaud have contributed equally and should be considered as joint first authors.

Authors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

    Magali Kernaleguen, Christian Daviaud, Yimin Shen, Jean-François Deleuze, Florence Mauger & Jörg Tost

  2. Laboratory for Bio-analysis, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

    Eric Bonnet

  3. Laboratory for Bioinformatics, Fondation Jean Dausset – CEPH, Paris, France

    Victor Renault & Jean-François Deleuze

Authors
  1. Magali Kernaleguen
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  2. Christian Daviaud
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  3. Yimin Shen
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  4. Eric Bonnet
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  5. Victor Renault
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  6. Jean-François Deleuze
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  7. Florence Mauger
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  8. Jörg Tost
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Corresponding author

Correspondence to Jörg Tost .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, Stuttgart, Germany

    Albert Jeltsch

  2. Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

    Marianne G. Rots

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Kernaleguen, M. et al. (2018). Whole-Genome Bisulfite Sequencing for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution. In: Jeltsch, A., Rots, M. (eds) Epigenome Editing. Methods in Molecular Biology, vol 1767. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7774-1_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7774-1_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7773-4

  • Online ISBN: 978-1-4939-7774-1

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