ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

DNA Methylation Protocols

Third Edition

Edited by

Jörg Tost

Laboratory for Epigenetics and EnvironmentCentre National de Recherche en Génomique Humaine,

CEA—Institut de Biologie Francois JacobEvry, France

EditorJorg TostLaboratory for Epigenetics and EnvironmentCentre National de Recherche en Genomique HumaineCEA—Institut de Biologie Francois JacobEvry, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-7479-5 ISBN 978-1-4939-7481-8 (eBook)https://doi.org/10.1007/978-1-4939-7481-8

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Cover caption: Merged immunostaining of a mouse brain for 5-methylcytosine, single-stranded DNA and total DNA –Image taken from Chapter 4 of this volume: “Antibody Based Detection of Global Nuclear DNA Methylation in Cells,Tissue Sections and Mammalian Embryos” by Sari Pennings and Nathalie Beaujean et al.

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Preface

It is my great pleasure to introduce the third edition of DNA Methylation: Methods andProtocols to the scientific and medical community.

DNA methylation at cytosines forms one of the multiple layers of epigenetic mechan-isms controlling and modulating gene expression through chromatin structure. It closelyinteracts with histone modifications and chromatin remodeling complexes to form the localgenomic and higher-order chromatin landscape. DNA methylation is essential for propermammalian development, crucial for imprinting, and plays a role in maintaining genomicstability. DNA methylation patterns are susceptible to change in response to environmentalstimuli whereby the epigenome seems to be most vulnerable during early life. Changes ofDNA methylation patterns have been widely studied in several diseases, especially cancer,where interest has focused on biomarkers for early detection of cancer development,accurate diagnosis, and response to treatment, but have also been shown to occur in manyother complex diseases. Recent advances in epigenome engineering technologies allow nowfor the large-scale assessment of the functional relevance of DNA methylation. As a stablenucleic acid-based modification that is technically easy to handle and which can be analyzedwith great reproducibility and accuracy by different laboratories, DNA methylation is apromising biomarker for many applications.

Although by no means complete, this edition of DNA methylation: Methods and Proto-cols gives a comprehensive overview of available technologies together with a detailed step-by-step protocol for all experimental procedures required to successfully perform DNAmethylation analysis. As the degree of difficulty associated with performing these molecularassays is nowadays generally outweighed by the challenges associated with their analysis,many chapters have therefore also included detailed protocols for the analysis of data downto the details of the command lines to be used.

This is the third edition of the DNA methylation protocols, and the field has—again—dramatically changed since the second edition, which I edited six years ago. DNA methyla-tion technologies and our knowledge of DNA methylation patterns have been advancing ata breathtaking pace over the past few years, and many important discoveries have been madedue to technological advances. Most of the techniques described in the second edition havebeen further optimized and/or replaced by novel easier, refined, and/or more quantitativeand resolutive technologies, many of which can now be (and have already been) performedon large cohorts. I have therefore again entirely remodeled and expanded the contents ofthis book, which in this third edition consists now of 35 chapters. Only three chapters havebeen retained from the last edition, and these have been completely rewritten by the authorsto accommodate the changes and improvements made in the last years (Pyrosequencing,MethyLight, and the HELP assays). The selection of different technologies presented in thisvolume enables the analysis of the global DNA methylation content as well as precisequantification of DNA methylation levels on single CpG positions. Methods for the high-resolution analysis of CpG positions within a target region identified by one of the multipleavailable genome-wide technologies are presented, and many of the technologies includedin a recent international study comparing the reliability and performance of locus-specificDNAmethylation assays (Bock et al., Nat. Biotech. 2016) are included in this volume.Whilethe second edition contained a large number of microarray-based analysis methods, they

v

have been completely superseded by sequencing-based approaches. With sequencing costsfurther decreasing, whole genome bisulfite sequencing (WGBS) will soon become afford-able in large cohorts and several protocols with different approaches such as MethylC-seq,PBAT, or tagmentation-based WGBS are presented in this volume. Special focus has beengiven to protocols that enable to start with low amounts of starting material allowing toanalyze the cell-type or developmental-stage-specific DNA methylation patterns. Genome-wide sequencing approaches will lead to a large number of potential candidate genes, andtherefore this volume now includes a novel section detailing methods analyzing a largenumber of target regions in parallel and the interested reader can choose from a variety ofcapture or amplification-based technologies.

For the first time, a chapter is dedicated to the experimental design of DNAmethylationstudies as well as the statistical considerations on their analysis, while a second chapterfocuses on analysis strategies for the highly popular Illumina BeadArrays with the differentparameters to be considered during data analysis and interpretation. Analysis of cell-freeDNA at both the genetic and epigenetic level has attracted recently much interest for thediagnosis and clinical management of cancer and other complex diseases, and this volumeprovides a protocol for the DNA methylation analysis in body fluids. This collection alsocontains a protocol for DNA methylation analysis from archived Guthrie cards potentiallyenabling longitudinal studies and identifying DNA methylation changes prior to diseaseonset. With the increasing interest in analyzing several epigenetic modifications on the samesamples, this volume contains also two chapters describing the integrated analysis of DNAmethylation patterns in immunoprecipitated DNA and thus associated with specific histonemodifications or transcription factor profiles as well as with nucleosome occupancy. As in thelast edition, an introductory chapter summarizes briefly the different biological processesDNA methylation is involved in, the changes that have been described in diseases as well aspotential clinical applications for which DNA methylation analysis might prove to beimportant allowing scientists to catch up with the current level of knowledge and learnabout recent trends.

This volume of the Methods in Molecular Biology series contains widely used methodssuch as Pyrosequencing and methylation-specific PCR as well as protocols for specialapplications such as the hairpin bisulfite sequencing which allows for the assessment of thesymmetry of DNA methylation in specific regions. Furthermore, research of the last yearshas shown that cytosine methylation is not the only DNA modification and that especially5-hydroxymethylation is not only an intermediate in oxidative DNA demethylation, butconstitutes a distinct layer in the complex process of epigenetic regulation with its owndistribution and regulatory functions. As bisulfite-based technologies are not able to distin-guish between these two modifications, special protocols have been developed and several ofthem are presented in the last section of this book.

This book is addressed to postdoctoral investigators and research scientists that areimplicated in the different aspects of genetics and cellular and molecular biology as well as toclinicians involved in diagnostics or choice of treatment of diseases that have an epigeneticcomponent, which nowadays means most biological and clinical questions. The presentationin this volume is equally suited for laboratories that already have a great deal of expertise in acertain technology to analyze DNA methylation, but might want to obtain other orcomplementary data using an orthogonal technique, and for genetics/genomics/biologygroups that want to initiate research in this exciting area and want to identify the methodbest suited to answer their question. Notes and tips from experts of the different methodswill enable a rapid implementation of the different protocols in the laboratory and avoid

vi Preface

time-consuming and cost-intensive mistakes. With the tools and protocols available, ourknowledge and understanding of DNA methylation will continue to increase rapidly withnew groundbreaking and exciting discoveries to come, and this book will contribute tospreading of the “savoir faire” to analyze DNA methylation.

I am indebted to all the authors for their hard work and outstanding contributions tothis third edition of DNAMethylation Protocols. It was a pleasure to work with them on thisproject. I hope that the protocols described in detail in this volume will help to accelerate theanalysis and description of the “methylome” of different species, enhance our understandingof the molecular processes that determine the genomic DNA methylation landscape as wellas provide robust technologies for the clinical implementation of DNA methylation-basedbiomarkers.

Evry, France Jorg Tost

Preface vii

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I INTRODUCTION

1 A Summary of the Biological Processes, Disease-Associated Changes,and Clinical Applications of DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Gitte Brinch Andersen and Jorg Tost

2 Considerations for Design and Analysis of DNA Methylation Studies . . . . . . . . . 31Karin B. Michels and Alexandra M. Binder

PART II GLOBAL DNA METHYLATION LEVELS

3 Quantification of Global DNA Methylation Levels by MassSpectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Agustin F. Fernandez, Luis Valledor, Fernando Vallejo, Maria Jesus Canal,and Mario F. Fraga

4 Antibody-Based Detection of Global Nuclear DNA Methylation in Cells,Tissue Sections, and Mammalian Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Nathalie Beaujean, Juliette Salvaing, Nur Annies Abd Hadi,and Sari Pennings

PART III GENOME-WIDE DNA METHYLATION ANALYSIS

5 Whole-Genome Bisulfite Sequencing Using the Ovation® UltralowMethyl-Seq Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Christian Daviaud, Victor Renault, Florence Mauger, Jean-FrancoisDeleuze, and Jorg Tost

6 Tagmentation-Based Library Preparation for Low DNA InputWhole Genome Bisulfite Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105Dieter Weichenhan, Qi Wang, Andrew Adey, Stephan Wolf, Jay Shendure,Roland Eils, and Christoph Plass

7 Post-Bisulfite Adaptor Tagging for PCR-Free Whole-Genome BisulfiteSequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Fumihito Miura and Takashi Ito

8 Multiplexed Reduced Representation Bisulfite Sequencing with MagneticBead Fragment Size Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137William P. Accomando Jr. and Karin B. Michels

9 Low Input Whole-Genome Bisulfite Sequencing Using a Post-BisulfiteAdapter Tagging Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Julian R. Peat and Sebastien A. Smallwood

ix

10 Methyl-CpG-Binding Domain Sequencing: MBD-seq. . . . . . . . . . . . . . . . . . . . . . . 171Karolina A. Aberg, Robin F. Chan, Linying Xie, Andrey A. Shabalin,and Edwin J.C.G. van den Oord

11 The HELP-Based DNA Methylation Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191John M. Greally

12 Comprehensive Whole DNA Methylome Analysis by IntegratingMeDIP-seq and MRE-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Xiaoyun Xing, Bo Zhang, Daofeng Li, and Ting Wang

13 Digital Restriction Enzyme Analysis of Methylation (DREAM). . . . . . . . . . . . . . . 247Jaroslav Jelinek, Justin T. Lee, Matteo Cesaroni, Jozef Madzo,Shoudan Liang, Yue Lu, and Jean-Pierre J. Issa

14 Nucleosome Occupancy and Methylome Sequencing (NOMe-seq) . . . . . . . . . . . 267Fides D. Lay, Theresa K. Kelly, and Peter A. Jones

15 Bisulphite Sequencing of Chromatin Immunoprecipitated DNA(BisChIP-seq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285Clare Stirzaker, Jenny Z. Song, Aaron L. Statham, and Susan J. Clark

16 A Guide to Illumina BeadChip Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303Michael C. Wu and Pei-Fen Kuan

PART IV ANALYSIS OF HIGHLY MULTIPLEXED TARGET REGIONS

17 Microdroplet PCR for Highly Multiplexed Targeted BisulfiteSequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333H. Kiyomi Komori, Sarah A. LaMere, Traver Hart, Steven R. Head,Ali Torkamani, and Daniel R. Salomon

18 Multiplexed DNA Methylation Analysis of Target Regions UsingMicrofluidics (Fluidigm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349Martyna Adamowicz, Klio Maratou, and Timothy J. Aitman

19 Large-Scale Targeted DNA Methylation Analysis Using BisulfitePadlock Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365Dinh Diep, Nongluk Plongthongkum, and Kun Zhang

20 Targeted Bisulfite Sequencing Using the SeqCap Epi EnrichmentSystem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383Jennifer Wendt, Heidi Rosenbaum, Todd A. Richmond, Jeffrey A. Jeddeloh,and Daniel L. Burgess

21 Multiplexed and Sensitive DNA Methylation Testing UsingMethylation-Sensitive Restriction Enzymes “MSRE-qPCR” . . . . . . . . . . . . . . . . . 407Gabriel Beikircher, Walter Pulverer, Manuela Hofner, Christa Noehammer,and Andreas Weinhaeusel

PART V LOCUS-SPECIFIC DNA METHYLATION ANALYSIS

22 Quantitative DNA Methylation Analysis at Single-Nucleotide Resolutionby Pyrosequencing® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427Florence Busato, Emelyne Dejeux, Hafida El abdalaoui, Ivo Glynne Gut,and Jorg Tost

x Contents

23 Methylation-Specific PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447Joao Ramalho-Carvalho, Rui Henrique, and Carmen Jer�onimo

24 Quantitation of DNA Methylation by Quantitative MultiplexMethylation-Specific PCR (QM-MSP) Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Mary Jo Fackler and Saraswati Sukumar

25 MethyLight and Digital MethyLight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497Mihaela Campan, Daniel J. Weisenberger, Binh Trinh, and Peter W. Laird

26 Quantitative Region-Specific DNA Methylation Analysis by theEpiTYPER™ Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515Sonja Kunze

27 Methylation-Specific Multiplex Ligation-Dependent Probe Amplification(MS-MLPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537Cathy B. Moelans, Lilit Atanesyan, Suvi P. Savola, and Paul J. van Diest

28 Methylation-Sensitive High Resolution Melting (MS-HRM). . . . . . . . . . . . . . . . . 551Dianna Hussmann and Lise Lotte Hansen

29 Hairpin Bisulfite Sequencing: Synchronous Methylation Analysison Complementary DNA Strands of Individual Chromosomes . . . . . . . . . . . . . . . 573Pascal Giehr and Jorn Walter

30 Helper-Dependent Chain Reaction (HDCR) for Selective Amplificationof Methylated DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587Susan M. Mitchell, Keith N. Rand, Zheng-Zhou Xu, Thu Ho,Glenn S. Brown, Jason P. Ross, and Peter L. Molloy

PART VI DNA METHYLATION ANALYSIS OF SPECIFIC BIOLOGICAL SAMPLES

31 DNA Methylation Analysis from Blood Spots: Increasing Yieldand Quality for Genome-Wide and Locus-Specific Methylation Analysis . . . . . . . 605Akram Ghantous, Hector Hernandez-Vargas, and Zdenko Herceg

32 DNA Methylation Analysis of Free-Circulating DNA in Body Fluids . . . . . . . . . . 621Maria Jung, Glen Kristiansen, and Dimo Dietrich

PART VII HYDROXYMETHYLATION

33 Tet-Assisted Bisulfite Sequencing (TAB-seq) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645Miao Yu, Dali Han, Gary C. Hon, and Chuan He

34 Multiplexing for Oxidative Bisulfite Sequencing (oxBS-seq). . . . . . . . . . . . . . . . . . 665Kristina Kirschner, Felix Krueger, Anthony R. Green, and Tamir Chandra

35 Affinity-Based Enrichment Techniques for the Genome-WideAnalysis of 5-Hydroxymethylcytosine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679John P. Thomson and Richard R. Meehan

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 697

Contents xi

Contributors

KAROLINA A. ABERG � Center for Biomarker Research and Precision Medicine, VirginiaCommonwealth University, Richmond, VA, USA

WILLIAM P. ACCOMANDO JR � Department of Epidemiology, Harvard School of Public Health,Boston, MA, USA; Department of Obstetrics, Gynecology, and Reproductive Biology;Obstetrics and Gynecology Epidemiology Center, Brigham and Women’s Hospital, Boston,MA, USA

MARTYNA ADAMOWICZ � MRC Clinical Sciences Centre, Faculty of Medicine, ImperialCollege London, London, UK

ANDREWADEY � Department of Molecular and Medical Genetics, Oregon Health and ScienceUniversity, Portland, OR, USA; The Knight Cardiovascular Institute, Oregon Health andScience University, Portland, OR, USA

TIMOTHY J. AITMAN � MRC Clinical Sciences Centre, Faculty of Medicine, Imperial CollegeLondon, London, UK; Institute of Genetics and Molecular Medicine, University ofEdinburgh, Edinburgh, UK

GITTE BRINCH ANDERSEN � Department of Biomedicine, Aarhus University, Aarhus,Denmark; Laboratory for Epigenetics and Environment, Centre National de Recherche enGenomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

LILIT ATANESYAN � MRC-Holland, Amsterdam, The NetherlandsNATHALIE BEAUJEAN � INRA, UMR1198 Biologie du Developpement et Reproduction, Jouy-

en-Josas, France; Univ Lyon, Universite Claude Bernard Lyon 1, Inserm, INRA, Stem Celland Brain Research Institute U1208, USC1361, Bron, France

GABRIEL BEIKIRCHER � Molecular Diagnostics Unit, AIT–Austrian Institute of TechnologyGmbH, Vienna, Austria

ALEXANDRA M. BINDER � Department of Epidemiology, Fielding School of Public Health,University of California, Los Angeles, CA, USA

GLENN S. BROWN � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaDANIEL L. BURGESS � Roche Sequencing Solutions, Madison, WI, USAFLORENCE BUSATO � Laboratory for Epigenetics and Environment, Centre National de

Recherche en Genomique Humaine, CEA–Institut de Biologie Francois Jacob, Evry, FranceMARIA JESUS CANAL � Area de Fisiologıa Vegetal, Dpto. Biologıa de Organismos y Sistemas,

Universidad de Oviedo, Oviedo, SpainMIHAELA CAMPAN � Clinical Sciences Building, University Hospital, Keck School of Medicine,

University of Southern California, Los Angeles, CA, USAMATTEO CESARONI � Fels Institute for Cancer Research and Molecular Biology, Temple

University School of Medicine, Philadelphia, PA, USAROBIN F. CHAN � Center for Biomarker Research and Precision Medicine, Virginia

Commonwealth University, Richmond, VA, USATAMIR CHANDRA � Epigenetics ISP, The Babraham Institute, Cambridge, UK; The Wellcome

Trust Sanger Institute, Cambridge, UK; The Institute of Genetics andMolecular Medicine,University of Edinburgh, Edinburgh, UK

SUSAN J. CLARK � Epigenetics Research Laboratory, Genomics and Epigenetics Division,Garvan Institute of Medical Research, Sydney, NSW, Australia; St. Vincent’s ClinicalSchool, University of NSW, Sydney, NSW, Australia

xiii

CHRISTIAN DAVIAUD � Laboratory for Epigenetics and Environment, Centre National deRecherche en Genomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

EMELYNE DEJEUX � Laboratory for Epigenetics and Environment, Centre National deRecherche en Genomique Humaine, CEA–Institut de Biologie Francois Jacob, Evry, France

JEAN-FRANCOIS DELEUZE � Laboratory for Epigenetics and Environment, Centre National deRecherche en Genomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France;Laboratory for Bioinformatics, Fondation Jean Dausset – CEPH, Paris, France

DINH DIEP � Department of Bioengineering, University of California at San Diego, La Jolla,CA, USA

DIMO DIETRICH � Institute of Pathology, University Hospital Bonn (UKB), Bonn, GermanyPAUL J. VAN DIEST � Department of Pathology, University Medical Centre Utrecht, Utrecht,

The NetherlandsROLAND EILS � Theoretical Bioinformatics, German Cancer Research Center (DKFZ),

Heidelberg, GermanyHAFIDA EL ABDALAOUI � Laboratory for Epigenetics and Environment, Centre National de

Recherche en Genomique Humaine, CEA–Institut de Biologie Francois Jacob, Evry, FranceMARY JO FACKLER � Breast and Ovarian Cancer Program, Sidney Kimmel Comprehensive

Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USAAGUSTIN F. FERNANDEZ � Cancer Epigenetics Laboratory, Institute of Oncology of Asturias

(IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain; Fundacion para laInvestigacion Biosanitaria de Asturias (FINBA), Grupo de Epigenetica del Cancer yNanomedicina, Instituto de Investigacion Sanitaria del Principado de Asturias (IISPA),Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain

MARIO F. FRAGA � Cancer Epigenetics Laboratory, Institute of Oncology of Asturias(IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain; Department of Immunologyand Oncology, National Center for Biotechnology, CNB-CSIC, Madrid, Spain; Fundacionpara la Investigacion Biosanitaria de Asturias (FINBA), Grupo de Epigenetica del Cancery Nanomedicina, Instituto de Investigacion Sanitaria del Principado de Asturias (IISPA),Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain

AKRAM GHANTOUS � Epigenetics Group, International Agency for Research on Cancer(IARC), Lyon, France

PASCAL GIEHR � Department of Biological Sciences, Genetics/Epigenetics, SaarlandUniversity, Saarbrucken, Saarland, Germany

JOHN M. GREALLY � Department of Genetics, Albert Einstein College of Medicine of YeshivaUniversity, Bronx, NY, USA

ANTHONY R. GREEN � Cambridge Institute for Medical Research, University of Cambridge,Cambridge, UK; Department of Haematology, University of Cambridge, Cambridge, UK;Stem Cell Institute, University of Cambridge, Cambridge, UK; Department ofHaematology, Addenbrooke’s Hospital, Cambridge, UK

IVO GLYNNE GUT � Biomedical Genomics Group, Centro Nacional de Analisis Genomico,CNAG-CRG, Center for Genomic Regulation, Barcelona Institute for Science andTechnology, Barcelona, Spain

NUR ANNIES ABD HADI � Centre for Cardiovascular Science, Queen’s Medical ResearchInstitute, University of Edinburgh, Edinburgh, UK

DALI HAN � Department of Chemistry and Institute for Biophysical Dynamics, TheUniversity of Chicago, Chicago, IL, USA; Howard Hughes Medical Institute, TheUniversity of Chicago, Chicago, IL, USA

LISE LOTTE HANSEN � Institute of Biomedicine, Aarhus University, Aarhus C, Denmark

xiv Contributors

TRAVER HART � Donnelly Centre and Banting and Best Department of Medical Research,University of Toronto, Toronto, ON, Canada

CHUAN HE � Department of Chemistry and Institute for Biophysical Dynamics,The University of Chicago, Chicago, IL, USA; Howard Hughes Medical Institute,The University of Chicago, Chicago, IL, USA

STEVEN R. HEAD � Next Generation Sequencing Core, The Scripps Research Institute,La Jolla, CA, USA

RUI HENRIQUE � Cancer Biology and Epigenetics Group, Research Center (CI-IPOP),Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal; Department ofPathology, Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal;Department of Pathology andMolecular Immunology, Institute of Biomedical Sciences AbelSalazar, University of Porto (ICBAS-UP), Porto, Portugal

ZDENKO HERCEG � Epigenetics Group, International Agency for Research on Cancer(IARC), Lyon, France

HECTOR HERNANDEZ-VARGAS � Epigenetics Group, International Agency for Research onCancer (IARC), Lyon, France

THU HO � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaMANUELA HOFNER � Molecular Diagnostics Unit, AIT–Austrian Institute of Technology

GmbH, Vienna, AustriaGARY C. HON � University of Texas Southwestern Medical Center, Dallas, TX, USADIANNA HUSSMANN � Institute of Biomedicine, Aarhus University, Aarhus C, DenmarkJEAN-PIERRE J. ISSA � Fels Institute for Cancer Research and Molecular Biology, Temple

University School of Medicine, Philadelphia, PA, USATAKASHI ITO � Department of Biochemistry, Kyushu University Graduate School of Medical

Sciences, Fukuoka, Japan; Core Research for Evolutional Science and Technology (CREST),Japan Agency for Medical Research and Development (AMED), Fukuoka, Japan

JEFFREY A. JEDDELOH � Roche Sequencing Solutions, Madison, WI, USAJAROSLAV JELINEK � Fels Institute for Cancer Research and Molecular Biology, Temple

University School of Medicine, Philadelphia, PA, USACARMEN JERONIMO � Cancer Biology and Epigenetics Group; Research Center (CI-IPOP),

Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal; Department ofPathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar,University of Porto (ICBAS-UP), Porto, Portugal; Portuguese Oncology Institute of Porto,Porto, Portugal

PETER A. JONES � Department of Biochemistry and Molecular Biology, Norris ComprehensiveCancer Center, Keck School of Medicine, University of Southern California, Los Angeles,CA, USA; Van Andel Institute, Grand Rapids, MI, USA

MARIA JUNG � Institute of Pathology, University Hospital Bonn (UKB), Bonn, GermanyTHERESA K. KELLY � Active Motif, Carlsbad, CA, USAKRISTINA KIRSCHNER � Cambridge Institute for Medical Research, University of Cambridge,

Cambridge, UK; Department of Haematology, University of Cambridge, Cambridge, UK;Stem Cell Institute, University of Cambridge, Cambridge, UK

H. KIYOMI KOMORI � Department of Molecular and Experimental Medicine, The ScrippsResearch Institute, La Jolla, CA, USA

GLEN KRISTIANSEN � Institute of Pathology, University Hospital Bonn (UKB), Bonn,Germany

FELIX KRUEGER � Bioinformatics, The Babraham Institute, Cambridge, UK

Contributors xv

PEI-FEN KUAN � Department of Applied Mathematics and Statistics, Stony Brook University,Stony Brook, NY, USA

SONJA KUNZE � Research Unit of Molecular Epidemiology/Institute of Epidemiology II,Helmholtz Zentrum Munchen, Deutsches Forschungszentrum fur Gesundheit und Umwelt(GmbH), Neuherberg, Germany

PETER W. LAIRD � Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI,USA

SARAH A. LAMERE � Department of Molecular and Experimental Medicine, The ScrippsResearch Institute, La Jolla, CA, USA

FIDES D. LAY � Department of Biochemistry and Molecular Biology, Norris ComprehensiveCancer Center, Keck School of Medicine, University of Southern California, Los Angeles,CA, USA; Program in Genetic, Molecular and Cellular Biology, Keck School of Medicine,University of Southern California, Los Angeles, CA, USA; Department of Molecular, Celland Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA

JUSTIN T. LEE � Fels Institute for Cancer Research and Molecular Biology, Temple UniversitySchool of Medicine, Philadelphia, PA, USA

DAOFENG LI � Department of Genetics, The Edison Family Center for Genome Sciences andSystems Biology, Washington University, St. Louis, MO, USA

SHOUDAN LIANG � Department of Bioinformatics and Computational Biology, TheUniversity of Texas MD Anderson Cancer Center, Houston, TX, USA

YUE LU � Department of Molecular Carcinogenesis, The University of Texas MD AndersonCancer Center, Smithville, TX, USA

JOZEF MADZO � Fels Institute for Cancer Research and Molecular Biology, Temple UniversitySchool of Medicine, Philadelphia, PA, USA

KLIO MARATOU � MRC Clinical Sciences Centre, Faculty of Medicine, Imperial CollegeLondon, London, UK

FLORENCE MAUGER � Laboratory for Epigenetics and Environment, Centre National deRecherche en Genomique Humaine, CEA-Institut de Biologie Francois Jacob, Evry, France

RICHARD R. MEEHAN � MRC Human Genetics Unit, Institute of Genetics and MolecularMedicine, University of Edinburgh, Edinburgh, UK

KARIN B. MICHELS � Department of Epidemiology, Fielding School of Public Health,University of California, Los Angeles, CA, USA

SUSAN M. MITCHELL � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaFUMIHITO MIURA � Department of Biochemistry, Kyushu University Graduate School of

Medical Sciences, Fukuoka, Japan; Core Research for Evolutional Science and Technology(CREST), Japan Agency for Medical Research and Development (AMED), Fukuoka,Japan; Precursory Research for Embryonic Science and Technology (PRESTO), JapanScience and Technology Agency (JST), Fukuoka, Japan

CATHY B. MOELANS � Department of Pathology, University Medical Centre Utrecht, Utrecht,The Netherlands

PETER L. MOLLOY � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaCHRISTA NOEHAMMER � Molecular Diagnostics Unit, AIT–Austrian Institute of Technology

GmbH, Vienna, AustriaEDWIN J.C.G. VAN DEN OORD � Center for Biomarker Research and Precision Medicine,

Virginia Commonwealth University, Richmond, VA, USAJULIAN R. PEAT � Epigenetics Programme, Babraham Institute, Cambridge, UKSARI PENNINGS � Centre for Cardiovascular Science, Queen’s Medical Research Institute,

University of Edinburgh, Edinburgh, United Kingdom

xvi Contributors

CHRISTOPH PLASS � Division of Epigenomics and Cancer Risk Factors, German CancerResearch Center (DKFZ), Heidelberg, Germany

NONGLUK PLONGTHONGKUM � Department of Bioengineering, University of California atSan Diego, La Jolla, CA, USA

WALTER PULVERER � Molecular Diagnostics Unit, AIT–Austrian Institute of TechnologyGmbH, Vienna, Austria

JOAO RAMALHO-CARVALHO � Cancer Biology and Epigenetics Group; Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal; BiomedicalSciences Graduate Program, Institute of Biomedical Sciences Abel Salazar, University ofPorto (ICBAS-UP), Porto, Portugal

KEITH N. RAND � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaVICTOR RENAULT � Laboratory for Bioinformatics, Fondation Jean Dausset – CEPH, Paris,

FranceTODD A. RICHMOND � Roche Sequencing Solutions, Madison, WI, USAHEIDI ROSENBAUM � Roche Sequencing Solutions, Madison, WI, USAJASON P. ROSS � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaDANIEL R. SALOMON (DECEASED) � Department of Molecular and Experimental Medicine,

The Scripps Research Institute, La Jolla, CA, USAJULIETTE SALVAING � INRA, UMR1198 Biologie du Developpement et Reproduction, Jouy-

en-Josas, France; Univ. Grenoble Alpes, INRA, CEA, CNRS, BIG-LPCV, Grenoble,France

SUVI P. SAVOLA � MRC-Holland, Amsterdam, The NetherlandsANDREY A. SHABALIN � Center for Biomarker Research and Precision Medicine, Virginia

Commonwealth University, Richmond, VA, USAJAY SHENDURE � Department of Genome Sciences, University of Washington, Seattle, WA,

USASEBASTIEN A. SMALLWOOD � Friedrich Miescher Institute for Biomedical Research, Basel,

Switzerland; Epigenetics Programme, Babraham Institute, Cambridge, UKJENNY Z. SONG � Epigenetics Research Laboratory, Genomics and Epigenetics Division,

Garvan Institute of Medical Research, Sydney, NSW, AustraliaAARON L. STATHAM � Epigenetics Research Laboratory, Genomics and Epigenetics Division,

Garvan Institute of Medical Research, Sydney, NSW, AustraliaCLARE STIRZAKER � Epigenetics Research Laboratory, Genomics and Epigenetics Division,

Garvan Institute of Medical Research, Sydney, NSW, Australia; St. Vincent’s ClinicalSchool, University of NSW, Sydney, NSW, Australia

SARASWATI SUKUMAR � Breast and Ovarian Cancer Program, Sidney Kimmel ComprehensiveCancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

JOHN P. THOMSON � MRC Human Genetics Unit, Institute of Genetics and MolecularMedicine, University of Edinburgh, Edinburgh, UK

ALI TORKAMANI � Department of Molecular and Experimental Medicine, The ScrippsResearch Institute, La Jolla, CA, USA

JORG TOST � Laboratory for Epigenetics and Environment, Centre National de Recherche enGenomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

BINH TRINH � UCSF School of Medicine, University of California, San Francisco, CA, USALUIS VALLEDOR � Area de Fisiologıa Vegetal, Dpto. Biologıa de Organismos y Sistemas,

Universidad de Oviedo, Oviedo, SpainFERNANDO VALLEJO � Centro de Edafologıa y Biologıa Aplicada del Segura (CEBAS-CSIC),

Campus Universitario de Espinardo, Murcia, Spain

Contributors xvii

JORN WALTER � Department of Biological Sciences, Genetics/Epigenetics, SaarlandUniversity, Saarbrucken, Saarland, Germany

QI WANG � Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg,Germany; Division of Applied Bioinformatics, German Cancer Research Center (DKFZ),and German Cancer Consortium (DKTK), Heidelberg, Germany

TING WANG � Department of Genetics, The Edison Family Center for Genome Sciences andSystems Biology, Washington University, St. Louis, MO, USA

DIETER WEICHENHAN � Division of Epigenomics and Cancer Risk Factors, German CancerResearch Center (DKFZ), Heidelberg, Germany

ANDREAS WEINHAEUSEL � Molecular Diagnostics Unit, AIT–Austrian Institute of TechnologyGmbH, Vienna, Austria

DANIEL J. WEISENBERGER � Department of Biochemistry and Molecular Biology, USC NorrisComprehensive Cancer Center, Keck School of Medicine, University of Southern California,Los Angeles, CA, USA

JENNIFER WENDT � Roche Sequencing Solutions, Madison, WI, USASTEPHAN WOLF � Core Facility High Throughput Sequencing, German Cancer Research

Center (DKFZ), Heidelberg, GermanyMICHAEL C. WU � Public Health Sciences Division, Fred Hutchinson Cancer Research

Center, Seattle, WA, USALINYING XIE � Center for Biomarker Research and Precision Medicine, Virginia

Commonwealth University, Richmond, VA, USAXIAOYUN XING � Department of Genetics, The Edison Family Center for Genome Sciences and

Systems Biology, Washington University, St. Louis, MO, USAZHENG-ZHOU XU � CSIRO Food and Nutrition Flagship, North Ryde, NSW, AustraliaMIAO YU � Ludwig Institute for Cancer Research, La Jolla, CA, USABO ZHANG � Department of Genetics, The Edison Family Center for Genome Sciences and

Systems Biology, Washington University, St. Louis, MO, USAKUN ZHANG � Department of Bioengineering, University of California at San Diego, La

Jolla, CA, USA

xviii Contributors