batman

ABayesiandeconvolutionstrategyforimmunoprecipitation-basedDNAmethylomeanalysisThomasADown1,8,VardhmanKRakyan2,8,DanielJTurner3,PaulFlicek4,HengLi3,EugeneKulesha4,StefanGra¨f4,NathanJohnson4,JavierHerrero4,EleniMTomazou3,NataliePThorne5,LiselotteBa¨ckdahl6,MarlisHerberth7,KevinLHowe5,DavidKJackson3,MarcosMMiretti3,JohnCMarioni5,EwanBirney4,TimJPHubbard3,RichardDurbin3,SimonTavare´5&StephanBeck6DNAmethylationisanindispensibleepigeneticmodificationrequiredforregulatingtheexpressionofmammaliangenomes.Immunoprecipitation-basedmethodsforDNAmethylomeanalysisarerapidlyshiftingthebottleneckinthisfieldfromdatagenerationtodataanalysis,necessitatingthedevelopmentofbetteranalyticaltools.Inparticular,anin-abilitytoestimateabsolutemethylationlevelsremainsamajoranalyticaldifficultyassociatedwithimmunoprecipitation-basedDNAmethylationprofiling.Toaddressthisissue,wedevelopedacross-platformalgorithm—Bayesiantoolformethylationanalysis(Batman)—foranalyzingmethylatedDNAimmunoprecipitation(MeDIP)profilesgeneratedusingoligonucleotidearrays(MeDIP-chip)ornext-generationsequencing(MeDIP-seq).Wedevelopedthelatterapproachtoprovideahigh-resolutionwhole-genomeDNAmethylationprofile(DNAmethylome)ofamammaliangenome.Strongcorrelationofourdata,obtainedusingmaturehumanspermatozoa,withthoseobtainedusingbisulfitesequencingsuggestthatcombiningMeDIP-seqorMeDIP-chipwithBatmanprovidesarobust,quantitativeandcost-effectivefunctionalgenomicstrategyforelucidatingthefunctionofDNAmethylation.Modulationoftheepigenome—thecombinationofDNA-andchromatin-associatedepigeneticmodificationsthatexistwithinacell—isoneofthekeymechanismsbywhichcellsgeneratefunctionaldiversityfromanessentiallystaticgenome1.Theepigenomeisadynamicentityinfluencedbypredeterminedgeneticprogramsorexternalenvironmentalcues.Giventhediversityofcelltypeswithincomplexorganismssuchasmammals,itisstaggeringtothinkofhowmanyepigenomesexist,orarepossible,andunravelingthiscomplex-ityremainsanimportantchallenge.DNAmethylationistheonlyknownepigeneticsystemthatmodi-fiestheDNAmoleculeitself.Inmammals,itoccurspredominantlyatCpGdinucleotidesandisinvolvedindiverseprocessessuchasdevelopment,genomicintegrity,X-inactivationandimprinting2.Furthermore,perturbedDNAmethyationisahallmarkofseveralhumandiseases,includingcancer.Consequently,thereisgreatinterestinexperimentalandanalyticaltoolsforgenome-wide(thatis,alimitednumberofgenomicsitesthatarerepresentativeofthegenome)orwhole-genomeDNAmethylationprofiling.Inthelastfewyears,avarietyofexperimentalapproacheshaveemergedforgenome-wide,andveryrecentlywhole-genome,DNAmethylationprofiling(reviewedinref.3).Thesecanbeclassifiedintothreemaincategories.Thefirstofthese,restrictionenzyme–basedmethods,usesoneormoreenzymesthatrestrictDNAonlyifitisunmethylated(e.g.,HpaIIorNotI)ormethylated(e.g.,McrBC).Thesemethods,coupledwitheithermicroarrays4–10orcapillarysequencing11,havebeenappliedtogenome-wideDNAmethylationprofilingofseveralorgan-ismsbutarelimitedtotheanalysisofCpGsiteslocatedwithintheenzymerecognitionsite(s).ThesecondgroupoftechniquesisbasedonthereactionbetweengenomicDNAandsodiumbisulfite,whichconvertsunmethylatedcytosinestouracil(andeventuallythyminefollowingamplification),whileleavingmethylatedcytosinesuncon-verted12.Bisulfiteconversion–basedapproachesoffersingleCpGresolutionandhavebeenappliedtomicroarrays13–16,high-through-putPCRsequencing17,18and,morerecently,tonext-generationbisulfitesequencing(BS-seq)19,resultinginanalmostcompleteDNAmethylationprofile(DNAmethylome)fortheB120-MbgenomeofArabidopsisthaliana.However,thereductionofsequencecomplexityfollowingbisulfiteconversionmeansthatitisdifficulttodesignenoughuniqueprobestoanalyzebisulfite-convertedDNAcomprehensivelyonagenome-widescaleonmicroarrays,whereastheBS-seqapproachiscurrentlyprohibitivelyexpensivefortheroutineanalysisoflargegenomessuchashuman.Methodsofthethirdclassuseeither5-methylcytosine-specificantibodies(methylatedDNAimmunoprecipitation20,MeDIPormDIP21)ormethyl-bindingdomainproteins22–24toenrichforthemethylated(orunmethylated25)fractionofthegenomebyimmunopreciptation.MeDIP/mDIP,com-binedwithmicroarrays(MeDIP-chip),wasusedtodelineatethefirsthigh-resolutionwhole-genomeDNAmethylationprofileofanyPublishedonline8July2008;doi:10.1038/nbt14141WellcomeTrustCancerResearchUKGurdonInstitute,andDepartmentofGenetics,UniversityofCambridge,TennisCourtRoad,CambridgeCB21QR,UK.2InstituteofCellandMolecularScience,BartsandTheLondon,4NewarkStreet,London,E12AT,UK.3WellcomeTrustSangerInstitute,Hinxton,Cambridge,CB101SA,UK.4EuropeanBioinformaticsInstitute(EMBL-EBI),WellcomeTrustGenomeCampusHinxton,CambridgeCB101SD,UK.5DepartmentofOncology,UniversityofCambridge,CancerResearchUKCambridgeResearchInstitute,LiKaShingCentre,RobinsonWay,CambridgeCB20RE,UK.6UCLCancerInstitute,UniversityCollegeLondon,72HuntleyStreet,London,WC1E6BT,UK.7InstituteofBiotechnology,UniversityofCambridge,TennisCourtRoad,CambridgeCB21QT,UK.8Theseauthorscontributedequallytothiswork.CorrespondenceshouldbeaddressedtoV.K.R.(v.rakyan@qmul.ac.uk),T.A.D.(thomas.down@gurdon.cam.ac.uk)orS.B.(s.beck@ucl.ac.uk).NATUREBIOTECHNOLOGYVOLUME26NUMBER7JULY2008779ANALYSIS