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Sequence analysis BiQ Analyzer visualization and quality control for DNA methylation data f

Summary: Manual processing of DNA methylation data from bisulfite sequencing is a tedious and error-prone task. Here we present an interactivesoftwaretool that providesstart-to-endsupportfor thisprocess.In an easy-to-use manner, the tool helps the user to

BIOINFORMATICSAPPLICATIONSNOTE

Sequenceanalysis

Vol.21no.212005,pages4067–4068

doi:10.1093/bioinformatics/bti652

BiQAnalyzer:visualizationandqualitycontrolforDNAmethylationdatafrombisul tesequencing

ChristophBock1,Ã,SabineReither2,ThomasMikeska2,MartinaPaulsen2,¨rnWalter2andThomasLengauer1Jo

1

¨tdesSaarlandes,Max-Planck-Institutfu¨rInformatik,Saarbru¨cken,Germanyand2UniversitaFR8.3Biowissenschaften,Genetik/Epigenetik,Saarbru¨cken,Germany

ReceivedonJune9,2005;revisedonAugust17,2005;acceptedonAugust26,2005AdvanceAccesspublicationSeptember1,2005

ABSTRACT

Summary:ManualprocessingofDNAmethylationdatafrombisulfitesequencingisatediousanderror-pronetask.Herewepresentaninter-activesoftwaretoolthatprovidesstart-to-endsupportforthisprocess.Inaneasy-to-usemanner,thetoolhelpstheusertoimportthesequencefilesfromthesequencer,toalignthem,toexcludeorcorrectcriticalsequences,todocumenttheexperiment,toperformbasicstatisticsandtoproducepublication-qualitydiagrams.

Emphasisisputonqualitycontrol:Theprogramautomaticallyassessesdataqualityandprovideswarningsandsuggestionsfordealingwithcriticalsequences.TheBiQAnalyzerprogramisimple-mentedintheJavaprogramminglanguageandrunsonanyplatformforwhicharecentJavavirtualmachineisavailable.

Availability:Theprogramisavailablewithoutchargefornon-commercialusersandcanbedownloadedfromhttp://biq-analyzer.bioinf.mpi-inf.mpg.de/

Contact:cbock@mpi-inf.mpg.de

1INTRODUCTION

DNAmethylationisafrequentbiochemicalmodi cationofeuka-ryoticDNA.Invertebrates,italmostexclusivelyaffectstheC5positionofcytosinesthatbelongtoCpGdinucleotides(i.e.acyto-sineisdirectlyfollowedbyaguanine).Althoughthisphenomenonhasbeenknownforseveraldecades,ithasrecentlywitnessedaboostofattention.DNAmethylationisassumedtoplayanimport-antroleincancer(FeinbergandTycko,2004)andageing(Issa,2003).Itisthecauseforseveraldevelopmentaldiseases(WalterandPaulsen,2003).Ithasbeenbroughtintoconnectionwithchromatinremodeling(Reiketal.,2003),lowsuccessratesinmammaliancloning(Reiketal.,2003)andRNAinterference(KawasakiandTaira,2004).

Themostaccurateandprobablythemostwidelyusedexperi-mentalprotocolforanalyzingDNAmethylationmakesuseofaselectiveconversionofunmethylatedcytosinestouracilsbybisul- tetreatment(Frommeretal.,1992;Hajkovaetal.,2002).Sub-sequentampli cation,cloning,sequencingandcomparisontothegenomicsequenceallowsforidentifyingtheunmethylatedcytosines,whichthenappearasthyminesinamultiplesequence

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alignment.Althoughthisprotocolisgenerallyreliable,itgivesrisetosomepotentialerrorsources,whichweaddresswithourprogram.Currently,fewsoftwaretoolsexistthataretailoredtosupportDNAmethylationresearch.Ontheonehand,severalprimerdesignwebsites(LiandDahiya,2002;Tusnadyetal.,2005)helptheexperimentertoprepareDNAmethylationexperiments,aproblemthatisupstreamofthedataprocessingtaskthatweconsiderhere.Ontheotherhand,thereisabasicMicrosoftExceltemplate(Anbazhaganetal.,2001),whichcanassistwiththecalculationofaveragemethylationandsimilarstatisticswhenmethylationdatahavealreadybeengeneratedandcleanedup(downstreamofourtask).TheonlysoftwarethatpartiallyoverlapsinscopewithBiQAnalyzerisMethTools(Grunauetal.,2000),asetofPerlscriptsthatgeneratepublication-qualitydiagrams(lollipopsandlogos)frommethylationdata.BiQAnalyzerdiffersfromMethToolsinseveralrespects.First,BiQAnalyzerimportssequence lesdirectlyfromthesequencerwithouttheneedforanymanualinterventionandassiststheuserwithallstepsofalignmentandqualitycontrol.Second,BiQAnalyzerdoesnotonlycalculatesummarystatisticsbutcanexportthemethylationdatainfulldetailandinaformatthatmakesiteasytoimportthemintoanystatisticspackageorspread-sheetprogram.Third,BiQAnalyzersupportsstandardizedexperi-mentdocumentation.Finally,BiQAnalyzerprovidesaninteractivegraphicalinterfacethatguidestheuserthroughqualitycontrolandgivescontinuousfeedbackonproblematicsequences.

2QUALITYCONTROLMETHODS

Potentialerrorsourcesinbisul tesequencingarisefromthreephasesoftheexperimentalprotocol:bisul teconversion,PCRandsequencing.Eachofthesestepscangiverisetocharacteristicerrorsinthesequences,whichtheexperimentermustaddressbeforederivingmethylationpro les.

Herewedescribetheseerrortypes,theirimpactonmethylationdataandthequalitycontrolmethodsthatBiQAnalyzerappliestoidentifythecriticalsequences.

Incompleteconversion.Inbisul tesequencingweassumethatallunconvertedCswereoriginallymethylated.Therefore,whenthebisul tetreatmentfailstoconvertunmethylatedCs,methylationwillbeoverestimated.Fortunately,forvertebratesitispossibletoidentifythosesequenceswithalowconversionrate.AssumingthatCsoutsideaCpGcontextarealwaysunmethylated(Reiketal.,

Towhomcorrespondenceshouldbeaddressed.

ÓTheAuthor2005.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:journals.permissions@http://www.wenkuxiazai.com

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