TheEpiNext™High-SensitivityBisulfite-SeqKit(Illumina) isdesignedtobisulfite-convertDNA andprepareanIllumina-based libraryforbisulfitesequencing,allinonekit.Intendedapplicationsincludewholegenome(WGBS),oxidative(oxBS-seq),reducedrepresentation(RRBS),andotherbisulfite-nextgenerationsequencing.TheoptimizedprotocolandcomponentsofthekitallowsubnanogramamountsofDNAtobe bisulfiteconvertedandfragmentedsimultaneously followedbyquicknon-barcoded(singleplexed)andbarcoded(multiplexed)libraryconstruction inlessthan7hours.Thekithasthefollowingadvantages:
- Innovativemethod-Allowsforsimultaneousbisulfiteconversionandsize-appropriateDNAfragmentation.ThebisulfiteDNAcanbedirectlyligatedtoadaptorstherebyeliminatingthepossibilityofbreakingadaptor-ligatedfragments,whichoftenoccurswithcurrentlyusedWGBSandRRBSmethods.
- Fastandstreamlinedprocedure-TheprocedurefromDNAbisulfitetreatmenttoready-to-uselibraryDNAcanbecompletedwithin6hoursand30minutes.
- Completeconversion-Completelyconvertsunmethylatedcytosineintouracil(>99%)withnegligIBLeinappropriate-orerror-conversionsofmethylcytosinetothymine.
- Highsensitivityandefficiency-InnovativeadaptorligationofbisulfiteDNAeliminateslossoffragmentsandselectionbias,whichenablesinputDNAtobeaslowas0.2ng,makingitidealformethylationprofilingofprecious,limitedsamples.Thekitcanbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation.
- Extremelyconvenient-ThekitcontainsallrequiredcomponentsforeachstepofDNAlibrarypreparation,whicharesufficientforbisulfiteconversion,ligation,clean-up,sizeselectionandlibraryamplification,therebyallowingthebisulfiteDNAlibrarypreparationtobestreamlinedforthemostreliableandconsistentresults.
- Minimalbias-UltraHiFiamplificationenablesachievementofreproduciblyhighyieldsofDNAlibrarieswithminimalsequencebiasandlowerrorrates.
- BroadsamplesuitABIlity-StartingmaterialscanbegenomicDNAisolatedfromvarioustissue/cellsamplessuchassuchasfreshandfrozentissue,culturedcellsfromaflaskormicroplate,microdissectionsamples,paraffin-embeddedtissue,biopsy,embryoniccells,plasma/serumsamples,andbodyfluidsamples,etc.DNAenrichedfromvariousenrichmentreactionssuchasChIP,MeDIP/hMeDIPorexoncapturemaybealsousedasstartingmaterial.
BackgroundInformation
DNAmethylationoccursbythecovalentadditionofamethylgroup(CH3)atthe5-carbonofthecytosinering,resultingin5-methylcytosine(5-mC).DNAmethylationisessentialinregulatinggeneexpressioninnearlyallBIOLOGicalprocessesincludingdevelopment,growth,anddifferentiation.AlterationsinDNAmethylationhavebeendemonstratedtocauseachangeingeneexpression.Forexample,hypermethylationleadstogenesilencingordecreasedgeneexpressionwhilehypomethylationactivatesgenesorincreasesgeneexpression.AberrantDNAmethylationisalsoassociatedwithpathogenesisofdiseasessuchascancer,autoimmunedisorders,andschizophrenia.Thus,genome-wideanalysisofDNAmethylationcouldprovidevaluableinformationfordiscoveringepigeneticMarkersusedfordiseasediagnosis,andpotentialtargetsusedfortherapeutics. Principle&Procedure
ThiskitincludesallreagentsrequiredforsuccessfullypreparingalibrarydirectlyusingbisulfiteDNAgeneratedfromatinyamountofinputDNA.Inthispreparation,DNAissimultaneouslybisulfiteconvertedandfragmentedtotheappropriatelengthduringthebisulfiteprocess.Thebisulfite-treatedDNA,whichisinsinglestrandedform,isthensimultaneouslyconvertedtodsDNAandadaptorligated.TheligatedfragmentsaresizeselectedandpurifiedusingMQbindingbeads,whichallowsforquickandprecisesizeselectionofDNA.Size-selectedDNAfragmentsareamplifiedusingahigh-fidelityPCRMixwhichensuresmaximumyieldsfromminimumamountsofstartingmaterialandprovideshighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimalbias.
StartingMaterials
StartingmaterialscanbegenomicDNAisolatedfromvarioustissue/cellsamplessuchasfreshandfrozentissue,culturedcellsfromaflaskormicroplate,microdissectionsamples,FFPEtissue,plasma/serum,andbodyfluidsamples,etc.DNAenrichedfromvariousenrichmentreactionssuchasChIP,MeDIP/hMeDIPorexoncapturemayalsobeusedasstartingmaterial.DNAshouldbewithoutanypreviousrestrictiondigestionstep.PlasmidDNAcanbeusedforbisulfitetreatmentwithorwithoutpreviouslinearization,asthekitallowsforDNAdenaturationstatustoremainduringtheentireDNAbisulfiteconversionprocessanddirectligationofadaptorstobisulfiteDNA.InputamountofDNAcanbefrom0.2ngto200ng.Foroptimalpreparation,theinputamountshouldbe10-50ng.
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 Fig.1. WorkflowoftheEpiNext™High-SensitivityBisulfite-SeqKit(Illumina).
Fig.2. The EpiNext™ High-SensitivityBisulfite-SeqKit(Illumina)wasusedtobisulfiteconvertDNAandpreparealibrary tobesequencingonanIlluminaHiSeq2500.Datatrackswerealignedtoa30kbregionofchromosome1andshowedunmethylated(blue)andmethylated(red)areasandthecorrespondinggeneregionsintreatedandcontrolsamples. 
Fig.3. Sizedistributionoflibraryfragments.Post-bisulfiteDNAlibrarieswerepreparedfromhumanplacentaDNAusingtheEpiNext™High-SensitivityBisulfite-SeqKit(Illumina):A:10ng;B:50ng. |
