TheEpiNext™Post-BisulfiteDNALibraryPreparationKit isacompletesetofoptimizedreagentstoprepareaDNAlibrary--aftersuccessfulbisulfiteconversion--forvariousIlluminaplatform-basedbisulfitesequencing(bisulfite-seq)assays,suchaswholegenomebisulfitesequencing(WGBS),oxidativebisulfitesequencing(oxBs-seq),reducedrepresentationbisulfitesequencing(RRBS),andotherbisulfite-basednextgenerationsequencingapplications.Theoptimizedprotocolandcomponentsofthekitallowbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrariestobequicklyconstructedusingsub-nanograminputconcentrationsofDNAsincetheDNAisfirstbisulfite-convertedandthenusedforlibrarypreparation. Thekithasthefollowingadvantagesandfeatures: - Allowsbisulfite-convertedDNAtobeuseddirectlyforligation,therebyeliminatingthepossibilityofbreakingadapter-ligatedfragments,whichcanoftenoccurincurrentlyusedWGBSandRRBSmethods.
- Fast5-hourprocedure,frominputstartingmaterialtolibraryamplification.
- Gel-freesizeselection/purificationsavestimeandpreventshandlingerrors,aswellaslossofvaluablesamples.
- Highsensitivtyandefficiency--directligationofadaptertobisulfite-convertedDNAfragmentsreduceslossoffragmentsandselectionbias,whichenablespre-bisulfiteinputDNAtobeaslowas1ng. Thekitcanbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation.
- ComprehensivesetofcomponentstoaccommodateeachstepofDNAlibrarypreparation--ligation,clean-up,sizeselection,andlibraryamplification-- forconvenience,consistency,andreliABIlity.
- UltraHiFiamplificationenablesachievementofreproducIBLyhighyieldsofDNAlibrarieswithminimalsequencebiasandlowerrorrates.
BackgroundInformation
Severalmethods,includingwholegenomebisulfitesequencing(WGBS)andreducedrepresentationbisulfitesequencing(RRBS),arecurrentlyusedforgenome-wideDNAmethlyationanalysis.Thesemethodsconvertunmethylatedcytosinestouracilwhile5-methylcytosinesremainunchangedbythebisulfitetreatment.Thisallowsepigeneticdifferencestobecomegeneticdifferences,whichcanbesubsequentlydetectedviasequencingatthesingle-basedresolutionlevelandonagenome-widescale.However,practicalusewithsuchcurrentmethodsarenotidealasthey(1)needlargeamountsofDNA(>1 µg)asinputmaterial,whichisdifficulttopreparefromlimitedBIOLOGicalsamplessuchastumorbiopsy,earlyembyros,embyronictissues,andcirculatingDNA;(2)requireDNAtofirstbeshearedandthenligatedtoadapters,followedbybisulfiteconversion(post-ligationbisulfiteconversion),whichcausessubstantialamountsofDNAfragmentsontainedintheadapter-DNAfragmentconstructstobebrokenandtherebyformsmono-taggedtemplatesthatbecomeremovedduringlibraryenrichment;and(3)aretime-consuming(2days).The EpiNextPost-BisulfiteDNALibraryPreparationKitisdesignedtoovercometheseweaknesses. Principle&Procedure
Withthiskit,bisulfite-treatedDNA(whichisinsingle-strandedform),isconvertedtodouble-strandedDNAanddirectlyusedforligationwithBisDNA-specificadapterswhicharenecessaryforamplificationandsequencing.ThefragmentsarethensizeselectedandpurifiedwithMQbeads,whichallowsforquickandprecisesizeselectionsofDNA.Size-selectedDNAfragmentsarethenamplifiedwithahigh-fidelityPCRMix,ensuringmaximumyieldsfromminimumamountsofstartingmaterialandprovidingahighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimalbias. StartingMaterials
Inputstartingmaterialmustbebisulfite-treatedDNAgeneratedfromvariousinputDNAamountsof1ngto1 µg.Foroptimalpreparation,theinputDNAamountforthebisulfiteconversionprocessshouldbe100ngto200ngsothatsufficientbisulfite-treatedDNAcanbeyielded. | 
 Fig.1.SchematicprocedurefortheEpiNext™Post-BisulfiteDNALibraryPreparationKit.
Fig.2.Sizedistributionoflibraryfragmentsasdemonstratedbyapost-bisulfiteDNAlibraryconstructedusingtheEpiNext™ Post-BisulfiteDNALibraryPreparationKitfrom10ngofinputDNA. 
Fig.3.The EpiNext™ Post-BisulfiteDNALibraryPreparationKitwasusedtopreparealibraryforMethyl-SeqwithanIlluminaHiSeq2500.Readalignmentdataat19kbandbasepairresolutioninchromosome7showsCpGmethylationdifferencesfortreatedandcontrolsamples.Controlsamplesareunmethylated(blue)whiletreatedsamplesaremethylated(red). |
