TheEpiNext™ DNALibraryPreparationKit (Illumina)isacompletesetofoptimizedreagentstocarryoutasuccessfulDNAlibrarypreparation.ThekitissuitableforpreparingaDNAlibraryfornextgenerationsequencingapplicationsusinganIlluminasequencer,whichincludesgenomicDNA-seq,ChIP-seq,MeDIP/hMeDIP-seq,bisulfite-seq,andtargetedre-sequencing.Theoptimizedprotocolandcomponentsofthekitallowbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrariestobeconstructedquicklywithreducedbias.Thekithasthefollowingadvantages: - Fastandstreamlinedprocedure-TheprocedurefromfragmentedDNAtosizeselectionislessthan2h30min. Onlyoneclean-upbetweeneachstep,therebysavingtimeandpreventinghandlingerrors,aswellaslossofvaluablesamples.Gel-freesizeselectionfurtherreducesthepreparationtime.
- HighlyConvenient-ThekitcontainsallrequiredcomponentsforeachstepofDNAlibrarypreparation,whicharesufficientforendrepair,dAtailing,ligation,clean-up,sizeselectionandlibraryamplification,therebyallowingthelibrarypreparationtobestreamlinedwiththemostreliableandconsistentresults.
- Minimizedbias-UltraHiFiamplificationandanoptionalPCR-freestepenabletheusertoachievereproducIBLyhighyieldsofDNAlibrarywithminimalsequencebiasandlowerrorrates.
- Flexibility-Canbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation. UsesvariousdsDNAincludingfragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromChIPreactions,MeDIP/hMeDIPreactions,orexoncapture.BroadrangeofinputDNAfrom10ngto1µg.PCR-freelibrarypreparationcanbeperformedwithuseof500ngormoreinputDNA.
BackgroundInformation
DNAlibrarypreparationisacriticalstepfornextgenerationsequencing(NGS).TogenerateaccuratesequencingdataforNGS,thepreparedlibraryDNAshouldbesufficientinyieldandofhighquality.Also,asNGStechnologyiscontinuouslyimproving,DNAlibrarypreparationisrequiredtobeoptimizedaccordingly. Mostofthecurrentlyusedmethodsareunfortunatelytime-consuming,expensive,andinconvenient.SomeofthemethodsarerelativelyquickbycombiningendrepairanddAtailingorevenligationinone-step,buthavebeenshowntogeneratesignificantGtailingorformconcatmersattheligationsteporhavehighinsertionbias.ThesesidereactionseventuallyresultinthepreparedDNAlibrarybeinglessefficientandinaccurate.AnidealDNAlibrarypreparationmethodshouldbebalancedinspeed,convenience,smallsample-suitABIlity,cost-effectiveness,andaccuracy.Toaddressthisissue,EpigentekofferstheEpiNextDNALibraryPreparationKit. Principle&Procedure
ThiskitincluldesallreagentsrequiredateachsteptocarryoutasuccessfulDNAlibrarypreparation.Inthelibrarypreparation,DNAisfirstfragmentedtotheappropriatesize(about300bppeaksize).TheendrepairoftheDNAfragmentsisperformedandanA-overhangisaddedatthe3"-endofeachstrand. Adaptorsarethenligatedtobothendsoftheendrepaired/dAtailedDNAfragmentsforamplificationandsequencing.FragmentsarethensizeselectedandpurifiedwithMQbeads,whichallowsforquickandprecisesizeselectionofDNA.Size-selectedDNAfragmentsarethenamplifiedwithahigh-fidelityPCRMixwhichensuresmaximumyieldsfromminimumamountsofstartingmaterialandprovideshighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimumbias. StartingMaterials
StartingmaterialscanincludefragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromChIPreactions,MeDIP/hMeDIPreaction,orexoncapture.DNAshouldberelativelyfreeofRNAsincelargefractionsofRNAwillimpairendrepairanddAtailing,resultinginreducedligationcapabilities.InputamountofDNAcanbefrom5ngto1µg.Foroptimalpreparation,theinputamountshouldbe100ngto200ng.Foramplification-free,500ngormoreisneeded. | 
 Fig.1. WorkflowoftheEpiNext™DNALibraryPreparationKit(Illumina).
Fig.2. Sizedistributionoflibraryfragments:humanplacentaDNAwasshearedto210bpspeaksizeand20ngofshearedDNAwasusedforDNAlibrarypreparationusingtheEpiNext™DNALibraryPreparationKit(Illumina). |
