TheEpiNext™ High-SensitivityDNALibraryPreparationKit(Illumina) isacompletesetofoptimizedreagentstoprepareaDNAlibraryfromverysmallamountofsamplesforuseinnext-generationsequencingapplications.ThiskitissuitableforpreparingaDNAlibraryusingsub-nanogramamountsofDNAinputfornextgenerationsequencingapplicationsusinganIlluminasequencer.TheseapplicationsincludegenomicDNA-seq,ChIP-seq,MeDIP/hMeDIP-seq,trADItionalbisulfite-seq,andtargetedre-sequencing.Theoptimizedprotocolandcomponentsofthekitallowbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrariestobeconstructedquicklywithreducedbias.Thekithasthefollowingadvantages: - Highsensitivityandflexibility -Canbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation.TheamountofinputDNAcanbeaslowas0.2ngwitharangefrom0.2to100ng.VariousdsDNAcanbeused,whichincludeslimitedamountsoffragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromChIPreactions,anddsDNAenrichedfromMeDIP/hMeDIPreactionsorexoncapture.
- Fastandstreamlinedprocedure-TheprocedurefromfragmentedDNAtosizeselectionislessthan1hourand30minutes.Noclean-upisrequiredbetweeneachstepandallreactionstakeplaceinthesametube,therebysavingtimeandpreventinghandlingerrorsaswellaslossofvaluablesamples.Gel-freesizeselectionfurtherreducesthepreparationtime.
- Highlyconvenient-ThekitcontainsallrequiredcomponentsforeachstepofDNAlibrarypreparation,whicharesufficientforendpolishing,ligation,clean-up,sizeselection,andlibraryamplification,therebyallowingthelibrarypreparationtobestreamlinedwiththemostreliableandconsistentresults.
- Minimizedbias-UltraHiFiamplificationandoptionalPCR-freestepenabletheusertoachievereproducIBLyachievehighyieldsofDNAlibrarywithminimalsequencebiasandlowerrorrates.
BackgroundInformation
DNAlibrarypreparationisacriticalstepfornextgenerationsequencing(NGS).TogenerateaccuratesequencingdatainNGS,thepreparedlibraryDNAshouldbesufficientinyieldandofhighquality.AsNGStechnologyiscontinuouslyimproving,DNAlibrarypreparationisrequiredtobeoptimizedaccordingly. Mostofthecurrentlyusedmethodsaretime-consuming,expensive,inconvenient,andspecificallyneedlargeamountsofDNA.ThesefactorsresultinaDNAlibrarypreparationwhichcannotbeusedforBIOLOGicalsampleswithlimitedamountsofstartingmaterialsuchastumorbiopsy,earlyembryos,andembryonictissuesandcirculatingDNA.Inaddition,theamountofDNAenrichedbyChIPorMeDIP/hmeDIPisoftenatloworsub-nanogramlevelswhichcausesinsufficientDNAlibraryyields.Toaddressthisissue,EpigentekofferstheEpiNext™High-SensitivityDNALibraryPreparationKit. Principle&Procedure
Thiskitincludesallreagentsrequiredateachstepoftheworkflowtocarryout asuccessfulDNAlibrarypreparation.Inthelibrarypreparation,DNAisfirstfragmentedtoanappropriatesize(about300bpsinpeaksize).Theendrepair/dAtailing(endpolishing)oftheDNAfragmentsareperformedsimultaneously.AdaptorsarethenligatedtobothendsofthepolishedDNAfragmentsforamplificationandsequencing.LigatedfragmentsaresizeselectedandpurifiedwithMQbeads,whichallowsforquickandprecisesizeselectionofDNA.Size-selectedDNAfragmentsareamplifiedwithahigh-fidelityPCRMixthatensuresmaximumyieldsfromminimumamountsofstartingmaterialandprovideshighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimumbias. StartingMaterials
StartingmaterialscanincludefragmenteddsDNAisolatedfromvarioustissueorcellsamples,dsDNAenrichedfromaChIPreaction,MeDIP/hMeDIPreaction,orexoncapture.DNAshouldberelativelyfreeofRNAbecauselargefractionsofRNAwillimpairendrepairanddA-tailing,resultinginreducedligationcapABIlities.TheinputamountofDNAcanbefrom0.2ngto100ng.Foroptimalpreparation,theinputamountshouldbe10ngto50ng. | 
 Fig.1. WorkflowoftheEpiNext™High-SensitivityDNALibraryPreparationKit.
Fig.2. Sizedistributionoflibraryfragments.HumanplacentaDNAwasshearedtoaround300bpsinpeaksizeand0.2ngofDNAwasusedforDNAlibrarypreparationusingEpiNext™High-SensitivityDNALibraryPreparationKit. |
