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TheUltraIIDNALibraryPrepKitforIlluminameetsthechallengeofconstructinghighqualitylibrariesfromever-decreasinginputquantities. Thereagentsforeachstepinthelibrarypreparationworkflowhavebeenreformulatedtoenablehighyieldpreparationofhighqualitylibrariesfrom500picogramsto1microgramofinputDNA. ThisnewgenerationofNEBNextreagentsusesafast,streamlined,automatableworkflowandenablesuseoffewerPCRcycleswhilealsoimprovingGCcoverage.ThekitisalsocompatIBLewithPCR-freeworkflowsandiseffectivewithchallengingsamplessuchasFFPEDNA.Nowalsoavailablewith optionalSPRIselect®beads forclean-upandsize-selectionsteps.

Advantages

  • Getmoreofwhatyouneed,withthehighestlibrary yields

  • Usetogenerate highqualitylibraries evenwhenyouhaveonlylimitedamountsofDNA,withinputsaslowas500pg

  • PreparelibrariesfromALLofyoursamples,including GC-richtargets and FFPEDNAsamples

  • Improveyieldsandqualityfor targetenrichment applications

  • Savetimewith streamlinedworkflows ,reducedhands-ontimeandautomationcompatibility,andenjoytheflexibilityofkitormoduleformatsTolearnmoreabouthowNEBNextUltraIIaddresseslowerinputamountsandchallengingsampletypes,downloadour technicalnote.

    • NEBNextUltraIIproducesthehighestlibraryyieldsandconversionratesfromabroadrangeofinputamounts

      A: LibrarieswerepreparedfromHumanNA19240genomicDNAusingtheinputamountsandnumbersofPCRcyclesshown.Manufacturers’recommendationswerefollowed,withtheexceptionthatsizeselectionwasomitted.

      B: LibrarieswerepreparedfromHumanNA19240genomicDNAusingtheinputamountsandlibraryprepkitsshownwithoutanamplificationstep,followingmanufacturers'recommendations.qPCRwasusedtoquantitateadaptor-ligatedmolecules,andquantitationvalueswerethennormalizedtotheconversionrateforUltraII.TheUltraIIKitproducesthehighestrateofconversiontoadaptor-ligatedmolecules,forabroadrangeofinputamounts.Viewadditionaldataonlibraryyield
       NEBNextUltraIIprovidesuniformGCcoverageformicrobialgenomicDNAoverabroadrangeofGCcompositionandinputamounts

      Librariesweremadeusing500pg,1ngand100ngofthegenomicDNAsshownandtheUltraIIDNALibraryPrepKitandsequencedonanIlluminaMiSeq®.ReadsweremappedusingBowtie2.2.4andGCcoverageinformationwascalculatedusingPicard'sCollectGCBiasMetrics(v1.117).Expectednormalizedcoverageof1.0isindicatedbythehorizontalgreyline,thenumberof100bpregionsateachGC%isindicatedbytheverticalgreybars,andthecoloredlinesrepresentthenormalizedcoverageforeachlibrary.Viewadditionaldataonlibraryquality
       NEBNextUltraIIprovidessuperioryieldsinPCR-freeworkflows

      Librariesweregeneratedfrom100ngofHumanNA19240genomicDNAusingthelibraryprepkitsshown,followingmanufacturers'recommendations,andwithnoamplificationstep.LibraryyieldsweredeterminedbyqPCRusingtheNEBNextLibraryQuantKitforIllumina.TheNEBNextUltraIIKitproducesthehighestyields.


      NEBNextUltraIIlibrariesprovidethehighestqualitysequencedatainPCR-freeworkflows


      PCR-freelibrariesweregeneratedfrom100ngofHumanNA19240genomicDNAusinglibraryprepkitsshown,followingmanufacturers’recommendations,andwithnoamplificationstep.LibrariesweresequencedontheIlluminaNextSeq500.ReadsweremappedtotheGRCh37referenceusingBowtie2.2.4. ThisdataillustratesthatNEBNextUltraIIDNALibraryPrepKitenableshighqualitysequencedatainPCR-freeworkflows,evenwithlowinputamounts.

      %Mapped: ThepercentageofreadsmappedtohumanGRCh37reference. 
      %Duplication: Thepercentageofmappedsequencethatismarkedasduplicate. 
      %Chimeras: Thepercentageofreadsthatmapoutsideofamaximuminsertsizeorthathavethetwoendsmappingtodifferentchromosomes. 


      NEBNextUltraIIenablesconstructionofhighqualitylibrariesfromFFPEDNAsamples
       





      LIBRARYKITTOTALREADS%DUPLICATION%MAPPED%CHIMERAS
      UltraII3,685,0290.0297.001.10
      KapaHyper3,679,1360.0297.002.00
      TruSeqPCR-Free3,681,6430.0596.651.38

      Librarieswerepreparedfrom17–30ngofhumanDNAextractedfromtheFFPEtissuesampleslisted,amplifiedusing10cyclesofPCRandsequencedontheIlluminaMiSeq. 

      ReadsweremappedtotheGRCh37referencegenomeusingBowtie2.2.4.
      %Mapped: ThepercentageofreadsmappedtoHumanGRCh37reference. 
      %MappedinPairs: Thepercentageofreadswhosematepairwasalsoalignedtothereference.
      %Duplication: Thepercentageofmappedsequencethatismarkedasduplicate. 
      %Chimeras: Thepercentageofreadsthatmapoutsideofamaximuminsertsizeorthathavethetwoendsmappingtodifferentchromosomes.

      ThisdataillustratesthattheNEBNextUltraIIDNALibraryPrepKitforIlluminaenableshighqualitysequencedata,evenwithlowinputamountsofFFPEDNA. 


      ViewadditionaldatafromFFPEDNAsamples
       

      NEBNextUltraIIprovidessuperioryieldsfortargetenrichmentapplications.ForNimblegen® enrichment,librarieswerepreparedwith100ngofHumanHG02922genomicDNA,usingNEBNextUltraIIwiththeNEBNextAdaptorandindexprimers,ortheKapaLibraryPreparationKitIlluminaPlatformswiththeSeqCap® AdapterKit(Roche).TargetenrichmentwasperformedwithNimbleGenSeqCapHumanExomev3.

      ForSureSelectXT enrichment,librarieswerepreparedwith200ngofHumanHG02922genomicDNA,usingNEBNextUltraIIorKapaHyperwiththeNEBNextAdaptorandindexprimers,ortheSureSelectXT ReagentKit,Illumina(ILM)platformswithHerculase® IIFusionDNAPolymerase.TargetenrichmentwasperformedwithSureSelect® HumanAllExonV6.

      Manufacturers’recommendationswerefollowed,includinguseoftherecommendednumberofPCRcyclesforpre-andpost-capture.NEBNextUltraIIlibrariesresultedinthehighestpost-captureyieldsforbothenrichmentworkflows.Viewadditionaldataontargetenrichment 
       

      LotControl

      Thelotsprovidedaremanagedseparatelyandqualifiedbyadditionalfunctionalvalidation.Individualreagentsundergostandardenzymeactivityandqualitycontrolassays,andalsomeetstringentcriteriaintheadditionalqualitycontrolslistedoneachindividualcomponentpage

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:








      FFPEDNADNAINPUT(ng)LIBRARYYIELDSINng%MAPPED%MAPPEDINPAIRS%DUPLICATION%CHIMERAS
      KidneyTumor1713291.596.10.483.0
      LungTumor2023290.194.90.424.1
      LiverNormal2069192.694.70.338.6
      BreastTumor3051491.995.10.374.5
       Storeat(°C)Concentration
      NEBNextUltraIIEndPrepEnzymeMix-20 
      NEBNextUltraIIEndPrepReactionBuffer-20 
      NEBNextLigationEnhancer-20 
      NEBNextUltraIILigationMasterMix-20 
      NEBNext®UltraIIQ5®MasterMix-202X