Description: TheNEB Next® Ultra™DNALibraryPrepKitforIllumina containsenzymesandbuffersthatareidealtoconvertasmallamountofDNAinputintoindexedlibrariesfornext-generationsequencingontheIlluminaplatform(Illumina,Inc).TheworkflowofNEBNextUltraDNALibraryPrepKitforIlluminaisveryuser-friendlyandfastwithminimalhands-ontime.Eachofthesecomponentsmustpassrigorousqualitycontrolstandardsandarelotcontrolled,bothindividuallyandasasetofreagents.
UltraDNALibraryPreparationWorkflowforIllumina NEBNextUltra™DNAprovideshighlibraryyieldsevenwithlow inputsanddifficultsamples Alllibrarieswerepreparedwith5ngofinputDNAandrunonanAgilent Bioanalyzer®. A.E.coli genomicDNAlibraries.1:Ladder;2:LibrarypreparedusingNEBNextDNA LibraryPrepMasterMixSet(E6040);3:LibrarypreparedusingNEBNextUltraDNA LibraryPrepKit(E7370).With5nginputs,libraryyieldsaresignificantlyhigherusing theNEBNextUltra™DNAKit. B.FFPEsample.HumanlungtumorgenomicDNAfroma66-yearoldmale,extractedfrom a10-yearoldFFPEblock(Biochain,Inc.).1:LibrarypreparedusingNEBNextUltraDNA LibraryPrepKit(E7370).2:InputDNA(highlydegradedFFPEgenomicDNA).Readdepthcorrelationshowsconsistenthighcoveragefor5ng-1µginputamounts LibrarieswerepreparedwithvariousamountsofhumanIMR90gDNA(5,50,and1000ng)andPCRamplified.APCR-freelibrarywasalsopreparedwith1000nghumanIMR90gDNA.Sequencecoverageof10kbwindowsofthehumangenomewasanalyzedonGalaxy,andPearsoncorrelationcoefficient(R2values)werecalculatedusingMicrosoft®Excel®.Alllibrariesarehighlycorrelatedwitheachother,independentofDNAinputaswellasPCRamplification.HighcoveragewasachievedforallDNAinputamounts. NEBNextUltraDNAKitsdisplaystrongperformancewithinputamountsaslowas5ng InputDNA:IMR90genomicDNA(humanfibroblast). PFMismatchRate:Therateofbasesmismatchingthereferenceforallbasesalignedtothereferencesequence. PercentDuplication:Thepercentageofmappedsequencethatismarkedasduplicate. PercentChimeras:Thepercentageofreadsthatmapoutsideofamaximuminsertsizeorthathavethetwoendsmappingtodifferentchromosomes. ThisdataillustratesthestrongperformanceoftheNEBNextUltraDNALibraryPrepKitforIllumina,evenwith5ngofhumangenomicDNAinput.Mismatchrate,%duplicationand%chimerasarealllow,andsimilar,regardlessofinputamount.FunctionalValidation EachsetofreagentsisfunctionallyvalidatedtogetherthroughconstructionandsequencingofaDNAlibraryontheIlluminasequencingplatform.LotControl Thelotsprovidedaremanagedseparatelyandqualifiedbyadditionalfunctionalvalidation.Individualreagentsundergostandardenzymeactivityandqualitycontrolassays,andalsomeetstringentcriteriaintheadditionalqualitycontrolslistedoneachindividualcomponentpage
ReagentsSupplied Thefollowingreagentsaresuppliedwiththisproduct:
Storeat(°C) Concentration NEBNextEndPrepEnzymeMix -20 NEBNextEndRepairReactionBuffer -20 10X Blunt/TALigaseMasterMix -20 NEBNext® LigationEnhancer -20 NEBNext®Q5®HotStartHiFiPCRMasterMix -20