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NEB//E7000S/8 reactions

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货号: E7000S-8reactions
品牌: NEB
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    • Description:







      Targetenrichment,coupledwithnextgenerationsequencing(NGS),enableshighthroughput,deepsequencingofgenomicregionsofinterest.NEBNextDirectisanovel,hybridization-basedcapturemethodofferingsignificantadvantagesovertrADItionalin-solutionhybridizationandmultiplexPCRprotocols.
       
      IntheNEBNextDirecttargetenrichmentapproach(Figure1),fragmentedDNAisrapidlyhybridizedtobiotinylatedoligonucleotidebaitsthatdefinethe3´endofeachtargetofinterest.Thebait-targethybridsareboundtostreptavidinbeadsandany3´offtargetsequenceisremovedenzymatically.Thiscombinationofashorthybridizationtimewiththeenzymaticremovalof3´offtargetsequenceenablesgreatersequencingefficiencyrelativetoconventionalhybridization-basedenrichmentmethods.ThetrimmedtargetsarethenconvertedintoIllumina-compatIBLelibrariesthatincludeuniquemolecularidentifiers(UMI)andasamplebarcode.Sequence-readylibrariesaregeneratedwithinoneday.Theprocedureiscompatiblewithmostautomatedliquidhandlinginstruments.

      TheNEBNextDirectHotSpotCancerPanelcontainsbaitsthatcapturebothstrandsofDNAacross190commoncancertargetsfrom50genes,encompassingapproximately40kbofsequenceandincludingover18,000COSMICfeatures(Table1).Thepanelisdesignedtogeneratetargetsofroughly150bp,compatiblewithPE75Illuminasequencing.

      Advantages
      • Generateahigherpercentageofyoursequencingreadsaligningtoyourtargets
      • Eliminatetheneedtoover-sequence,reducingcostpersample
      • Obtainuniformsequencingofalltargets,regardlessofGCcontent
      • Savetimewitha1-dayworkflowthatcombinesenrichmentwithlibrarypreparation
      • GeneratehighqualitylibrarieswithlimitedinputamountsanddegradedDNAsamples,includingFFPEandctDNA
      • Distinguishmolecularduplicates,reducingfalsepositivevariantsandimprovingsensitivity


      Figure1.NEBNextDirectemploysafasthybridization-basedworkflowthatcombinescapturewithlibrarypreparation.

      fast hybridization workflow


      Table1.Targetsincluderegionsfromthefollowingcancer-relatedgenes:

      Targets include regions from the following cancer-related genes


      Figure2.TheNEBNextDirectCancerHotSpotPaneldemonstratestheABIlitytoaccuratelydetectarangeofnucleicacidvariants.

      Allele Frequency
      Thisfigureshowstheexpectedversusobservedvariantallelefrequencies(VAF)acrosstherangeofwell-characterizedvariantspresentinapoolof24HapMapsamplesscreenedagainsttheNEBNextDirectCancerHotSpotPanel.100ngofinputDNAwasused,samplesweresequencedontheIllumina®MiSeq®using2x75bpsequencing,andstandarddataanalysisandvariantcallingalgorithmswereused.Wewereabletosuccessfullydetect100%ofthe168truthvariantspresentacrossarangeof2-100%VAF.ThehighdegreeoflinearityacrossthisbroaddynamicrangedemonstratestheabilityoftheNEBNextDirectCancerHotSpotPaneltoaccuratelypredictvariantallelefrequenciesacrossabroaddynamicrange.


      Figure3.TheNEBNextDirectCancerHotSpotPaneldeliversahighpercentageofsequencereadsmappingtotargets,evenwithchallengingsampletypes.

      The NEBNext Direct Cancer HotSpot Panel delivers a high percentage of sequence reads mapping to targets, even with challenging sample types.

      • Graphshowsthepercentageofalignedsequencereadsthatmaptothetargets
      • 100ngofDNAwasusedforeachlibrarypreparation
      • ReadsweregeneratedonanIllumina®MiSeq®with2x75bpreads,8bpsampleID,and12bpuniquemoleculeID
      • AlignmentswereperformedwithBWA-MEMandPCRduplicateswerefilteredusingtheuniquemoleculeIDs

      Figure4.TheNEBNextDirectCancerHotSpotPaneldisplayshighuniformityofcoverageacrosstargets.

      Cancer HotSpot Panel displays high uniformity of coverage across targets.

      • Graphshowsthepercentageoftargetbasessequencedtoatleast50%,33%,and25%ofthemeanreaddepth
      • 100ngofDNAwasusedforeachlibrarypreparation
      • ReadsweregeneratedonanIlluminaMiSeqwith2x75bpreads,8bpsampleID,and12bpuniquemoleculeID
      • AlignmentswereperformedwithBWA-MEMandPCRduplicateswerefilteredusingtheuniquemoleculeIDs


      Figure5.TheNEBNextDirectCancerHotSpotPaneloffersminimizedbiasacrosssequencecontent.

      minimized bias across sequence content

      • GraphshowsthenormalizeddepthofcoverageoftargetsofvaryingGCcontent
      • 100ngofDNAwasusedforeachlibrarypreparation
      • ReadsweregeneratedonanIlluminaMiSeqwith2x75bpreads,8bpsampleID,and12bpuniquemoleculeID
      • AlignmentswereperformedwithBWA-MEMandPCRduplicateswerefilteredusingtheuniquemoleculeIDs

      ILLUMINA®andMISEQ®areregisteredtrademarksofIllumina®,Inc.

      LotControl

      Thelotsprovidedaremanagedseparatelyandqualifiedbyadditionalfunctionalvalidation.Individualreagentsundergostandardenzymeactivityandqualitycontrolassays,andalsomeetstringentcriteriaintheadditionalqualitycontrolslistedoneachindividualcomponentpage

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      NEBNextDirect®FFPEPhosphorylationEnzyme-20
      NEBNextDirect®FFPEPhosphorylationBuffer-20
      NEBNextDirect®HybridizationAdditive-20
      NEBNextDirect®dA-TailingEnzyme-20
      NEBNextDirect®3´Adaptor-20
      NEBNextDirect®Ligase-20
      NEBNextDirect®5´BluntingEnzymeMix-20
      NEBNextDirect®5´UMIAdaptor-20
      NEBNextDirect®CleavingEnzymeMix-20
      NEBNextDirect®Q5MasterMix-20
      NEBNextIndexPrimerMixD01-D08(E7020-E7027)-20
      NEBNextDirect®BeadWash125
      NEBNextDirect®StreptavidinBeads4
      NEBNextDirect®HybridizationWash(HW)4
      NEBNextDirect®BeadPrepBuffer4
      NEBNextDirect®dA-TailingBuffer4
      NEBNextDirect®AdaptorLigationBuffer4
      NEBNextDirect®CleavingBuffer4
      NEBNextDirect®BeadWash24
      NEBNextDirect®3´BluntingEnzymeMix-20
      NEBNextDirect®5´BluntingBuffer4
      NEBNextDirectCancerHotSpotBaits-20
      NEBNextSamplePurificationBeads4
      NEBNextDirectHybridizationBuffer4
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