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NEB//E7000S/8 reactions

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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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