NEB//E7000S/8 reactions

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.

Table1.Targetsincluderegionsfromthefollowingcancer-relatedgenes:

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.

Figure5.TheNEBNextDirectCancerHotSpotPaneloffersminimizedbiasacrosssequencecontent.

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®BeadWash1	25
NEBNextDirect®StreptavidinBeads	4
NEBNextDirect®HybridizationWash(HW)	4
NEBNextDirect®BeadPrepBuffer	4
NEBNextDirect®dA-TailingBuffer	4
NEBNextDirect®AdaptorLigationBuffer	4
NEBNextDirect®CleavingBuffer	4
NEBNextDirect®BeadWash2	4
NEBNextDirect®3´BluntingEnzymeMix	-20
NEBNextDirect®5´BluntingBuffer	4
NEBNextDirectCancerHotSpotBaits	-20
NEBNextSamplePurificationBeads	4
NEBNextDirectHybridizationBuffer	4

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NEB//E7000S/8 reactions

Description:

LotControl

ReagentsSupplied