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NEB/NEBNext® Microbiome DNA Enrichment Kit/E2612S/6 reactions
  • NEB/NEBNext® Microbiome DNA Enrichment Kit/E2612S/6 reactions

NEB/NEBNext® Microbiome DNA Enrichment Kit/E2612S/6 reactions

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货号: E2612S-6reactions
品牌: NEB
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    • Description:











      TheNEBNextMicrobiomeDNAEnrichmentKitfacilitatesenrichmentofmicrobialDNAfromsamplescontainingmethylatedhostDNA(includinghuman),byselectivebindingandremovaloftheCpG-methylatedhostDNA.Importantly,microbialdiversityremainsintactafterenrichment.

      InhumanDNA,4–6%ofcytosinesaremethylated,and60–90%ofthesemethylatedcytosinesareatCpGsites(1,2).Incontrast,methylationatCpGsitesinmicrobialspeciesisrare.TheNEBNextMicrobiomeDNAEnrichmentKitusesasimpleandfastmagneticbead-basedmethodtoselectivelybindandremoveCpG-methylatedhostDNA.ThemethodusestheMBD2-Fcprotein,whichiscomposedofthemethylatedCpG-specificbindingproteinMBD2,fusedtotheFcfragmentofhumanIgG.TheFcfragmentbindsreADIlytoProteinA,enablingeffectiveattachmenttoProteinA-boundmagneticbeads.TheMBD2domainofthisproteinbindsspecificallyandtightlytoCpGmethylatedDNA.ApplicationofamagneticfieldthenpullsouttheCpG-methylated(eukaryotic)DNA,leavingthenon-CpG-methylated(microbial)DNAinthesupernatant(3).Ifdesired,thehostDNAcapturedinthemagneticbeadpelletcanbeeluted,andaprotocolisprovidedforthis.

      TheNEBNextMicrobiomeDNAEnrichmentKitissuitableforawiderangeofsampletypes,includingsampleswithhighlevelsofcontaminatinghostDNA,(3,4)andiscompatIBLewithdownstreamapplicationsincludingnextgenerationsequencingonallplatforms,qPCRandend-pointPCR.

      MethodOverview:

      StepI—PrepareGenomicDNA
      DNAshouldbefreeofproteins,proteinaseA,SDSandorganicsolvents;sizeshouldbe≥15kbforoptimalperformance.

      StepII—CombineMBD2-FcandMagneticBeadsin1XBind/washBuffer.
      Incubatethereactionfor10minutesatroomtemperature.WashbeadstwotimesinBind/washReactionBuffer.

      StepIII—AddDNAtoMBD2-FcMagneticBeads.
      Incubatethereactionfor15minutesatroomtemperaturewithgentlemixing.

      StepIV—CollectSupernatantFractioncontainingenrichedmicrobialDNAandBeadFraction-ContainingHostDNA. 

      Figure1.Separationworkflow
      Figure 1. Separation workflow

      SalivaryMicrobiomeDNAEnrichment
      Salivary Microbiome DNA Enrichment
      DNAwaspurifiedfrompooledhumansalivaDNA(InnovativeResearch)andenrichedusingtheNEBNextMicrobiomeDNAEnrichmentKit.LibrarieswerepreparedfromunenrichedandenrichedsamplesandsequencedontheSOLiD4platform.Thegraphshowspercentagesof500M-537MSOLiD450bpreadsthatmappedtoeithertheHumanreferencesequence(hg19)ortoamicrobelistedinHumanOralMicrobiomeDatabase(HOMD)[1].(BecausetheHOMDcollectionisnotcomprehensive,~80%ofreadsintheenrichedsamplesdonotmaptoeitherdatabase.)ReadsweremappedusingBowtie0.12.7[2]withtypicalsettings(2mismatchesina28bpseedregion,etc.).
      MicrobiomeDiversityisRetainedafterEnrichmentwiththeNEBNextMicrobiomeDNAEnrichmentKit
      Microbiome Diversity is Retained after Enrichment with the NEBNext Microbiome DNA Enrichment Kit
      DNAwaspurifiedfrompooledhumansalivaDNA(InnovativeResearch)andenrichedusingtheNEBNextMicrobiomeDNAEnrichmentKit.Librarieswerepreparedfromunenrichedandenrichedsamples,followedbysequencingontheSOLiD4platform.ThegraphshowsacomparisonbetweenrelativeabundanceofeachbacterialspecieslistedinHOMD[1]beforeandafterenrichmentwiththeNEBNextMicrobiomeDNAEnrichmentKit.AbundanceisinferredfromthenumberofreadsmappingtoeachspeciesasapercentageofallreadsmappingtoHOMD.Highconcordancecontinueseventoverylowabundancespecies(inset).Wecompared501M50bpSOliD4readsintheenricheddatasetto537M50bpSOLiD4readsintheunenricheddataset.ReadsweremappedusingBowtie0.12.7[2]withtypicalsettings(2mismatchesina28bpseedregion,etc).
      *Niesseriaflavescens–Thisorganismmayhaveunusualmethylationdensity,allowingittobindtheenrichingbeadsatalowlevel.OtherNiesseriaspecies(N.mucosa,N.siccaandN.elognata)arerepresented,butdonotexhibitthisanomalousenrichment.

       
      EachkitcontainssufficientreagentsfortheeffectiveseparationofCpGmethylatedDNAfromamixedpoolcontainingmicrobialorviralDNA.Ifstartingwith1μgofinputDNAperexperiment,thevolumesprovidedaresufficientforpreparationsofupto6reactions(NEB#E2612S)and24reactions(NEB#E2612L).Allreactionsshouldbestoredat-20°.

      Box1:Storeat-20°C.
      Box2:Storeat4°C.Donotfreeze.

      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      NEBNextMBD2-FcProtein-20
      NEBNextBind/washBuffer-205X
      16srRNAUniversalGeneBacteriaControlPrimers-20
      NEBNextProteinAMagneticBeads4
      RPL30HumanDNAControlPrimers-20
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