Description: The
NEB NextMicrobiomeDNAEnrichmentKitfacilitatesenrichmentofmicrobialDNAfromsamplescontainingmethylatedhostDNA(includinghuman),byselectivebindingandremovaloftheCpG-methylatedhostDNA.Importantly,microbialdiversityremainsintactafterenrichment.
InhumanDNA,4–6%ofcytosinesaremethylated,and60–90%ofthesemethylatedcytosinesareatCpGsites(1,2).Incontrast,methylationatCpGsitesinmicrobialspeciesisrare.TheNEBNextMicrobiomeDNAEnrichmentKitusesasimpleandfastmagneticbead-basedmethodtoselectivelybindandremoveCpG-methylatedhostDNA.ThemethodusestheM
BD 2-Fcprotein,whichiscomposedofthemethylatedCpG-specificbindingproteinMBD2,fusedtotheFcfragmentofhumanIgG.TheFcfragmentbindsre
ADI lytoProteinA,enablingeffectiveattachmenttoProteinA-boundmagneticbeads.TheMBD2domainofthisproteinbindsspecificallyandtightlytoCpGmethylatedDNA.ApplicationofamagneticfieldthenpullsouttheCpG-methylated(eukaryotic)DNA,leavingthenon-CpG-methylated(microbial)DNAinthesupernatant(3).Ifdesired,thehostDNAcapturedinthemagneticbeadpelletcanbeeluted,andaprotocolisprovidedforthis.
TheNEBNextMicrobiomeDNAEnrichmentKitissuitableforawiderangeofsampletypes,includingsampleswithhighlevelsofcontaminatinghostDNA,(3,4)andiscompat
IBL ewithdownstreamapplicationsincludingnextgenerationsequencingonallplatforms,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 SalivaryMicrobiomeDNAEnrichment DNAwaspurifiedfrompooledhumansalivaDNA(InnovativeResearch )andenrichedusingtheNEBNextMicrobiomeDNAEnrichmentKit.LibrarieswerepreparedfromunenrichedandenrichedsamplesandsequencedontheSOLiD4platform.Thegraphshowspercentagesof500M-537MSOLiD450bpreadsthatmappedtoeithertheHumanreferencesequence(hg19)ortoamicrobelistedinHumanOralMicrobiomeDatabase(HOMD)[1].(BecausetheHOMDcollectionisnotcomprehensive,~80%ofreadsintheenrichedsamplesdonotmaptoeitherdatabase.)ReadsweremappedusingBowtie0.12.7[2]withtypicalsettings(2mismatchesina28bpseedregion,etc.). MicrobiomeDiversityisRetainedafterEnrichmentwiththeNEBNextMicrobiomeDNAEnrichmentKit 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 -20 5X 16srRNAUniversalGeneBacteriaControlPrimers -20 NEBNextProteinAMagneticBeads 4 RPL30HumanDNAControlPrimers -20