16S Sample Preparation Guide - Illumina, Inc.

1 , # Up8 IndexPCR10 PCRC lean Up213[Optional]ValidateLibrary15 LibraryQuantification,Normalization,andP ooling16 LibraryDenaturingandMiSeqSampleLoading17 MiSeqReporterMetagenomicsWorkflow20 SupportingInformation21 IntroductionMetagenomicstudiesarecommonl yperformedbyanalyzingtheprokaryotic16 SribosomalRNAgene(16 SrRNA),whichisapproximately1, ,andyourregionofinterestmightvarydependi ngonthingssuchasexperimentalobjectives,d esign, ,on boardprimaryanalysis,andsecondaryanalysi susingMiSeqReporterorBaseSpace, :1 Orderampliconprimers Theprotocolincludestheprimerpairsequence sfortheV3andV4regionthatcreateasingleamp liconofapproximately~ , Theprotocoldescribesthestepstoamplifythe V3andV4regionandusingalimitedcyclePCR,ad dIlluminasequencingadaptersanddual , Usingpaired300 bpreads,andMiSeqv3reagents,theendsofeach readareoverlappedtogeneratehigh quality,full lengthreadsoftheV3andV4regioninasingle65 >20millionreadsand,assuming96indexedsamp les,cangenerate>100,000readspersample.

2 TheMetagenomicsworkflowisasecondaryanaly sisoptionbuiltintotheMiSeqReporter(on systemsoftware)oravailableonBaseSpace(cl oud basedsoftware). ,usetheGenerateFASTQW orkflow(secondaryanalysisoption).Formore information,seeMiSeqReporterMetagenomics Workflow, ;insomecasesreagentsarerequiredtobepurch asedfromnon authorizedthird authorizedthird definedforwardandreverseprimersthatareco mplementaryupstreamanddownstreamofthereg ionofinterestaredesignedwithoverhangadap ters, , Thegene (KlindworthA,PruesseE,SchweerT,PepllesJ, QuastC,etal.(2013)Evaluationofgeneral16 SribosomalRNAgenePCRprimersforclassicala ndnext generationsequencing (1).)

3 ,usingstandardIUPAC nucleotidenomenclature,tofollowtheprotoc oltargetingthisregionare:16 SAmpliconPCRF orwardPrimer=5'TCGTCGGCAGCGTCAGATGTGTATA AGAGACAGCCTACGGGNGGCWGCAG16 SAmpliconPCRR eversePrimer=5'GTCTCGTGGGCTCGGAGATGTGTAT AAGAGACAGGACTACHVGGGTATCTAATCC Thismethodcanalsobeutilizedtotargetother regionsonthegenome(eitherfor16 Swithothersetsofprimerpairs,ornon 16 Sregionsthroughoutthegenome;ieanyamplico n).Theoverhangadaptersequencemustbeadded tothelocus specificprimerfortheregiontobetargeted(F igure1).TheIlluminaoverhangadaptersequen cestobeaddedtolocus specificsequencesare:Forwardoverhang:5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG [locus specificsequence]Reverseoverhang:5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG [locus specificsequence]IntroductionPage3 Thefollowingconsiderationsarerecommended fordesigningotherlocus specificprimers.

4 AIlluminarecommendstargetingregionsthatr esultinanampliconthatwhensequencedwithpa ired endreadshasatleast~ ,ifrunning2x300bppaired specificportionofprimer(notincludingover hangsequence)musthaveameltingtemperature (Tm)of60 65 ( ) ,thegene ,thefully assembledprimersequence(includingtheover hang) ,seeConsumablesandEquipment, ,seeAmpliconPrimers, , (5ng/ ) lpersample 15 to 25 CAmpliconPCRR eversePrimer(1 M)5 lpersample 15 to 25 CAmpliconPCRF orwardPrimer(1 M)5 lpersample 15 to 25 lpersample 15 to 25 CMicroseal'A'film96 [Optional]Bioanalyzerchip(AgilentDNA1000 kitcatalog#5067 1504)Procedure1 SetupthefollowingreactionofDNA,2xKAPAHiF iHotStartReadyMix,andprimers:VolumeMicro bialDNA(5ng/ l) lAmpliconPCRF orwardPrimer1 M5 lAmpliconPCRR eversePrimer1 M5 lTotal25 lAmpliconPCRPage62 SealplateandperformPCRinathermalcyclerus ingthefollowingprogram: 95 Cfor3minutes 25cyclesof.

5 95 Cfor30seconds 55 Cfor30seconds 72 Cfor30seconds 72 Cfor5minutes Holdat4 C3[Optional]Run1 ,theexpectedsizeonaBioanalyzertraceafter theAmpliconPCRstepis~ lpersample 15 to 25 CAMPureXPbeads20 lpersample2 to8 CFreshlyPrepared80%Ethanol(EtOH)400 lpersample96 [Optional]Microseal'B'film[Optional]96 wellMIDI plate1platePreparation ,000 gat20 Cfor1minutetocollectcondensation, [Optional-forusewithshakerformixing]Usin gamultichannelpipettesetto25 l, ,thesamplecanremaininthe96 ,add20 , ,washthebeadswithfreshlyprepared80%ethan olasfollows:aUsingamultichannelpipette,a dd200 loffreshlyprepared80% ,performasecondethanolwashasfollows:aUsi ngamultichannelpipette,add200 loffreshlyprepared80% ,allowthebeadstoair , ,changingtipsaftereachcolumn(orsealplate andshakeat1800rpmfor2minutes).

6 ,carefullytransfer50 lofthesupernatantfromtheAmpliconPCRplate toanew96 ,sealplatewithMicroseal B adhesivesealandstoreitat 15 to 25 lpersample 15 to 25 CNexteraXTIndex1 Primers(N7XX)fromtheNexteraXTIndexkit(FC 131 1001orFC 131 1002)5 lpersample 15 to 25 CNexteraXTIndex2 Primers(S5XX)fromtheNexteraXTIndexkit(FC 131 1001orFC 131 1002)5 lpersample 15 to 25 CPCRG radeWater10 lpersampleTruSeqIndexPlateFixture(FC 130 1005)196 'A'film1 Procedure1 Usingamultichannelpipette,transfer5 lfromeachwelltoanew96 ( )usingthefollowingarrangementsasneeded:a ArrangeIndex2primertubes(whitecaps,clear solution)vertically, (orangecaps,yellowsolution)horizontally, ,seeDualIndexingPrinciple, (whitecaps)BIndex1primers(orangecaps)C96 wellplate3 Placethe96 wellPCRplatewiththe5 ,Index1and2primers,2xKAPAHiFiHotStartRea dyMix,andPCRG radewater:VolumeDNA5 lNexteraXTIndexPrimer1(N7xx)5 lNexteraXTIndexPrimer2(S5xx)5 l2xKAPAHiFiHotStartReadyMix25 lPCRG radewater10 lTotal50 'A'.

7 7 Centrifugetheplateat1,000 gat20 : 95 Cfor3minutes 8cyclesof: 95 Cfor30seconds 55 Cfor30seconds 72 Cfor30seconds 72 Cfor5minutes Holdat4 CIndexPCRPage12 PCRC lean lpersample 15 to 25 CAMPureXPbeads56 lpersample2 to8 CFreshlyPrepared80%Ethanol(EtOH)400 lpersample96 [Optional]Microseal'B'film[Optional]96 wellMIDI plate1plateProcedure1 CentrifugetheIndexPCRplateat280 gat20 [Optional-forusewithshakerformixing]Usin gamultichannelpipettesetto50 l, ,thesamplecanremaininthe96 ,add56 , ,washthebeadswithfreshlyprepared80%ethan olasfollows:aUsingamultichannelpipette,a dd200 loffreshlyprepared80% ,performasecondethanolwashasfollows.

8 AUsingamultichannelpipette,add200 loffreshlyprepared80% ,allowthebeadstoair , wellPCRplate,gentlypipettemixupanddown10 timesuntilbeadsarefullyresuspended, plate, ,carefullytransfer25 lofthesupernatantfromtheIndexPCRplatetoa new96 ,Normalization,andPooling,onpage16,sealt heplatewithMicroseal B 15 to 25 [Optional]ValidateLibraryRun1 lofa1 ,theexpectedsizeonaBioanalyzertraceofthe finallibraryis~ [Optional]ValidateLibraryPage15 LibraryQuantification,Normalization, ,basedonthesizeofDNAampliconsasdetermine dbyanAgilentTechnologies2100 Bioanalyzertrace:(concentrationinng/ l)(660g/mol averagelibrarysize) 106=concentrationinnMForexample.

9 15ng/ l(660g/mol 500) 106=45nMDiluteconcentratedfinallibraryus ingResuspensionBuffer(RSB) , ,>100, ,giventheMiSeqoutputof> ,Normalization,andPoolingPage16 LibraryDenaturingandMiSeqSampleLoadingIn preparationforclustergenerationandsequen cing,pooledlibrariesaredenaturedwithNaOH ,dilutedwithhybridizationbuffer, (ResuspensionBuffer)6 l 15 to 25 CHT1(HybridizationBuffer)1540 l 15 to 25 (lessthanaweekold)10 l 15 to 25 CPhiXControlKitv3(FC 110 3001)4 l 15 to 25 CMiSeqreagentcartridge1cartridge 15 to 25 (screwcaprecommended) C2 RemoveaMiSeqreagentcartridgefrom 15 Cto 25 ,prepareanice : 4nMpooledlibrary(5 l) (5 l) ,andthencentrifugethesamplesolutionat280 gat20 chilledHT1tothetubecontainingdenaturedDN A: DenaturedDNA(10 l)LibraryDenaturingandMiSeqSampleLoading Page17 Pre chilledHT1(990 l) :NOTEI lluminarecommendstargeting800 1000K/mm l120 l180 l240 l300 lPre chilledHT1540 l480 l420 l360 l300 : 10nMPhiXlibrary(2 l) (3 l) : 4nMPhiXlibrary(5 l) (5 l) chilledHT1tothetubecontainingdenaturedPh iXlibrarytoresultina20pMPhiXlibrary.

10 DenaturedPhiXlibrary(10 l) Pre chilledHT1(990 l)6 Dilutethedenatured20pMPhiXlibrarytothesa meloadingconcentrationastheAmpliconlibra ryasfollows:LibraryDenaturingandMiSeqSam pleLoadingPage18 FinalConcentration2pM4pM6pM8pM10pM20pMde naturedlibrary60 l120 l180 l240 l300 lPre chilledHT1540 l480 l420 l360 l300 inof 5% , ,updatetov3software( ).IfyouareusinganolderversionoftheMiSeqs oftwareorsequencingtheselibrariesontheGA orHiSeq,Illuminarecommendsusing 25%PhiXcontrolspike : DenaturedanddilutedPhiXcontrol(30 l) Denaturedanddilutedampliconlibrary(570 l) ,incubatethecombinedlibraryandPhiXcontro ltubeat96 ,invertthetube1 2timestomixandimmediatelyplaceintheice ,theMiSeqsystemprovideson instrumentsecondaryanalysisusingtheMiSeq Reportersoftware(MSR).