Choice of 16S ribosomal RNA primers affects the microbiome ...

Bacterial 16S rRNA contains 9 hypervariable regions used to calculate evolutionary relationships and similarities between species, that are ... Skiptomaincontent Thankyouforvisitingnature.com.YouareusingabrowserversionwithlimitedsupportforCSS.Toobtain thebestexperience,werecommendyouuseamoreuptodatebrowser(orturnoffcompatibilitymodein InternetExplorer).Inthemeantime,toensurecontinuedsupport,wearedisplayingthesitewithoutstyles andJavaScript. Advertisement nature scientificreports articles article Choiceof16SribosomalRNAprimersaffectsthemicrobiomeanalysisinchickenceca DownloadPDF DownloadPDF Subjects MicrobiologyMolecularbiology AbstractWeevaluatedtheeffectofapplyingdifferentsetsof16SrRNAprimersonbacterialcomposition,diversity,andpredictedfunctioninchickenceca.CecalcontentsfromRoss708birdsat1,3,and5 weeksofagewerecollectedforDNAisolation.Eightdifferentprimerpairstargetingdifferentvariableregionsofthe16SrRNAgenewereemployed.DNAsequenceswereanalyzedusingopen-sourceplatformQIIME2andtheGreengenesdatabase.PICRUSt2wasusedtodeterminethepredictedfunctionofbacterialcommunities.Changesinbacterialrelativeabundancedueto16SprimersweredeterminedbyGLMs.TheaveragePCRampliconsizerangedfrom315 bp(V3)to769 bp(V4–V6).Alpha-andbeta-diversity,taxonomiccomposition,andpredictedfunctionsweresignificantlyaffectedbytheprimerchoice.BetadiversityanalysisbasedonUnweightedUniFracdistancematrixshowedseparationofmicrobiotawithfourdifferentclustersofbacterialcommunities.Basedonthealpha-andbeta-diversityandtaxonomiccomposition,variableregionsV1–V3(1)and(2),andV3–V4andV3–V5wereinmostconsensus.Ourdatastronglysuggestthatselectionofparticularsetsofthe16SrRNAprimerscanimpactmicrobiotaanalysisandinterpretationofresultsinchickenaswasshownpreviouslyforhumansandotheranimalspecies. IntroductionBacteriaarethemajorcomponentofchickengastrointestinaltract(GIT)microbiotathatplaysimportantroleinhealth,nutrition,hostphysiologyregulation,GITdevelopment,andgrowth.Microbiotacompositionandfunctioncanbeaffectedbyage,hostgenotypeandsex,dietcompositionandform,dietaryingredientssuchasprobiotics,prebiotics,synbiotics,phytobioticsandbacteriophages,stress,antibiotics,andGITlocation1,2,3.Recently,thenumberofavailabledatacharacterizingtheavianmicrobiotahassignificantlyincreased4.Publishedpapersmostlyfocusedontheimpactofdiet5,6,disease7,8,antibiotics5,probiotics7,9,10,prebiotics11,12andenvironmentalexposures13onthechickenmicrobiota.Analysisofthemicrobiotaisbelievedtobeimportanttoimproveanimalnutritionstrategies,animalhealth,andwell-being.Inchickens,adiversemicrobiotaisfoundthroughouttheGITwiththemostdiversityinthececumwhichservesasakeyorganforfermentationofvariousformsofpolysaccharidestoshort-chainfattyacids14,15.Historically,microbiotainGITwasdetectedbybiochemical,microbiological,immunological,andmolecularbiologytechniques16.BecausemostofthemicrobiotainGITisstrictlyanaerobic,itwasdifficulttoidentifyandcharacterizeindividualspeciesusingclassicmethodology16.Withtime,moresophisticatedmolecularbiologymethodsweredevelopedtocharacterizemicrobiota,includingPCR,denaturinggradientgelelectrophoresis(DGGE),temperaturegradientelectrophoresis(TGGE),microarrays,andnext-generationsequencing(NGS)17,18.Recently,themicrobialcommunityprofilingmethodbasedonthe16SribosomalRNA(rRNA)sequencingapproach(NGS)hasbecomethemostpopulartodeterminethetaxonomiccompositionanddiversityofchickenmicrobiota19.Bacterial16SrRNAcontains9hypervariableregionsusedtocalculateevolutionaryrelationshipsandsimilaritiesbetweenspecies,thatareflankedbyhighlyconservedregionswhicharegenerallyusedtodesignpolymerasechainreaction(PCR)primers20.The16SrRNAprofilingconsistsofmanystepssuchas:samplecollectionsandstorage,DNAisolation,16Sprimerselections,16SrRNAPCRs,librariespreparationsandindexing,sequencing,rawdataanalysis(pipelineorsoftwareselection),OTU/ASV(OperationalTaxonomicUnit/AmpliconSequenceVariant)picking,databaseselection,diversityanalysis,andstatisticalanalysis.SeveralbioinformaticspipelinesforrawsequencesanalysishasbeendevelopedandusedtoprovideataxonomiccompositionandpopulationdiversityincludingMothur21,22andQIIME23,24,25.Inthecaseoftaxonomiccomposition,mostanalysesareperformedusingdatabasessuchasGreengenes26,theRibosomalDatabaseProject27,andSILVA28.Eventhough16Sprofilingisthemostpopularapproachtostudymicrobialdiversity,itischaracterizedbyseverallimitationsincludingampliconsize,primersensitivity,amplificationerrors,andcontamination29.Ithasbeenalreadyshownthatprimerdesign30,31,32,librarypreparation33,DNAisolationmethods34,35,andPCRamplificationartifactscanintroduceuniquebiasesthatcanaffectcommunitystructure,richness,andmicrobialpopulationanalysis36andleadtoover-orunder-representationofindividualbacteriawithincommunities37.Moreover,differentsequencingplatformsandbioinformaticspipelinescanaffecttheaveragerelativeabundanceofmicrobiotaandshapethetaxonomiccommunityprofiles31,38.Additionally,theMicrobiomeQualityControlproject(MBQC)inhumanmicrobiomestudyreportedthattheDNAisolationmethod,aswellas16SrRNAprimersused,arethemajorsourcesofvariation,withsequencingdepthandsamplestoragehavingasmallerbutdetectableinfluenceonthedata39.Inchickenmicrobiotastudies,eventhoughtheexperimentsarecommonlystandardizedandbasedonidenticalbreeds,theresultsareoftencontradictoryandtheresultsdependonusedanimal(breed,age,gender,etc.),theexperimentaldesign(feedingandsampling),andDNAextractionandsequencingmethods.Therefore,itishardtocomparethosedataandcorrelatethemwitheachother19,40.Thedevelopmentofastandardizedprotocolformicrobiotaprofilinginchickens,similartotheoneusedinhumanmicrobiotaresearch,hasbeenproposedbyBorda-Molinaandcolleagues40toobtaincomparabledatasetsforpoultrymicrobiota.Followingtheaboverecommendationandtakingintoaccountthefactthatmicrobialstudiesinpoultry,covered,sofar,theV1–V3,V3–V4,V4–V5,V4–V6,V1,V3orV4regionofthe16SrRNAgene3,41,42,43,44,45,thepresentstudyaimedtoexploretheinfluenceofapplyingthedifferentsetsof16SrRNAprimersonchickenmicrobiotadiversity,taxonomiccompositionandpredictedfunction.ResultsSequencingAtotalof12samplesobtainedfromchickencecalcontentatthreedifferentages(n = 4forage)wereusedin16SrRNAhigh-throughputsequencingusingeightdifferent16SrRNAprimersets.ChickenDNAamplificationwiththeseprimersetsresultedinaveragedindexedPCRproductsizerangingfrom315 bp(V3)to769 bp(V4–V6)(datanotshown).Inallcases,asinglePCRbandwasvisibleontheelectropherogram,buttheintensityofthePCRbandwasdifferentamongprimerssets,withthelowestoneforV3andthehighestoneforV1–V3(1),V3–V4andV4–V5,andV3–V5(datanotshown).Sequencingof12samplesgenerated16,050,150sequenceswith15,776to939,976sequencespersample.Afterremovingchimericsequences,thetotalpoolofsequenceswasreducedto11,113,440readswith13,524to634,783sequencespersample. MicrobiotadiversityanalysisSignificant(P