Supplementary Materials The microbiomes of blowflies and houseflies as bacterial transmission reservoirs Ana Carolina M. Junqueira, Aakrosh Ratan , Enzo Acerbi, Daniela I. Drautz-Moses, Balakrishnan N. V. Premkrishnan, Paul I. Costea, Bodo Linz, Rikky W. Purbojati, Daniel F. Paulo, Nicolas E. Gaultier, Poorani Subramanian, Nur A. Hasan, Rita R. Colwell, Peer Bork, Ana Maria L. Azeredo-Espin, Donald A. Bryant, Stephan C. Schuster Figures S1 to S14 Tables S1, S7, and S8 Supplementary data - Tables S2, S3, S4, S5, and S6 Legends for the supplementary excel format tables: Supplementary Table S2. Metadata of 116 samples of blowflies and houseflies and details of the data generated, filtered, and assigned by each bioinformatics method. Bit-score cutoffs for taxonomic assignment based on read length are also provided. Supplementary Table S3. Species-level assignment of the microbiome of blowflies (C. megacephala) and houseflies (M. domestica) using dbAssign. Detailed information about number of normalized reads assigned to each bacterial species in each fly sample. Supplementary Table S4. Species-level assignment of the microbiome of blowflies (C. megacephala) and houseflies (M. domestica) using rapsearch2. Detailed information on number of normalized reads assigned to each microbial species in each fly sample. Supplementary Table S5. Species identification of the microbiomes of blowflies (C. megacephala) and houseflies (M. domestica) using specI. Detailed information about number of normalized reads assigned to each microbial species in each fly sample. Supplementary Table S6. Summary of the bacterial species assigned by the dbAssign toolset, indicating shared and unique species in blowflies and houseflies. 1
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Supplementary Materials - static-content.springer.com10.1038/s41598-017... · of the blowfly (Shannon-Weaver diversity index). Legs+wings harbor the highest bacterial diversity, despite
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SupplementaryMaterials
ThemicrobiomesofblowfliesandhousefliesasbacterialtransmissionreservoirsAnaCarolinaM.Junqueira,AakroshRatan,EnzoAcerbi,DanielaI.Drautz-Moses,BalakrishnanN.V.Premkrishnan,PaulI.Costea,BodoLinz,RikkyW.Purbojati,DanielF.Paulo,NicolasE.Gaultier, Poorani Subramanian, Nur A. Hasan, Rita R. Colwell, Peer Bork, Ana Maria L.Azeredo-Espin,DonaldA.Bryant,StephanC.Schuster
FiguresS1toS14TablesS1,S7,andS8Supplementarydata-TablesS2,S3,S4,S5,andS6Legendsforthesupplementaryexcelformattables:SupplementaryTableS2.Metadataof116samplesofblowfliesandhousefliesanddetailsofthedata generated, filtered, and assigned by each bioinformatics method. Bit-score cutoffs fortaxonomicassignmentbasedonreadlengtharealsoprovided.Supplementary Table S3. Species-level assignment of the microbiome of blowflies (C.megacephala)andhouseflies(M.domestica)usingdbAssign.Detailedinformationaboutnumberofnormalizedreadsassignedtoeachbacterialspeciesineachflysample.Supplementary Table S4. Species-level assignment of the microbiome of blowflies (C.megacephala)andhouseflies(M.domestica)usingrapsearch2.Detailedinformationonnumberofnormalizedreadsassignedtoeachmicrobialspeciesineachflysample.SupplementaryTableS5.Speciesidentificationofthemicrobiomesofblowflies(C.megacephala)andhouseflies(M.domestica)usingspecI.Detailedinformationaboutnumberofnormalizedreadsassignedtoeachmicrobialspeciesineachflysample.Supplementary Table S6. Summary of the bacterial species assigned by the dbAssign toolset,indicatingsharedanduniquespeciesinblowfliesandhouseflies.
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SupplementaryFigureS1.CategoriesofreadsassignedtoEukaryotesdomainusingrapsearch2againstthenon-redundantNCBIdatabase,showingthatmostmatchedtoflyspeciesoftheorderDiptera,thusrevealingthereadsthatpassedhost-genomefiltering.Blowfliesarehighlightedinblueandhousefliesinyellow.Supplementary Figure S2. Themost frequently identified twenty viruses assigned frommetagenomics datasets of 116 blowflies and houseflies using the rapsearch2againstthenon-redundantNCBIdatabase.Blowfliesarehighlightedinblueandhousefliesinyellow.Thebubblesizeislog-scaledandreferstothenormalizednumberofreads.
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Supplementary Figure S3. Phylum-level assignment of normalized metagenomic datasets withdifferent bioinformatics methods. (A) reads assigned against the complete bacterial genomesdatabasefromNCBIusingin-housedevelopedscriptdbAssign.(B)readsassignedusingspecIclustersof 40 single universal genes. Proteobacteria, Bacteroidetes and Firmicutes are themost abundantphyla described in the blowfly Chrysomyamegacephala (blue shadow) and in the houseflyMuscadomestica(yellowshadow).
SupplementaryFigureS4.Diversityofbacterialtaxainblowfliesandhouseflies.Boxplotsshowdistributionofalpha-diversity(Shannon-Weaverindex)foundin116samples.Solidlinereferstomediananddashedlinetomean values. Whiskers represent minimum and maximum values. Individual points represent outlier andasterisk indicates alpha-diversity of the lab-reared pool of blowflies serving as control (values of 3.004 forrapsearch2 and 0.576 for dbAssign). Full data are available in Supplementary Tables S2 and S3. Differentdatabases used for rapsearch2 (nr) and dbAssign (bacterial complete genomes) provided different bacterialdiversityforblowfliesandhouseflies.dbAssignrecoveredlessdiversity(blowflies=1.69;houseflies=3.15)thanrapsearch2(blowflies=5.18;houseflies=5.04)duetodifferentstringenciesusedforreadmappingparametersanddifferentdatabases.
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SupplementaryFigureS5.Rarefactioncurvesofallblowflies(bluelines)andhouseflies(yellowlines)analyzedwith rapsearch2 (A) and dbAssign (B). The OTU discovery levels off around 25,000 reads sampled withrapsearch2 and around 500 reads for dbAssign, reflecting differences in databases used and alignmentstringency.TheboxplotsintherightuppercornersshowstheOTUrichnessperhostspecies,compilingthetotalnumberofobservedbacterialspeciesassignedforthetwovectors.Solidlinereferstomediananddashedlinetomeanvalues.Whiskersrepresentminimumandmaximumvalues.Individualpointsrepresentoutliers.
A BSupplementaryFigureS9.PCoAplotsofthebeta-diversityofmicrobiomeofindividualblowfliesandhouseflies.Samplesarecolouredbasedongeographicaloriginofthesamples.Thegeographicoriginofsampleshadaminoreffect on the segregationof flies and explains a small proportionof thebeta-diversity variation foundwithrapsearch2(A)anddbAssign(B).R-squaredandp-valuesareshownabovetheplots.PCoAandPERMANOVAweregeneratedwiththenormalizeddatasetsusing‘species’taxonomicrankwithBray-Curtisindex.
Supplementary Figure 10. Comparison of methods used for OTU assignment. Venn diagram showing thenumberofOTUsassignedtospeciesbythreedifferentbioinformaticsapproaches.Onlyhigh-confidencepaired-endreadsthatweremappedabove97%identityarerepresentedbythespecIcircle(totalof50OTUs)andonlyOTUswithmorethan500mappedreadswereconsideredforanalysiswithrapsearch2(1,655microbialspecies)and dbAssign (316 prokaryotic species) comparison. A total of 33 bacterial specieswere identified by all 3methodsandarelistedinTableS7.
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SupplementaryFigureS11.Alpha-diversityofbacterialspeciesassignedtothemicrobiomeoffourbodypartsoftheblowfly(Shannon-Weaverdiversityindex).Legs+wingsharborthehighestbacterialdiversity,despitetheleastnumberof reads generated, compared tohead, thorax andabdomen. This is particularly important inestablishingtheroleoftheouterbodyasthemainrouteofbacterialdispersalbymechanicalvectors.Fungi,archaeaandvirusesshowalowdiversityandwerenotdetectedinmostofthefliesanalyzed,aswellasinthefourbodyparts.
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SupplementaryFigureS12.Helicobacterpylorigenomecoveragebymetagenomicreads.(A)ReadsassignedtothegenusHelicobacterspp.wereextractedfromthefourbodypartsinthedbAssignoutputandmappedagainstH.pyloristrain26695referencegenome(NC_000915.1).Atotalof5,890readsweremappedusingbowtie1.1.2(default parameters), covering 25.3% of the reference genome. (B) Metagenomic reads covering the geneclusterinthecagpathogenicityisland.Atotalof18,281identicalsitesweremappedwithpairwiseidentityof97.9%.(C)MetagenomicreadscoveringthevacAvirulencefactorofH.pylori.Thelengthoftheregionanalysedis3,019bp(partialvacAgene),with966identicalsitescoveredwithpairwiseidentityof95.6%.
Figure S14. Overlapping of bacterial species among the carrion fliesmicrobiomes, human gutmicrobiomesproject (data from the gastrointestinal tract downloaded from http://hmpdacc.org/catalog/) and the urbanmicrobiome conducted in the New York City subway system (NYC subway). The highlighted table lists 19bacterialspeciesthatoverlapallthreemicrobiomeprojects.
SupplementaryTableS8.Identificationandrelativeabundanceofthebacterialstrainsinthemetagenomesofthe four body parts (head, thorax, abdomen and legs+wings) using the CosmosID metagenomics softwarepackage.Onlythosebacterialstrainsidentifiedwithconfidenceandabovethresholdarelisted.