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TheDrosophilaGeneExpressionTool(DGET)forexpressionanalyses

YanhuiHu1,AramComjean1,NorbertPerrimon1,2,StephanieMohr1

1. Dept.ofGenetics,HarvardMedicalSchool,Boston,MA0211;2.HowardHughesMedicalInstitute

Correspondingauthor:StephanieMohr

Abstract

Background

Next-generationsequencingtechnologieshavegreatlyincreasedourabilitytoidentifygeneexpressionlevels,includingatspecificdevelopmentalstagesandinspecifictissues.Geneexpressiondatacanhelpresearchersunderstandthediversefunctionsofgenesandgenenetworks,aswellashelpinthedesignofspecificandefficientfunctionalstudies,suchasbyhelpingresearcherschoosethemostappropriatetissueforastudyofagroupofgenes,orconversely,bylimitingalonglistofgenecandidatestothesubsetthatarenormallyexpressedatagivenstageorinagiventissue.

Results

WereportaDrosophilaGeneExpressionTool(DGET,www.flyrnai.org/tools/dget/web/),whichstoresandfacilitatessearchofRNA-SeqbasedexpressionprofilesavailablefromthemodENCODEconsortiumandotherpublicdatasets.UsingDGET,researchersareabletolookupgeneexpressionprofiles,filterresultsbasedonthresholdexpressionvalues,andcompareexpressiondataacrossdifferentdevelopmentalstages,tissuesandtreatments.Inaddition,atDGETaresearchercananalyzetissueorstage-specificenrichmentforaninputtedlistofgenes(e.g.‘hits’fromascreen)andsearchforadditionalgeneswithsimilarexpressionpatterns.Weperformedanumberofanalysestodemonstratethequalityandrobustnessoftheresource.Inparticular,weshowthatevolutionaryconservedgenesexpressedathighormoderatelevelsinbothflyandhumantendtobeexpressedinsimilartissues.UsingDGET,wecomparedwholetissueprofileandsub-region/cell-typespecificdatasetsandestimatedthepotentialcauseoffalsepositivesinonedataset.WealsodemonstratedtheusefulnessofDGETforsynexpressionstudiesbyqueryinggeneswithsimilarexpressionprofiletothemesodermalmasterregulatorTwist.

Conclusion

Altogether,DGETprovidesaflexibletoolforexpressiondataretrievalandanalysiswithshortorlonglistsofDrosophilagenes,whichcanhelpscientiststodesignstage-ortissue-specificinvivostudiesanddoothersubsequentanalyses.

Keywords

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Drosophila,RNA-Seq,expressionprofile,synexpression

Background

Theapplicationofnext-generationsequencetechnologiestoRNAanalysishasopenedthedoortorelativelyrapid,large-scaleanalysesofgeneexpression.‘Standard’RNA-seqanalysis,forexample,canprovideasnapshotofgeneexpressioninspecificcelltypesortissues(Wang,Gersteinetal.2009),andrelatedtechnologiessuchasRibo-seq(MichelandBaranov2013)providemorerefinedviews,suchasasnapshotofwhatgenesareactivelytranscribedinagivencellortissue.ForDrosophila,effortssuchasthemodENCODEproject(mod,Royetal.2010,Cherbas,Willinghametal.2011,Graveley,Brooksetal.2011,Boley,Wanetal.2014)haveprovidedabaselineoverviewofexpressionunderstandardlaboratoryconditionsforvariousculturedcelltypes,developmentalstages,andtissues,aswellastreatmentconditions.Moreover,studiessuchasthoseinvestigatingexpressioninsub-regionsoftheflygut(MarianesandSpradling2013,Dutta,Dobsonetal.2015)areprovidingincreasinglydetailedviewsofthebaselineexpressionlevelsofvariousgenesinvarioustissues,celltypesandsub-regions.Altogether,theseRNAseqdataresourcesprovidehelpfulstartingpointsforanalysisofothergenelists.

ResourcessuchasFlyBase(dosSantos,Schroederetal.2015)makeitpossibletoquicklyviewmodENCODEdataforagivengeneandmakethesedatagenerallyaccessibletothecommunity.Thevalueofthesedatatothecommunitycanbefurtherincreasedbyfacilitatingsearchoflistsofgenes.Forexample,forgenelistsoriginatingfromwhole-animalorculturedcellstudies,orforstudiesbasedonalistoforthologsofgenesfromanotherspecies,itcanbeveryhelpfultogetapictureofwhatstagesortissuesnormallyexpressthosegenes,asthatwillhelpfocusstage-ortissue-specificinvivostudiesandothersubsequentanalyses.WeimplementedDGETtohelpscientistsretrievemodENCODEexpressiondatainbatchmode.DGETalsohostsotherrelevantRNA-Seqdatasetspublishedinindividualstudies,suchasprofilesofspecificsub-regionsandcelltypesoftheDrosophilagut(MarianesandSpradling2013,Dutta,Dobsonetal.2015).Here,wedescribeDGETandperformanumberofanalysestodemonstratethequalityandrobustnessoftheresource.

ResultsandDiscussion

Databasecontentandfeaturesoftheuserinterface(UI)

TheDGETdatabasecontainsprocessedRNA-SeqdatafromthemodENCODEconsortium(mod,Royetal.2010,Cherbas,Willinghametal.2011,Graveley,Brooksetal.2011,Boley,Wanetal.2014),asreleasedbyFlyBase(dosSantos,Schroederetal.2015),aswellasotherpublisheddatasetsweobtainedfromsupplementaltables(MarianesandSpradling2013,Dutta,Dobsonetal.2015,CloughandBarrett2016).TheDGETUIhastwotabs(Figure1).

Atthe“SearchGeneExpression”tab,userscanenteralistofgenesorchooseoneofthepredefinedgeneclassesfromGLAD(Hu,Comjeanetal.2015),e.g.kinases,thenspecifythedatasetstobedisplayed.Therearetwosearchoptions,“lookatexpression”and“enrichmentanalysis.”Theresultspagefor“lookatexpression”displaysexpressionvaluesinaheatmapformat.Atthisresultspage,usershavetheoptiontodownloadtherelevantexpressionvalues;downloadtheheatmap;andfurtherfilterthelistbydefiningacutoff,limittospecificdataset(s),orfilteringoutgenes,forexamplewithlessthan

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1RPKMvaluebasedoncarcassand/ordigestionsystemexpressionof1dayadult.WeusedanRPKMcutoffof1becausethisisconsideredthecutofffor‘noorextremelylowexpression’atFlyBase.Theresultspageforanenrichmentanalysisdisplaysthedistributionofgenesatdifferentexpressionlevelsusingabargraphandheatmap.ThecutoffvaluesfordifferentlevelsaredefinedbasedonFlyBaseguidelines.

Usingthe“SearchSimilarGenes”tab,userscanenterageneofinterestandsearchforothergeneswithsimilarexpressionpatternbasedonPearsoncorrelationscore.Usershavetheoptionstodownloadthelistofgeneswithsimilarexpressionpatterns,aheatmap,andanormalizedheatmap.

ExpressionpatternofDrosophilaregulatorygenes

Whengenome-scalescreeningisnotpracticaltodo,acommonapproachistoselectaspecificsubsetofgenestostartwith,suchasagroupofgeneswithrelatedactivities.Themostfrequentlyscreenedsub-setsofgenesareimportantregulatorygenesincludinggenesthatencodekinases,phosphatases,transcriptionfactors,orcanonicalsignaltransductionpathwayscomponents.Ourexpectationisthattheseregulatorygenes,whichappeartobere-usedinmanycontexts,willbeexpressedinmanytissues.Totestthis,weanalyzedtheexpressionpatternsofseveralDrosophilaregulatorygeneclassesdefinedbyGLAD(Hu,Comjeanetal.2015).Theseincludedcanonicalsignaltransductionpathwaygenes,kinases,phosphatases,transcriptionfactors,secretedproteins,andreceptors.ThepercentagesofexpressedgeneswerecalculatedacrossalltissuesprofiledusingaRPKMof1oraboveasacutoffforexpressedversusnotexpressed(Figure2).About70-90%ofthegenescategorizedasencodingcanonicalsignaltransductionpathwaycomponents,kinases,phosphatases,ortranscriptionfactorsareexpressedineachofthemajortissuecategoriesprofiled,whereasonly30-60%ofreceptororsecretedproteinsaredetectedinanygiventissue.Correlationofexpressionwithconfidenceinanorthologrelationship

Itiswellestablishedthattheevolutionaryconservationofproteinscorrelateswithconservationatthelevelofbiologicaland/orbiochemicalfunctions.Drosophilaisamodelorganismofparticularinterestforwhichawidevarietyofmoleculargenetictoolsarereadilyavailable.Particularly,Drosophilamodelshavebeendevelopedforanumberofhumandiseases(Perrimon,Boninietal.2016).AccordingtoDIOPT,9,705of13,902protein-codinggenesinDrosophilaarepredictedtohavehumanortholog(s)(Hu,Flockhartetal.2011).UsingDGETweanalyzedtheexpressionlevelsofthesubsetofDrosophilagenesforwhichthereisevidencethattheyareconservedinthehumangenome.Specifically,weanalyzedsubsetsofgenesscoringasputativehumanorthologsofflygenesatdifferentlevelsofconfidence,asdefinedbytheDIOPTscore(Hu,Flockhartetal.2011).WefoundastrongcorrelationofpercentexpressedgeneswithDIOPTscore(Figure3).Forexample,forgenesthathaveahigh-confidenceorthologrelationship(DIOPTscoreof7orabove),almostallareexpressedacrossalltissues.Bycontrast,forgenesforwhichDIOPTanalysissuggeststhatthereisnoevidenceofahumanortholog(i.e.noneofthe10orthologalgorithmsqueriedwithDIOPTpredictanortholog),only20-50%areexpressedineachofthemajortissuecategoriesprofiled.Wesuspectthatthiscorrelationisdrivenby

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essentialgenes,whicharemoreconservedevolutionary.Wealsonotethatgenesetenrichmentforthesetofhigh-confidenceorthologsindicatesthat“kinases”and“nucleotidebinding”amongthetop20enrichedsets,indicatingthatthesetofregulatorygenesanalyzedabovehasoverlapwiththisset.

Wenextanalyzedthe418DrosophilaessentialgenesidentifiedbySpradlingetal(Spradling,Sternetal.1999)usingalarge-scalesingleP-elementinsertionflystockcollection.TheproportionsofessentialgenesexpressedatdetectablelevelsinvarioustissuesareverysimilartothegeneswithDIOPTscore7-10(Figure3,lightpurpleanddarkpurplebars)withaPearsoncorrelationcoefficientequalto0.92.ExpressionpatternsofDrosophilaorthologsofhumangenesthatarehighlyexpressedinspecifictissues

Next,weaskedwhethergenesconservedbetweenhumanandDrosophilaarealsoexpressedinsimilarpatterns.Weusedthetissue-basedhumanproteomeannotationavailableattheHumanProteinAtlas(HPA)(www.proteinatlas.org)(Uhlen,Fagerbergetal.2015),asthesourcefortissue-specificexpression,andretrievedthesetofhumangenesthatareexpressedinspecifictissues.Next,wemappedthesehumangenestoDrosophilaorthologsusingDIOPT(Hu,Flockhartetal.2011),filteringoutlowrankorthologpairs(seeMaterialsandMethods),andanalyzedtheexpressionpatternsofthesehigh-confidenceorthologsinDrosophilatissuesusingDGET(Figure4).Theresultsofouranalysisusingallannotatedproteinswithoutafilterdidnotclearlydemonstrateconservationofexpressionpatterns.However,ananalysislimitedtogenesexpressedathighormoderatelevels(asannotatedbyHPA)fromhighconfidentannotation(i.e.excludingHPA“reliability”valueof“uncertain”),indicatesthatgeneexpressionpatternsareconservedinsimilartissuesinDrosophila.Forexample,asagroup,orthologsofgeneshighlyexpressedinthehumancerebellum,cerebralcortex,lateralventricleorhippocampusarehighlyexpressedintheDrosophilacentralnervoussystem(CNS)orhead,atbothlarvalandadultstages,andorthologsofgeneshighlyexpressedinhumantestisarealsohighlyexpressedintheDrosophilatestis.Moreover,orthologsofgenesfromsomeorgansofthehumandigestivesystem,suchasstomach,duodenumorsmallintestine,arealsohighlyexpressedintheDrosophiladigestivesystem.TofurthercomparetheexpressionpatternsofgenesexpressedinthehumanandDrosophila,digestivesystems,weanalyzedtheDrosophilagutsub-regiondatafromDuttaetal.(Dutta,Dobsonetal.2015)(Figure5).OrthologsofgeneshighlyexpressedinthehumansalivaryglandandesophagusarehighlyexpressedintheR1upstreamregion,andorthologsofgeneshighlyexpressedinthehumanrectum,colonorappendixaremorebiasedtowardsexpressionintheR5downstreamregion.Flyorthologsofgeneshighlyexpressedinthehumanstomach,duodenumandsmallintestinearedetectedthroughoutthesamplescorrespondingtoR1toR5.

Mininginformationfromdistinctbutrelatedflygutgeneexpressiondatasets

Wenextsoughttocomparetheresultsofwhole-gutprofilingwithresultsfromprofilingofspecificsub-regionsorcelltypeswiththegoalofidentifyinggenesonlyexpressedinspecificsub-populations.Ourrationalefortheanalysiswastodeterminethelikelihoodthatgenesexpressedinasub-populationaremissedinexpressionanalysisofanentireorgan.Thistypeoffalsenegativeanalysisshouldprovidehelpfulinformationforinterpretingresultsofwhole-organorwhole-tissuestudies.Thus,

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wecomparedthewholegutprofilingdataobtainedbymodENCODEconsortiumfor20dayoldadultflies(mod,Royetal.2010)withdatageneratedbyprofilingsub-regionsofthemidgutin16-20dayoldadultflies(MarianesandSpradling2013).Wholegutprofilingindicatesthat9,109genesareexpressedinthegutof20dayoldadultflies(RPKMcutoffvalueof1).Amongthe4,790protein-codinggenesnotdetectedasexpressedinthewhole-gutstudy,136genesaredetectedinatleast3sub-regionsofthegut(RPKM>=3).Thesegenesareeitherfalsenegativeinwholegutprofilingorfalsepositiveinsub-regionprofiling.Wenextdidagenesetenrichmentanalysiswiththese136genesandfoundthatstressresponsegenes,suchasheat-shockgenes(Hsp70Aa,Hsp70Ab,Hsp70Ba,Hsp70Bbb)areenriched(Pvalue=3.05E-07).Thissuggeststhatthesampleusedforsub-regionprofilingwasassociatedwithsomelevelofstress.Comparingthelistof136geneswiththeDrosophilaessentialgenelist,wefoundonlyoneoverlappinggene.Inaddition,only23ofthe136geneshaveDIOPTscore7-10whenmappingtohumangenes.Thus,smallfractionofthesegenesmightbethefalsenegativewithwholetissueprofilingwhilemajorityofthegenesarelikelytobethefalsepositivesnotnormallypresentinthegutundernon-stressconditions.

SynexpressionanalysisfortranscriptionfactorTwist

Expressionprofilingisapowerfulapproachtoidentifyfunctionallyrelatedgenes,asgenesshowingsynexpressionoftenoperateinsimilarpathwaysand/orprocesses(seeforexample(Dequeant,Fagegaltieretal.2015)).WetestedDGETforitsusefulnessforsynexpressionstudiesbyqueryinggeneswithsimilarexpressionprofiletothemesodermalmasterregulatorTwist.DGETpreferentiallyretrievedTwisttargetgeneswithcelllinedataaswellasdevelopmentdata.Forexample,amongthetop27genesthatsharesimilarexpressionwithTwistincelllines(Pearsoncorrelationco-efficiencycutoff=0.7),11ofthemareTwisttargetgenesbasedonChip-on-chipdata(Sandmann,Girardotetal.2007),and8ofthe11genesarehigh-confidence(Table1).Theenrichmentp-valueforTwisttargetgenesis8.70E-04and3.05E-05forhigh-confidencetargets.Weobservedalesssignificantenrichmentwithdevelopmentdata(p-value5.00E-02forallTwisttargetgenesandp-valueof2.70E-03forhigh-confidencetargets),likelyreflectingthediversityofcelltypespresentinthedevelopmentaldataandthatnotenoughcellsexpresstwist.Thus,DGETwillbeverypowerfulwhenappliedtoRNA-seqdatasetsfromsinglecellorgroupsofhomogeneouscellpopulations.

ConcludingRemarks

Insummary,DGETmakesitpossibletoretrieveandcompareDrosophilageneexpressionpatternsgeneratedbyvariousgroupsusingRNA-Seq.Thetoolcanhelpscientistsdesignexperimentsbasedonexpressionandanalyzeexperimentresults.ThebackenddatabaseforDGETisdesignedtoeasilyaccommodatetheadditionofnewhighqualityRNA-Seqdatasetsastheybecomeavailable.Finally,althoughtheanatomyofhumanandDrosophilaarequitedifferent,byusingDGET,wedemonstratethatexpressionpatternsofgenesthatareconservedandhighlyexpressedareconservedbetweenhumanandDrosophilainmanymatchingtissues,underscoringtheutilityoftheDrosophilamodeltounderstandtheroleofhumangeneswithunknownfunctions.

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Methods

Dataretrieval

ProcessedmodENCODEdatawereretrievedfromFlyBase(ftp://ftp.flybase.net/releases/current/precomputed_files/genes/gene_rpkm_report_fb_2015_05.tsv.gz).DatapublishedbyMarianesandSpradling(MarianesandSpradling2013)wereretrievedfromNCBIGeneExpressionOmnibusat(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47780).DatapublishedbyDuttaetal(Dutta,Dobsonetal.2015)wereretrievedfromtheflygut-seqwebsite(http://flygutseq.buchonlab.com/resources).DataretrievedweremappedtoFlyBaseidentifiersfromrelease2015_5andformattedforuploadintotheFlyRNAidatabase(Hu,Flockhartetal.2011).

Expressionpatternanalysis

Humanproteinexpressiondatawereretrievedfromproteinatlas.organdtissue-specificgeneswereselectedusingthefile“ProteinAtlas_Normal_tissue_vs14.”Proteinswithhighormediumexpressionlevelswithareliabilityvalueof“supportive”wereselected.Proteinsexpressedinabroadrangeoftissues(i.e.morethan5tissues)werefilteredout.DIOPTvs5wasusedtomapgenesfromhumantoDrosophila(Hu,Flockhartetal.2011).‘Orthologpairrank’wasaddedatrecentDIOPTrelease5.2.1(http://www.flyrnai.org/DRSC-ORH.html#versions).Drosophilageneswithhighormoderaterankwereselected.Thehigh/moderaterankmappingincludethegenepairsthatarebestscoreineitherforwardorreversemapping(andDIOPTscore>1)aswellasgenepairswithDIOPTscore>3ifnotbestscoreeitherway.

Implementation

DGETwasimplementedusingphpandJavaScriptwithMySQLdatabasefordatastore.ItishostedonaserverprovidedbytheResearchITGroup(RITG)atHarvardMedicalSchool.TheMySQLdatabaseisalsohostedonaserverprovidedbyRITG.Plottingofheat-mapsforsvgdownloadisdoneinRusingthegplotheatmapfunction.Websitebarchartsaredrawnusingthe3C.jsplottingpackage.ThephpsymfonyframeworkscaffoldisusedtocreateDGETwebpagesandforms.

Declarations

FundingWorkattheDRSCissupportedbyNIGMSR01GM067761,NIGMSR01GM084947,andORIP/NCRRR24RR032668.S.E.M.isadditionallysupportedinpartbyNCICancerCenterSupportGrantNIH5P30CA06516(E.Benz,PI).N.PisanInvestigatoroftheHowardHughesMedicalInstitute.

Competinginterest

Theauthorsdeclarethattheyhavenocompetinginterests.

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Authors’contributions

YHdesignedandtestedtheapplication,implementedtheback-endoftheapplication,performedtheanalysisanddraftedthemanuscript.ACimplementedtheuserinterfaceandcontributedtotheback-endoftheapplication.NPprovidedcriticalinputonkeyfeaturesandtheanalysisaswellaseditedthemanuscript.SEMprovidedoversightandcriticalinputonkeyfeaturesandtheanalysis,andhelpeddraftthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.

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Table1.DGETsimilargenesearchresultsforTwistwithcelllinedataFBgn Gene Correlationscore Twisttarget?*FBgn0005636 nvy 0.910987 Yes,highconfidentFBgn0031738 CG9171 0.88094 Yes,highconfidentFBgn0015568 alpha-Est1 0.831094 FBgn0035733 CG8641 0.816603 FBgn0034997 CG3376 0.813417 Yes,lowconfidentFBgn0040091 Ugt58Fa 0.799835 FBgn0039827 CG1544 0.773761 FBgn0010389 htl 0.772568 Yes,highconfidentFBgn0001250 if 0.769353 Yes,highconfidentFBgn0039799 CG15543 0.765649 FBgn0038755 Hs6st 0.76281 Yes,highconfidentFBgn0265577 CR44404 0.76095 FBgn0037439 CG10286 0.745739 FBgn0025682 scf 0.744414 FBgn0003301 rut 0.74375 Yes,lowconfidentFBgn0036147 Plod 0.73896 FBgn0000723 FER 0.738927 Yes,lowconfidentFBgn0034804 CG3831 0.735346 FBgn0051075 CG31075 0.731916 FBgn0263144 bin3 0.72961 Yes,highconfidentFBgn0000575 emc 0.728894 Yes,highconfidentFBgn0038353 CG5399 0.724139 FBgn0085407 Pvf3 0.720044 Yes,highconfidentFBgn0036857 CG9629 0.716929 FBgn0039073 CG4408 0.714359 FBgn0037632 Tcp-1eta 0.702547 FBgn0038804 CG10877 0.701509 *Twisttargetsasdefinedin(Sandmann,Girardotetal.2007)

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Figure1.TheDGETuserinterface.

1a.Onthe“SearchGeneExpression”page,userscaninputagenelistbypastingDrosophilageneorproteinsymbolsorIDsintothetextbox,orbyuploadingafile.ThespecificidentifiersacceptedareFlyBaseGeneIdentifier(FBgn),genesymbol,CGnumber,andfullgenename.UserscanchoosetolookatexpressionpatternsorperformanenrichmentanalysisoftheinputtedlistascomparedwiththeunderlyingRNA-Seqdata.

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1b.Atthe“SearchSimilarGenes”page,userscanenteragenesymbol(orotheracceptedidentifier)tofindgeneswithsimilarexpressionpatterns.

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Figure2.ExpressionpatternsofgenesinmajorDrosophilaregulatorygenegroups.

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Figure3.Relationshipbetweenexpressionlevelsandgeneconservation.

Drosophilagenesthatareconservedinthehumangenomeatdifferentconfidencelevels(i.e.differentDIOPTscores)wereanalyzedbyDGET.Wefoundthatacrossalltissues,expressionlevelscorrelatewithconfidenceintheorthologrelationship.Thatis,ingeneral,geneswithhigherDIOPTscoresvs.humangeneshavehigherexpressionlevels.GeneswithDIOPTscoresof7-10(lightpurplebars)havesimilarexpressionpatternsascomparedwithDrosophilaessentialgenes(darkpurplebars);i.e.inbothcases,thegenesarelikelytobeexpressedinmanyoralltissues.

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Figure4.ComparisonofgeneexpressionpatternsinhumansandDrosophila.

High-confidenceDrosophilaorthologsofgenesthatarehighlyexpressedinthesmallintestine,ovary,testis,cerebellum,cerebralcortex,orothertissueswereanalyzedusingDGET.Foratleastsometissues,weseeacorrelationbetweengeneshighlyexpressedinspecifichumantissues(e.g.cerebellum,testis)andtheexpressionoforthologsincognatetissuesample(s)availableforDrosophila(e.g.CNSorhead,testis).

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Figure5.ComparisonofDrosophilagutsub-regiondatawiththehumandigestivesystem.

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References

Boley,N.,K.H.Wan,P.J.BickelandS.E.Celniker(2014)."NavigatingandminingmodENCODEdata."Methods68(1):38-47.Cherbas,L.,A.Willingham,D.Zhang,L.Yang,Y.Zou,B.D.Eads,J.W.Carlson,J.M.Landolin,P.Kapranov,J.Dumais,A.Samsonova,J.H.Choi,J.Roberts,C.A.Davis,H.Tang,M.J.vanBaren,S.Ghosh,A.Dobin,K.Bell,W.Lin,L.Langton,M.O.Duff,A.E.Tenney,C.Zaleski,M.R.Brent,R.A.Hoskins,T.C.Kaufman,J.Andrews,B.R.Graveley,N.Perrimon,S.E.Celniker,T.R.GingerasandP.Cherbas(2011)."Thetranscriptionaldiversityof25Drosophilacelllines."GenomeRes21(2):301-314.Clough,E.andT.Barrett(2016)."TheGeneExpressionOmnibusDatabase."MethodsMolBiol1418:93-110.Dequeant,M.L.,D.Fagegaltier,Y.Hu,K.Spirohn,A.Simcox,G.J.HannonandN.Perrimon(2015)."Discoveryofprogenitorcellsignaturesbytime-seriessynexpressionanalysisduringDrosophilaembryoniccellimmortalization."ProcNatlAcadSciUSA112(42):12974-12979.dosSantos,G.,A.J.Schroeder,J.L.Goodman,V.B.Strelets,M.A.Crosby,J.Thurmond,D.B.Emmert,W.M.GelbartandC.FlyBase(2015)."FlyBase:introductionoftheDrosophilamelanogasterRelease6referencegenomeassemblyandlarge-scalemigrationofgenomeannotations."NucleicAcidsRes43(Databaseissue):D690-697.Dutta,D.,A.J.Dobson,P.L.Houtz,C.Glasser,J.Revah,J.Korzelius,P.H.Patel,B.A.EdgarandN.Buchon(2015)."RegionalCell-SpecificTranscriptomeMappingRevealsRegulatoryComplexityintheAdultDrosophilaMidgut."CellRep12(2):346-358.Graveley,B.R.,A.N.Brooks,J.W.Carlson,M.O.Duff,J.M.Landolin,L.Yang,C.G.Artieri,M.J.vanBaren,N.Boley,B.W.Booth,J.B.Brown,L.Cherbas,C.A.Davis,A.Dobin,R.Li,W.Lin,J.H.Malone,N.R.Mattiuzzo,D.Miller,D.Sturgill,B.B.Tuch,C.Zaleski,D.Zhang,M.Blanchette,S.Dudoit,B.Eads,R.E.Green,A.Hammonds,L.Jiang,P.Kapranov,L.Langton,N.Perrimon,J.E.Sandler,K.H.Wan,A.Willingham,Y.Zhang,Y.Zou,J.Andrews,P.J.Bickel,S.E.Brenner,M.R.Brent,P.Cherbas,T.R.Gingeras,R.A.Hoskins,T.C.Kaufman,B.OliverandS.E.Celniker(2011)."ThedevelopmentaltranscriptomeofDrosophilamelanogaster."Nature471(7339):473-479.Hu,Y.,A.Comjean,L.A.Perkins,N.PerrimonandS.E.Mohr(2015)."GLAD:anOnlineDatabaseofGeneListAnnotationforDrosophila."JGenomics3:75-81.Hu,Y.,I.Flockhart,A.Vinayagam,C.Bergwitz,B.Berger,N.PerrimonandS.E.Mohr(2011)."Anintegrativeapproachtoorthologpredictionfordisease-focusedandotherfunctionalstudies."BMCBioinformatics12:357.Marianes,A.andA.C.Spradling(2013)."PhysiologicalandstemcellcompartmentalizationwithintheDrosophilamidgut."Elife2:e00886.Michel,A.M.andP.V.Baranov(2013)."Ribosomeprofiling:aHi-Defmonitorforproteinsynthesisatthegenome-widescale."WileyInterdiscipRevRNA4(5):473-490.mod,E.C.,S.Roy,J.Ernst,P.V.Kharchenko,P.Kheradpour,N.Negre,M.L.Eaton,J.M.Landolin,C.A.Bristow,L.Ma,M.F.Lin,S.Washietl,B.I.Arshinoff,F.Ay,P.E.Meyer,N.Robine,N.L.Washington,L.DiStefano,E.Berezikov,C.D.Brown,R.Candeias,J.W.Carlson,A.Carr,I.Jungreis,D.Marbach,R.Sealfon,M.Y.Tolstorukov,S.Will,A.A.Alekseyenko,C.Artieri,B.W.Booth,A.N.Brooks,Q.Dai,C.A.Davis,M.O.Duff,X.Feng,A.A.Gorchakov,T.Gu,J.G.Henikoff,P.Kapranov,R.Li,H.K.MacAlpine,J.Malone,A.Minoda,J.Nordman,K.Okamura,M.Perry,S.K.Powell,N.C.Riddle,A.Sakai,A.Samsonova,J.E.Sandler,Y.B.Schwartz,N.Sher,R.Spokony,D.Sturgill,M.vanBaren,K.H.Wan,L.Yang,C.Yu,E.Feingold,P.Good,M.Guyer,R.Lowdon,K.Ahmad,J.Andrews,B.Berger,S.E.Brenner,M.R.Brent,L.Cherbas,S.C.Elgin,T.R.Gingeras,R.Grossman,R.A.Hoskins,T.C.Kaufman,W.Kent,M.I.Kuroda,T.Orr-Weaver,N.Perrimon,V.Pirrotta,J.W.Posakony,B.Ren,S.Russell,P.Cherbas,B.R.Graveley,S.Lewis,G.Micklem,B.Oliver,P.J.Park,S.E.Celniker,S.Henikoff,G.H.Karpen,E.C.Lai,D.M.

.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted September 15, 2016. . https://doi.org/10.1101/075358doi: bioRxiv preprint

MacAlpine,L.D.Stein,K.P.WhiteandM.Kellis(2010)."IdentificationoffunctionalelementsandregulatorycircuitsbyDrosophilamodENCODE."Science330(6012):1787-1797.Perrimon,N.,N.M.BoniniandP.Dhillon(2016)."Fruitfliesonthefrontline:thetranslationalimpactofDrosophila."DisModelMech9(3):229-231.Sandmann,T.,C.Girardot,M.Brehme,W.Tongprasit,V.StolcandE.E.Furlong(2007)."AcoretranscriptionalnetworkforearlymesodermdevelopmentinDrosophilamelanogaster."GenesDev21(4):436-449.Spradling,A.C.,D.Stern,A.Beaton,E.J.Rhem,T.Laverty,N.Mozden,S.MisraandG.M.Rubin(1999)."TheBerkeleyDrosophilaGenomeProjectgenedisruptionproject:SingleP-elementinsertionsmutating25%ofvitalDrosophilagenes."Genetics153(1):135-177.Uhlen,M.,L.Fagerberg,B.M.Hallstrom,C.Lindskog,P.Oksvold,A.Mardinoglu,A.Sivertsson,C.Kampf,E.Sjostedt,A.Asplund,I.Olsson,K.Edlund,E.Lundberg,S.Navani,C.A.Szigyarto,J.Odeberg,D.Djureinovic,J.O.Takanen,S.Hober,T.Alm,P.H.Edqvist,H.Berling,H.Tegel,J.Mulder,J.Rockberg,P.Nilsson,J.M.Schwenk,M.Hamsten,K.vonFeilitzen,M.Forsberg,L.Persson,F.Johansson,M.Zwahlen,G.vonHeijne,J.NielsenandF.Ponten(2015)."Proteomics.Tissue-basedmapofthehumanproteome."Science347(6220):1260419.Wang,Z.,M.GersteinandM.Snyder(2009)."RNA-Seq:arevolutionarytoolfortranscriptomics."NatRevGenet10(1):57-63.

.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted September 15, 2016. . https://doi.org/10.1101/075358doi: bioRxiv preprint