Chicken interferome - avian interferon-stimulated genes ... et... · 1 Chicken interferome - avian...
Transcript of Chicken interferome - avian interferon-stimulated genes ... et... · 1 Chicken interferome - avian...
GiotisetalChISGVRsub_manuscript_R4.docx Page1of33
Chicken interferome - avian interferon-stimulated genes identified by microarray and RNA-1
seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α). 2
3
Efstathios S. Giotis1‡, Rebecca C. Robey1‡, Natalie G. Skinner2, Christopher D. Tomlinson3, 4
Stephen Goodbourn4, Michael A. Skinner1* 5
6
1Section of Virology, Faculty of Medicine, Imperial College London, 7
St. Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom 8
2 Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom 9
3 Bioinformatics Support Service, Sir Alexander Fleming Bldg, Imperial College London, 10
London SW7 2AZ, United Kingdom 11
4Institute for Infection and Immunity, St. George's, University of London, London SW17 12
0RE, United Kingdom 13
14
‡These authors contributed equally to the project and manuscript 15
16
Running title: Chicken Interferon-Stimulated Genes17
18
*Corresponding author:19
Dr. Michael A. Skinner 20
Section of Virology, 21
Imperial College London, Faculty of Medicine, 22
St. Mary's Campus, Norfolk Place, 23
London W2 1PG, United Kingdom 24
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ABSTRACT26
27
Virusesthatinfectbirdsposemajorthreats–totheglobalsupplyofchicken,themajor,28
universally-acceptablemeat,andaszoonoticagents(e.g.avianinfluenzavirusesH5N129
andH7N9).Controllingthesevirusesinbirdsaswellasunderstandingtheir30
emergenceinto,andtransmissionamongst,humanswillrequireconsiderable31
ingenuityandunderstandingofhowdifferentspeciesdefendthemselves.ThetypeI32
interferon-coordinatedresponseconstitutesthemajorantiviralinnatedefence.33
Althoughinterferonwasdiscoveredinchickencells,detailsoftheresponse,34
particularlytheidentityofhundredsofstimulatedgenes,arefarbetterdescribedin35
mammals.Virusesinduceinterferon-stimulatedgenesbuttheyalsoregulatethe36
expressionofmanyhundredsofcellularmetabolicandstructuralgenestofacilitate37
theirreplication.Thisstudyfocussesonthepotentiallyanti-viralgenesbyidentifying38
thoseinducedjustbyinterferoninprimarychickembryofibroblasts.Three39
transcriptomictechnologieswereexploited:RNA-seq,aclassical3’-biasedchicken40
microarrayandahighdensity,‘sensetarget’,wholetranscriptomechickenmicroarray,41
witheachrecognising120to150regulatedgenes(curatedforduplicationand42
incorrectassignmentofsomemicroarrayprobesets).Overall,theresultsare43
consideredrobustbecause128ofthecompiled,curatedlistof193regulatedgenes44
weredetectedbytwo,ormore,ofthetechnologies.45
46
KEYWORDS47
ChickenAvianTypeIinterferon-stimulatedgenes 48
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INTRODUCTION49
Theinterferon(IFN)responseisoneofthemostimportantarmsofhostinnate50
immunityagainstvirusinfection[1,2].Infectedcellsareabletorecogniseforeign51
nucleicacidsandinducethesynthesisandsecretionoftypeIIFN(IFN-αandIFN-β)52
andtypeIIIIFN(IFN-λ),whichbindtoreceptorsonthesurfaceofneighbouringcells53
andtriggerthetranscriptionalregulationofgenesinvolvedintheantiviralstate.54
StudiesinmammalshavedemonstratedthatthereareseveralhundredsuchIFN-55
regulatedgenes(IRGs).Becausethevastmajorityareup-regulatedtheyare56
overwhelminglyreferredtoasIFN-stimulatedgenes(ISGs)so,hereafter,theywillbe57
referredtogenericallyasISGs(orspecificallyaschickenISGs,ChISGs),exceptwhere58
themoregenerictermavoidsconfusion.InductionofISGsinvolvestheJAK/STAT59
signallingpathway:STAT1iseitherrecruiteddirectlytotargetpromotersfora60
relativelyweakactivationor,morecommonly,isrecruitedinacomplexcalledISGF3in61
associationwithSTAT2andIRF9[1,3].62
63
ISGsarethefocusofconsiderablecurrentattentionwithregardto:(i)theirantiviral64
activity,(ii)anincreasingappreciationofthecomplexityoftheirregulationand(iii)65
theirtargetingbyvirus-encodedmodulatorsofIFN-inducedresponses[1,3,4].These66
studiesrequirecomprehensivecataloguesoftheISGs,especiallywheresystem-wide67
approachesareundertaken.EventhoughmanykeymammalianISGshavebeenknown68
forsometime,itiswiththerelativelyrecentadventoftranscriptomictechnologiesthat69
thefullcomplementhasbeencatalogued(mainlyusingmicroarrays[5];seealso70
Schogginsetal.2011[6]).71
72
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IncontrasttothemammalianIFNsystemourequivalentknowledgeoftheavian73
systemhaslaggedbehind.AlthoughIFNwasdiscoveredinchickensin1957[7]the74
firstchickenIFNgenewascharacterisedin1994[8]andthekeychickenISG,PKR,was75
identifiedin2004[9].Thederivationofthechickengenomesequence,firstdraftedin76
2004[10],didnotgreatlyadvanceourunderstandingofchickenISGsbecauseofthe77
incompletenatureoftheGallusgallusgenomeassembly,evenatv4(Galgal4),which78
mightbepartlyduetothefactthatthechickenkaryotypehassixpairsof79
macrochromosomes(but33pairsofmicrochromosomes),andthedifficultiesin80
annotatingimmunitygenes,whicharesomeofthemostdivergentbetweenmammals81
andbirds[11].However,ithasbecomeapparentthatkeygenesoftheinnateimmune82
system,suchasthetranscriptionfactorsIRF9andonememberoftheIRF3/IRF7dyad83
[12,13;unpublished],areabsentfromavianspecies,indicativeofsignificantfunctional84
differencesbetweenthemandmammals.Moreover,forreasonsthatarenot85
understood,thecytosolicpatternrecognitionreceptor,RIG-I,appearstohavebeenlost86
fromchickenaswellasothergalliformspecies[13,14].87
88
TogenerateachickenISGdatabasewehavecompareddatafromthreetranscriptomic89
technologyplatforms:(i)theclassical3’-biasedGeneChipChickenGenomeArray(32K;90
Affymetrix), (ii) the Chicken Gene 1.0 Sense Target (ST)whole transcriptome Array91
(Affymetrix) and (iii) Illumina RNA-seq. This three-way comparison allowed a high92
levelofcross-validationofdatafromeachtechnology,beyondwhatwouldnormallybe93
achieved by qRT-PCR. It also allows subsequent studies, constrained to use any94
particulartechnology,tobemorebroadlycompared.WemonitoredIRGexpressionin95
chickenembryofibroblast(CEF)inducedfor6hwith1000urecombinantchickenIFN-96
α (rChIFN1; hereafter routinely referred to as IFN), a time chosen to reflect97
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predominantlyprimarysignallingtargets.Theexpressiondataforselectedgeneswere98
alsovalidatedbyPCRandqRT-PCR.Overlappingdatashowgenerallyhighdegreesof99
concordance in the identityof the IRGsand theirrelative levelsofregulationby IFN,100
with disparity mainly wheremultiple microarray probes exist for single genes. The101
studywaspresentedinapreliminaryformasaposterattheInternationalCytokine&102
InterferonSociety(ICIS)meeting(“Cytokines2015”;October11-14,2015)inBamberg,103
Germany[15].104
105
MATERIALSANDMETHODS106
Culture,infectionandharvestingofCEFformicroarray107
FreshlyisolatedCEFwereprovidedbytheformerInstituteforAnimalHealth,108
Compton,Berks.(nowThePirbrightInstitute,Pirbright,Surrey),UK.Cellswere109
seededinT25flasks(GreinerBioOne;5.6x106cells/flask)andculturedovernightin110
5.5ml199media(Gibco®,Invitrogen)supplementedwith8%heat-inactivated111
newbornbovineserum(NBCS;Gibco®,Invitrogen),10%tryptosephosphatebroth112
(TPB;Sigma),2%nystatin(Sigma)and0.1%penicillinstreptomycin(Gibco®,113
Invitrogen).114
115
TreatmentwithIFN116
RecombinantchickenIFN-α(rChIFN1)waspreparedaspreviouslyreported[16]and117
wasaddedinculturemediatoafinalconcentrationof1000u/ml.Confluentcellswere118
treatedwithIFNormock-treatedandincubatedforsixhoursbeforeharvesting.Cells119
werestoredat-80°CinRNAlater(Sigma)untilRNAextraction.Theexperimentwas120
repeatedintriplicatewiththreedifferentbatchesofCEF.121
122
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RNAextractionandprocessingofsamplesformicroarray123
TotalRNAwasextractedfromcellsusinganRNeasykit(Qiagen)accordingtothe124
manufacturer’sinstructions.On-columnDNAdigestionwasperformedusingRNase-125
freeDNase(Qiagen)toremovecontaminatinggenomicDNA.RNAsampleswere126
quantifiedusingaNanodropSpectrophotometer(ThermoScientific)andcheckedfor127
qualityusinga2100Bioanalyzer(AgilentTechnologies).AllRNAsampleshadanRNA128
integritynumber(RIN)≥9.6.129
130
RNAsampleswereprocessedformicroarraywiththeGeneChip®ChickenGenome131
Array(Affymetrix)usingtheGeneChip®3’IVTExpressKit(Affymetrix)orfor132
microarraywiththeChickenGene1.0STArray(Affymetrix)usingtheAmbion®WT133
ExpressionKitforAffymetrix®GeneChip®WholeTranscript(WT)ExpressionArrays134
(Ambion®)andtheGeneChipWTTerminalLabelingandControlsKit(Ambion®),135
followingthemanufacturers’instructions,asdescribedpreviously[17].136
TotalRNA(100ng)wasusedasinputandqualitycheckswereperformedusingthe137
2100Bioanalyzeratallstagessuggestedbythemanufacturer.RNAsampleswere138
processedintwobatchesof18butbatchmixingwasusedateverystagetoavoid139
creatingexperimentalbias.HybridisationofRNAtochipsandscanningofarrayswas140
performedbytheMedicalResearchCouncil’sClinicalSciencesCentre(CSC)Genomics141
Laboratory,HammersmithHospital,London,UK.RNAwashybridisedtoGeneChip142
ChickenGenomeArraychips(Affymetrix)inaGeneChipHybridizationOven143
(Affymetrix),thechipswerestainedandwashedonaGeneChipFluidicsStation450144
(Affymetrix),andthearrayswerescannedinaGeneChipScanner30007Gwith145
autoloader(Affymetrix).146
147
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ValidationofmicroarraydataforIFN-responsivegenesbyquantitativereal-time148
PCR(qRT-PCR)149
cDNAwassynthesisedfromRNAsamplesfromuntreatedandIFN-treatedCEFusing150
theQuantiTect®ReverseTranscriptasesystem(Qiagen)accordingtothe151
manufacturer’sinstructions.ThecDNAwasusedasatemplatein25μlRT-PCR152
reactionscontaining:19.35μlnuclease-freedistilledH20(Gibco®,Invitrogen),2.5μl153
10xbuffer(Invitrogen)0.75μlMgCl2(Invitrogen),0.2μlDNTPs(25mm;Sigma),0.5μl154
eachofforwardandreverseprimers(20pmol/μl;Invitrogen),0.2μlTaqDNA155
polymerase(Invitrogen)and1μltemplatecDNA.PrimersequencesareshowninTable156
1.157
158
[Table1nearhere]159
160
qRT-PCRwasperformedusingMESAGREENqPCRMasterMixPlusforSYBR®AssayI161
dTTP(Eurogentec)accordingtothemanufacturer’sinstructions.Afinalvolumeof10μl162
perreactionwasused,with1μlcDNAdiluted1:10innuclease-freeH2Oasatemplate.163
Primerswereusedatafinalconcentrationof300nM.Primersequencesareshownin164
Table1.ReactionswereperformedonanABI-7900HTFastReal-TimePCRSystem165
(AppliedBiosystems)usingthefollowingprogramme:95°Cfor5minutes;40cyclesof166
95°Cfor15seconds,57°Cfor20seconds,72°Cfor20seconds;95°Cfor15seconds;167
and60°Cfor15seconds.DatawereanalysedusingSDS2.3andRQManagersoftware168
(AppliedBiosystems).Glyceraldehyde3-phosphatedehydrogenase(GAPDH)wasused169
asareferencegene.Alltargetgeneexpressionlevelswerecalculatedrelativeto170
GAPDHexpressionlevelsandthetargetgeneexpressionlevelin-2huninfectedCEF171
usingthecomparativeCTmethod(alsoreferredtoasthe2–ΔΔCTmethod).172
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173
RNA-seq174
Triplicateuntreated(control)andIFN-treatedCEFwereprocessedfortranscriptome175
analysisbyRNA-seq.Thecellsamplesusedwereidenticaltothoseusedforthe176
microarrayanalyses.TotalRNAwasextractedasformicroarrays(above)andRNA177
librarieswerepreparedfordeepsequencingusingtheTruSeqRNASample178
PreparationKit(Illumina)accordingtothemanufacturer’sinstructions.TotalRNA179
(2.5μg)wasusedasaninputforeachlibrary.Atotalof6RNAadapterindiceswere180
randomlyassignedtothe12samplestoallowmultiplexingoflibraries.Attheendof181
theprotocol,librarieswerequantifiedusingaNanodropSpectrophotometer(Thermo182
Scientific)andcheckedforqualityusinga2100BioanalyzerHighSensitivityDNAchip183
(AgilentTechnologies).RNAlibraryqPCRquantification,multiplexingandsequencing184
wasperformedbytheMedicalResearchCouncil’sClinicalSciencesCentre(CSC)185
GenomicsLaboratory,HammersmithHospital,London,UK.Librarieswerequantified186
usingtheKAPABiosystemslibraryquantificationkit(KK4824)onanABi7500FAST187
qPCRmachine.Librarieswerethendilutedtoa2nMstocksolution,pooledfor188
multiplexing,denaturedanddilutedtoafinalmolarityof20pM.Librarieswereloaded189
ontotheflowcell(8-16pMperlane)forclusteringandclustergenerationwas190
performedbytheIlluminacBotusingversion3kits.Sequencingoftheflowcellwas191
thencarriedoutontheIlluminaHiSeq2000usingtheversion3kits.Datawere192
processedusingRTAversion1.12.4.2,withdefaultfilterandqualitysettings.Thereads193
weredemultiplexed(allowingnomismatchesintheindexsequence)withCASAVA194
1.8.1.195
196
BioinformaticAnalysis197
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MicroarraydatawereprocessedusingworkflowsinGENESPRINGTM(Agilent)and198
PARTEKTM(PartekInc.,StLouis,Missouri,USA)commercialsoftwaresuites.199
Data(.CELfiles)wereanalysedandstatisticallyfilteredusingeitherPartekGenomic200
Suite6.6(PartekGS)orGenespringversion7.2(AgilentTechnologiesInc.,SantaClara,201
CA)softwares.InputfileswerenormalizedwitheitherGCRMAorGeneSpring202
algorithmsforgenearrayoncoremetaprobesets.Aone-wayANOVAwasperformed203
usingeithersoftwareacrossallsamples.Statisticallysignificantgeneswereidentified204
usingmixedmodelanalysisofvariancewithafalsediscoveryrate(Benjamini–205
Hochbergtest)ofP<0.05.Fold-changevalues<±3.0wereremoved.206
RNA-seqdatawereimportedintoCLCbio’sGenomicsWorkbench(CLCBio,Aarhus,207
Denmark;nowQiagen),quality-controlledandthereafterprocessedusingthatpackage208
(versions6&7).209
210
Afterqualitycontrol,thereadsweresubjectedtoqualitytrimmingthenmapped211
againstENSEMBLGalgal4annotatedgenes(release75[18])forquantitativeanalysis212
ofexpression.FoldchangeandFalseDiscoveryRates(FDR)werecalculatedusingKal’s213
Ztest[19],withpooleddata,orBaggerly’stest[20],usingseparatetriplicates.214
215
RESULTS216
Initially,weusedthe32KGeneChip®ChickenGenomeArray(Affymetrix)because,as217
wellasdisplayingprobesfor32,773chickentranscripts,itdisplaysprobesfor684218
transcriptsfrom17differentviralpathogensofchickens,whichoffersadvantagesto219
thosestudyingvirusinfectionsinachickenbackground.Subsequently,weusedthe220
morerefinedChickenGene1.0STArray(Affymetrix)becauseitoffersahigherprobe221
densityagainst18,214chickengenesandshouldallowdetectionoftranscript222
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isoforms,includingnon-polyadenylatedandalternativelypolyadenylated,thoughit223
doesnotincludeprobesforviralgenes.224
225
SeparateweeklybatchesofCEF,producedfrompoolsofeggsfromthesameflock226
(RhodeIslandRed)heldinSPF-likeconditionsattheformerComptonLaboratoryof227
theInstituteforAnimalHealth(nowThePirbrightLaboratory,Pirbright,Surrey,UK)228
servedasbiologicalreplicates.Principalcomponentanalysisofthemicroarraydata229
(datanotshown)indicatedlimitedvariationbetweenbatchesso,thereafter,biological230
triplicateswereusedroutinely.231
232
IRGswereidentifiedfromexpressionanalysisdatadeterminedusingthe32K233
GeneChipfollowingIFNtreatment(1000u,6h)ofCEF.Afterquantilenormalization,234
significanthitswereidentifiedwithGENESPRINGusinganunpairedT-testwith235
asymptoticp-valuecomputationandBenjamini-Hochbergmultipletestingcorrection236
togeneratefalsediscoveryrates(FDR).AmatrixofFDR(from<0.001to1)plotted237
againstFoldChange(FC;from1.0to>3)isshowninTable2.Arelativelyconservative238
FDRof<0.01returned250differentiallyexpressedprobesets.Overlayingthiswitha239
valueforFCforwhichchangesinexpressionmightreasonablybeexpectedtobe240
readilyandreliablyassayedusingothertechnologies,namely> 3,reducedthe241
numberofselected,significantprobesetstoamanageable181(180up,1down).These242
settingswerethereforechosenforfurtheranalysis.For23oftheseprobesets,no243
currentlyrecognisedgeneswereautomaticallyassigned.Oftheremaining158probe244
sets,29wereassignedtogenesrecognisedinduplicatebyotherprobesets.245
Consequently129recognisedgeneswereidentifiedasdifferentiallyexpressed(the246
down-regulatedtranscriptwasnot,atthattime,assignedtoarecognisedgene).247
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248
[Table2nearhere]249
250
WiththeChickenGene1.0STArray,157probesetsdemonstrateddifferential251
expression(156up,1down)atthesamesettings(FC> 3,FDR<0.01).Amongstthese,252
therewere5duplicatedprobesetsand27thatwerenotautomaticallyassignedto253
recognisedgenestherefore125recognisedgeneswereuniquelyidentifiedas254
differentiallyregulated.255
256
IlluminaRNA-seqyieldedatotalof170millionreads(100bases;paired)forthemock-257
treatedCEFtriplicatesamplesand167millionfortheIFN-treatedsamples.Upon258
qualitytrimmingandmappingtoENSEMBLGalgal4annotatedgenes(release75),259
usingCLCBio’sGenomicWorkbench,138recognisedgeneswereidentifiedas260
differentiallyregulated(137up,1down)usingKal’sproportion-basedZ-test[19;as261
implementedintheCLCBiopackage]atthesamesettings(FC> 3,FDR<0.01).Kal’sis262
performedonthepooledreadsfromIFN-treatedanduntreatedsamples.Itisperhaps,263
therefore,morewidelyapplicable;italsoreturnedanumberofIRGscomparableto264
thosereturnedbythemicroarrays.Triplicate-basedanalysisusingBaggerly’s265
proportion-basedBeta-binomialtest[20;asimplementedintheCLCBiopackage]at266
thesamesettings(FC> 3,FDR<0.01)returnedanadditional37up-regulatedgenes.267
268
Comparisonofthecompleterawgenelistsfromthethreetechnologiesusingthemost269
compatibleidentifier(essentiallytheGeneSymbol)withanonlineVennDiagramtool270
(VennDiagramGenerator;[21])demonstratedthat233recognisedgeneswere271
identifiedasdifferentiallyregulated.Ofthese,51wereidentifiedincommonbyall272
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threetechnologiesandafurther57wereidentifiedbytwooutofthreetechnologies,273
meaningthat108wereidentifiedbyatleasttwotechnologies.Atotalof125were274
thereforeeachidentifiedonlybyindividualtechnologies(Fig.1A).275
276
Aswellascomparingtheidentitiesofthedifferentiallyregulatedgenes,thecorrelation277
ofexpressionofthegenesidentifiedbythedifferentplatformswasexaminedinterms278
ofbothlevelandrankofFC(Fig.2A&B).Forinstance,comparingRNA-seqdatawith279
the32KGeneChipdata,Spearmancorrelationvalueswere0.93forFClevelandrank.280
Consideringthecurrentstateofassemblyandannotationofthechickengenome,the281
correlationofISGsintermsofgeneidentityaswellasthelevelandrankofinductionas282
indicatedbyallthreetechnologyplatformsisreassuring.Neverthelesstheplatform283
transcriptomicdatawerevalidatedforselectedgenesbyRT-PCR(datanotshown)and284
byqRT-PCR(Fig.3A).285
286
A6htimepointwaschosenformicroarrayandRNA-seqanalysisofIFNtreatmentasit287
hasbeenwidelyusedandisknowntoresultinsignificantlevelsofabroadrangeof288
ISGsinmammals,makingitsuitablefordefiningthechickeninterferome.Useofthis289
singletimepointdoesnot,however,provideunequivocalinsightintomechanistic290
interpretationofISGinduction;forinstance,itdoesnotdiscriminatebetweenstrictly291
ISRE-dependentinductionofISGsandISRE-independentinductionofISGsby292
mechanismsthatmightincludeimmediatehigh-levelinductionofIRF1,whichhas293
beenobservedinmammaliansystems[22-24].Kineticanalysisoftheinductionof294
expressionofasubsetofISGswasthereforeconductedat45,90,180and360minpost295
applicationofIFN(seeFig.3B).Evenamonghighly-inducedISGs,differenttemporal296
profileswereobserved,fromtherapidaccumulationofIFIT5(1000foldby90min)297
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andRSAD2(whichremainatsteadylevelsto360min)tothesteadier,sustained298
accumulationofMxandthemoremodestlyinducedSTAT1;withLGP2andTRIM25299
peakingat180min.AlthoughdifferencesinmRNAstabilityandturnoverwill300
influencetheprofiles,thisidentificationoftheISGswillallowdetailedanalysisoftheir301
promoterstoinvestigateelements(andthefactorsthatbindthem)thatcontributeto302
thecomplexityoftheobservedinductionpatterns.303
304
DISCUSSION305
Ofthe51IRGsinitiallyidentifiedbyallthreetechnologies,47hadmammalian306
equivalentsthatareknownasISGsfromhumanormouseaccordingtothe307
“Interferome”database(v2.01;[25,26]).ThosenotlistedinInterferomewere:308
EPB41L3,IFI27L2,OLFML1andTMEM168.Ofthe57IRGsinitiallyidentifiedbytwo309
outofthethreetechnologies,29havemammalianequivalentsknownashumanor310
mouseISGs.Therefore,ofthe108ChISGsidentifiedinitiallybyatleasttwo311
technologies,76wereequivalenttoknownmammalianISGs.ForthoseChISGs312
identifiedbysingletechnologies,12ofthe55identifiedbyRNAseq(L1),10ofthe36313
identifiedbythe32KGenechip(L2)and12ofthe34identifiedbytheSTArray(L3)314
werelistedinInterferome.Thisaddedafurther34candidateChISGs(atotalof110)315
withknownmammalianISGequivalents(asrecognisedbytheInterferomedatabase).316
ThemajorityofChISGsforwhichmammalianequivalentscannotbefoundinthe317
Interferomedatabase(all4fromthe“common”ISGs,23of28identifiedbyatleasttwo318
technologiesaswellas21outof43forL1,15outof26forL2and13outof22forL3)319
havegeneequivalentsinthemammaliangenomedatabases(seeadditionaltableand320
spreadsheetfiles[Additionalfiles1and2,respectively];seealsothe“ChISGBrowser”321
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[Tomlinson,unpublished;27]).Thissuggestseitherthatthemammalianequivalents322
areISGsbutthattheyarenotincludedassuchinInterferomeorthattheyarenotISGs323
inmammals.324
325
Therawlistswererefinedbymanual“curation”,allowingforsynonymsofrecognised326
genes(forinstanceISG12-2versusISG12(2))and,afterbioinformaticanalysisusing327
BLAST,etc.,assigningrecognisedgeneidentifierstoprobesetsthatpreviouslylacked328
them.Attheendofthisprocess(seeFig.1B;Additionalfiles1and2),itwasapparent329
thatsome(n=12)differentiallyregulatedgenesidentifiedbythemicroarrayswerealso330
identifiedasdifferentiallyregulatedbyRNA-seqbutthattheyfelloutsideofthestrict331
FC>3andFDR<0.01parameters,reflectingunsurprisingdisparityinthesensitivityof332
thethreetechnologies.ThosegenesthatwereexpresseddowntoFC>2.5orwithan333
FDRupto<0.05were,therefore,alsoincorporatedtoproduceafinallist(Fig.1C;334
Additionalfiles1and2).335
336
Itisobviousthatthismanualcurationofthedata,toallowforalternativeGeneID337
nomenclatureusedbythethreetechnologiesandfordifferencesinsensitivity,338
introducedminorchangestothefiguresfromtheautomaticcomparisonscitedabove339
(Fig.1;Additionalfiles1and2).Curation,therefore,reducedthenumberofIRGsfrom340
233to193.Italsoincreasedthenumberofdifferentiallyexpressedgenesdetectedby341
twooutofthreetechnologiesfrom108to118(compareFig.1Aand1B).Relaxingthe342
criteriafordetectionofdifferentiallyregulatedgenesbyRNA-seq(toFC>2.5and/or343
FDR<0.05)furtherincreasedthenumberofgenesdetectedbyallthreetechnologies344
from70to72(representing37%)orbyatleasttwoofthetechnologiesfrom118to345
128(66%),leaving65genesdetectedbysingletechnologies(compareFig.1Band1C),346
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with29ofthosedetectedbyRNA-seqalone(usingtheKal’stest,atFC>3.0andFDR<347
0.01;Additionalfiles1and2).348
349
Ofthe37additionalISGsidentifiedbyRNA-seqassignificant(FC>3FDR<0.01)bythe350
moresensitiveBaggerly’stestbutnotbyKal’s(Table3),twowerealsoidentifiedas351
significantbyKal’susingtherelaxedcriteria(FDR<0.05).Baggerly’s,therefore,352
identified35ISGsadditionaltothosedescribedintheaboveanalysesusingRNA-seq353
(Kal’sanalysis)andthemicroarrays[Table3].354
355
[Table3nearhere]356
357
Comparisonoftechnologyplatforms.358
AnalysisofRNA-seqdatadependsdirectlyontheextantannotatedgenomesequence.359
Perhapsnotsurprisinglytherefore,RNA-seqidentifiedthelargestproportionofgenes360
amongstthesetof193uniqueIRGsthatwecompiled(150;78%).Nevertheless,the361
microarrayseachidentified63%ofthegenes(122and121).Congruencewashighest,362
andalmostidentical,betweenRNA-seqandeachmicroarray(98and95;51+/-1%;all363
percentagesreferringtothetotalof193uniqueIRGs).Betweenmicroarraysitfellto364
41%(79).Fortwo-way-onlycomparisons,thedistributionofuniquegenesbetween365
themicroarrayswassymmetrical(42and43;22%).BetweenRNA-seqandeach366
microarray,uniquegeneswerebiased>2-foldtowardsRNA-seq:52(27%)versus24367
(12%)againsttheGenechipand55(28%)versus26(13%)againsttheSTArray.368
369
ClearlyinsimpletermsofnumbersofIRGsidentified,RNA-seqoutperformsthe370
microarrays.Thisisprobablyattributabletothehistoricnatureofthearraydesign371
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basedonearliergenomeassembliesandannotations,withconsequenteffectson372
overallcoverage(whichmightdisproportionatelyaffectconditionallyexpressedgenes373
suchasthoseoftheinnateimmuneresponses).Nevertheless,theabilityofmicroarrays374
toquantifyexpressionof50%(about100)ofsuchalargepoolofimportantgeneswill375
oftenprovesufficientfortheexperimentalobjectiveswhereotherconsiderations376
mightaffectthechoiceoftechnology(seebelow).377
378
Movingawayfromactualnumbersofgenes,itisworthnotingthatdeeperanalysis(in379
theformofvalidationbyalternativeapproaches)will,bydefinition,berequiredto380
determinewhichofthegenesidentifieduniquelyasIRGsbyindividualtechnologies381
areactuallyIRGs.382
383
IdentificationofISGsnotannotatedonthecurrentgenome384
GenomiclociforeachofthepredictedISGswerevisuallyinspectedusingGenomic385
Workbench’sgenomebrowser,displayingtracksshowing:gene,transcript,exonand386
ORFannotationsforthecurrentchickengenomebuildaswellasread-mappingfor387
controlandIFN-treatedreads[27].Onoccasions,suchinspectionrevealedthe388
presenceofnon-annotated,inducibly-transcribedregions,representingexons,whole389
genesorevengenefamilies.Examplesincludethosepreviouslydescribedatthe390
chickenIFITMlocus[28;datanotshown],attheHERClocus(describedbelow)or391
downstreamofCCL19(LOC100857191;“C-Cmotifchemokine26-like”;Fig.4).392
SystematicanalysisoftheseISGsisoutsidethescopeofthismanuscriptbutthedata393
depositedfromthisstudywillfacilitateongoingstudyandimprovedannotation.In394
somecases,althoughnotcurrentlyannotatedontheENSEMBLchickengenome,the395
geneshaveIDsinNCBIandwereidentifiedasISGsbyoneofthemicroarrays.396
GiotisetalChISGVRsub_manuscript_R4.docx Page17of33
ExamplesoftheseincludeLOC415756,LOC415922(“guanylate-bindingprotein4-397
like”)andLOC422513(“hectdomainandRLD4-like”,amemberoftheHERCfamily,398
discussedbelow).399
400
IdentificationofISGsnotpresentinthecurrentgenome401
About10%ofthereadsfromCEFSdidnotmaptothecurrentchickengenome.The402
unmappedreadscombinedfromthecontrolandIFN-treatedsampleswereassembled403
intocontigsusingthedenovoassemblyfunctionofGenomicWorkbench.TheRNA-seq404
functionofGenomicWorkbenchwasthenusedtoquantitateexpressionofthecontigs405
incontrolandIFN-treatedsamples.Oneofthemosthighly-expressedcontigswasone406
which,whenanalysedbyBLAST,provedtorepresentahomologueofSTAT2,whichis407
missingfromthecurrentENSEMBLannotatedreferencechickengenomeassembly408
(Galgal4;release84),thoughatNCBIithasrecentlybeenplacedasaRefseqgeneon409
chromosome33inthenewassemblyGalgal5(anannotatedformofwhichhasnotyet410
beenreleasedandiscurrentlynotscheduledforrelease).Thedenovoassembled411
contigsequencewasusedtoderiveprimersforRT-PCR;characterisationofchicken412
STAT2willbereportedelsewhere.413
414
Interferondown-regulatedgeneexpression415
Thedataondifferentialexpressionshowedanoverwhelmingover-representationof416
genesup-regulatedbyIFN.Foreachofthetechnologies,onlyonegenewasdetectedas417
down-regulated.CorrespondingGeneIDswerePYURF(PIGYupstreamreadingframe;418
ENSGALG00000026229)forRNA-seqandPIGY(phosphatidylinositolglycananchor419
biosynthesis,classY;NCBIGeneID:101748971)fortheSTarray.Thedown-regulated420
GiotisetalChISGVRsub_manuscript_R4.docx Page18of33
32kGeneChipprobe(Gga.8802.1.S1_at),thoughnotmappedtoaknowngeneatthe421
timeofinitialprocessing,accordingtotheAffymetrixNetAffx™AnalysisCenter[29]is422
nowalsoassignedasPYURF.Inhumans,PIGYandPYURFrepresentdifferentopen423
readingframesonthesamesplicedtranscriptofageneonHschromosome4located424
downstreamofHERC6thenHERC5.ThePYURF/PIGYgeneisoverlappedonthe425
oppositestrandbyHERC3,whichextendsdownstreamtobefollowedbyFAM13A.426
Similarly,thechickenPIGY(NCBI)andPYURF(Ensembl)genesmaptoalocuslying427
upstreamofHERC3thenFAM13AonGgchromosome4(seeFig.4),withHERC-like428
LOC422513(“hectdomainandRLD4-like”)startingupstreambutspanningand429
extendingdownstreamofthechickenPYURF.OurRNA-seqdata(Fig.4)indicatethat430
thislocusispoorlyannotatedanddemonstratescomplexregulationofthecomponent431
genesbyIFN.Thus,althoughthePIGY/PYURFtranscriptisdown-regulatedbyIFN,as432
recordedbyallthreetechnologies,itappearstobecloselyflankedupstreamand433
downstreambystillunannotatedmultipleexonsthatareclearlystronglyinducedby434
IFN(Fig.4).Sequenceswithintheseupstreamanddownstreamregions(whichare435
representedbythesingleNCBIRefseq(Galgal5)gene,LOC422513,butappearas436
thoughtheymayrepresenttwoseparategenes,Fig.4)bearhomologywithgenesof437
theHERCfamily,consistentwiththefactthatHERC5neighboursthehuman438
PIGY/PYURFgeneandthatHERC3neighboursthechickenPIGY/PYURFgene.The439
chickenHERC3geneshowsnoevidenceofinductionbyIFN.440
441
Conclusions442
Descriptionoftheinterferon-inducibilityoftheChISGsservesasthefirststepin443
understandingtheregulationoftheirexpressionandtheirroleinanti-viral(and444
potentiallybroaderanti-microbial)activities.Thereisconsiderablecurrentinterestin445
GiotisetalChISGVRsub_manuscript_R4.docx Page19of33
theantiviralresponsesofparticularcelltypes,particularlythoseofthelymphoid,446
myeloidanddendriticlineages.However,thedefinitionofawidevarietyofthesecell447
typesisnotsoadvancedinavianspeciessowefeltitbesttoproducebaselinedatafor448
readilyavailable,primarycells,namelychickembryofibroblasts(CEF)astheyare449
highlyresponsivetoIFN.Theyalsoremainimportantforcommercialproductionof450
vaccineviruses(includinghumanvaccines)aswellasfortheroutineisolationand451
diagnosisofavianpathogens.452
453
Giventhecurrentlyincompletenatureofthechickengenomeassembly(evenat454
Galgal5)andofitsannotation(ascurrentlyavailableforGalgal4andevenasawaited455
forGalgal5)itisinevitablethatupdateswillcontinuetobereleasedbuttheprimary456
datareportedhere,andpublicly-available,formicroarraysandRNA-seq,canalwaysbe457
appliedtoupdatedmicroarrayassignmentsaswellastosubsequentgenome458
assembliesandannotations.459
460
Allthingsbeingequal,RNA-seqwouldseemtobethemethodofchoicefor461
transcriptomicanalysisofchickenIFNresponses,particularlygivenitsabilityto462
producehigh-resolutionquantitativeandqualitativedata.Moreoverthedataare463
readilyportableandcanbeeasilyminedbyotherswithdifferentresearchfocus.They464
canalsobeappliedimmediatelytonewlyreleasedgenomeassembliesandannotations465
(whetherglobalorlocal),whereasmicroarrayanalysismustawaitthegenerationof466
annotationupdatesforeachtechnology.467
468
However,althoughthecostofsequencinghasfallen,andwillprobablycontinuetodo469
so,thereremainconsiderableoverheadstohandlinglargedatasetsfromextensive,470
GiotisetalChISGVRsub_manuscript_R4.docx Page20of33
complicatedexperiments,especiallyintermsofcomputinganddatastoragecapacity,471
aswellasspeedofprocessingandarchiving.Forsuchexperiments,microarrays472
continuetoofferatractableapproach,capableofquicklyquantifyingandcomparing473
theexpressionofthecentralcoreofIRGsproducingrelativelycompactdataforrapid474
analysisandeasyarchiving.475
476
InductionofinnateresponseswithPAMPSwilltriggerdifferentorbroaderrangesof477
responsesbyvirtueofthefactthattheywilltriggerotherormorepathwaysthanjust478
theIFN-pathway.Forinstancewe(Giotisetal.,unpublished)andothers[12]have479
beguntoanalysetheresponsesinducedbythedsRNAanaloguepoly[I:C].Regulationof480
ISGexpressionmightaffecttheinnateresponsesobservedindifferentcelllinesor481
tissuessoitwillbeimportanttounderstandthemechanismsinvolved.Additionally,482
wehaveobservedsuppressionofISGinductioninthespontaneouslyimmortalized483
chickenfibroblastcellline,DF-1[30],duetotheirenhancedbasalexpressionofthe484
regulatoryISG,SOCS1(Giotisetal.,unpublished).IdentificationoftheISGsmeansthat485
theirpromoters,enhancersandotherregulatoryelementscanbesystematically486
analysedtohelpunderstandthecomplexkineticsofexpressionoftheirexpression487
(Fig.4).488
489
Severalstudieshaveinvestigatedchangesinhostgeneexpressioninresponseto490
infectioninvivoorinculturewithparticularavianviruses[31-39].Althoughmanyof491
thesegeneswillrepresentinnate(andpotentiallyantiviral)hostresponses,the492
majoritywillbeinvolvedinthemetabolic,cellcycleandultrastructuralchangesthat493
thevirushastoinducetofacilitatereplication.Furthermore,itisnotunusualfor494
virusestomodulatetheexpressionofsignallingmoleculeskeytotheantiviral495
GiotisetalChISGVRsub_manuscript_R4.docx Page21of33
responsesorofantiviraleffectorsthemselves.Forinstance,wehaveshownthateven496
anattenuatedstrainoffowlpoxvirusblocksinductionofIFN-β(ChIFN2)andishighly497
resistanttotheantiviralactivityinducedbyIFN[16,40].498
499
Theresultsofexistingandfuturestudiesofinfectioninvivoorinculturewith500
particularavianvirusescannowbecomparedwithdatapresentedhereforISG501
inductionbyIFNtolookforevidenceofmodulationofISGexpressionbyviruses,502
whetherthatbemodulationofindividualISGs,subsets[4]orthecompleteset.For503
instance,fowlpoxvirusblocksessentiallyallISGexpressionbutamutantdefectivein504
thefpv012ankyrinrepeat/F-boxproteinidentifiedbyLaidlawetal.[40]induces505
modestlevelsofasubsetoftheISGs(Giotisetal.,unpublished).Suchanalysescanbe506
extendedtoimportantavianzoonoticvirusesandpathogenswithhugeimpactonthe507
globalpoultryindustry.AlthoughthisstudyrelatestotypeIIFN,extensivecomparison508
withtheeffectsoftypeIIIIFNcouldnowbeconducted,extendingontheqRT-PCR509
comparisonmadebyMasudaetal,wholookedatinductionofMxandOASbyIFN-β,510
IFN-γandIFN-λ[41].511
512
LISTOFABBREVIATIONS513
IFN interferon
IRGs IFN-regulatedgenes
ISGs IFN-stimulatedgenes
CEF Chickenembryofibroblasts
rChIFN1 recombinantchickenIFN-α
RIN RNAintegritynumber
GiotisetalChISGVRsub_manuscript_R4.docx Page22of33
qRT-PCR quantitativereal-timePCR
GAPDH glyceraldehyde3-phosphatedehydrogenase
FC Foldchange
FDR Falsediscoveryrate
514
AVAILABILITYOFDATAANDMATERIALS515
516
Thedatasetssupportingtheconclusionsofthisarticleareavailablefromthefollowing517
repositories:518
519
EuropeanBioinformaticsInstitute(EBI)ArrayExpressaccessionnumbersE-MTAB-520
3711(forthe32KGeneChip;[42])andE-MTAB-3712(fortheSTarray;[43]).521
522
EuropeanNucleotideArchive(ENA)studynumberPRJEB7620(forIlluminaRNA-seq;523
[44]).524
525
COMPETINGINTERESTS526
Theauthorsdeclarethattheyhavenocompetinginterests.527
528
FUNDING529
WewishtoacknowledgetheUK’sBiotechnologyandBiosciencesResearchCouncil530
(BBSRC)forfundingviagrantsBB/K002465/1(“DevelopingRapidResponsesto531
EmergingVirusInfectionsofPoultry(DRREVIP)”),BB/H005323/1(“Correlationof532
immunogenicitywithmicroarrayanalysisofvectormutantstoimproveliverecombinant533
GiotisetalChISGVRsub_manuscript_R4.docx Page23of33
poxvirusvaccinesinpoultry”)andBB/G018545/1(“Theavianinterferonsystemandits534
evasionbyAvipoxviruses”).535
536
AUTHORS’CONTRIBUTIONS537
ESGandRCC(equalcontribution):designofthestudy,dataacquisitionandanalysis,538
draftingthemanuscript.NGS:datacompilationandanalysis,draftingthemanuscript.539
CDT:design,production,curationandmaintenanceofChISGBrowserwebsite.SG:540
designofthestudy,criticallyreviewingthemanuscript.MAS:designofthestudy,data541
analysis,finalizingmanuscript.Allauthorsreadandapprovedthefinalmanuscript.542
543
ACKNOWLEDGEMENTS544
WearegratefulfortheskilledsupportofLaurenceGame,NathalieLambieandAdam545
GiessoftheMedicalResearchCouncil’s(MRC)ClinicalSciencesCentre’s(CSC)546
GenomicsFacilityinconductingmicroarrayanalysisandIlluminasequencing.We547
gratefullyacknowledgeSarahButcherandGeraintBartonoftheBioinformatics548
SupportServiceatImperialCollegeLondonfortheiradvice.549
550
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689
690
691
692
693
694
695
696
697
698
GiotisetalChISGVRsub_manuscript_R4.docx Page27of33
699
700
701
Fig.1.CorrelationofISGsidentifiedassignificantbyRNA-seqandmicroarray.702
VenndiagramsshowingcorrelationofsignificantISGs(FC≥3;FDR≤0.01,unless703
statedotherwise)for:(i)Illumina100bpaired-endRNA-seq,(ii)Affymetrix32K704
GeneChipChickenGenomeArrayand(iii)ChickenGene1.0STArray,following705
inductionofCEFfor6hwith1000uofrChIFN1.(A)Totalhits(“n=“)shownforeach706
technology;thosecorrespondingtoGalgal4assemblyGeneIDsareshowninbrackets707
(“recognised”)–RNA-seqhitsallrepresentGalgal4mappedgenes.(B)Hitsfromarray708
technologiesweremanuallycreatedtomaximisenumbersofcorrespondinggenes.(C)709
Curatedarrayhitsshownin(B)thatarepresentamongstRNA-seqhits,butatlower710
levelsofsignificance,weretransferredtotherespectiveRNA-seq-overlappingsectors.711
Totalgenesareshownfor(A–C).712
713
Fig.2.ComparisonofexpressionlevelandrankofsignificantISGsidentifiedby714
RNA-seqandmicroarrays.715
SpearmancorrelationplotsforsignificantISGsfrom:(i)Illumina100bpaired-end716
RNA-seqand(ii)Affymetrix32KGeneChipChickenGenomeArray,followinginduction717
ofCEFfor6hwith1000uofrChIFN1,byFC(A)andbyRank(B).718
719
Fig.3.qRT-PCRvalidationandkineticanalysisofISGexpression.720
(A)ValidationofRNA-seqandMicroarrayISGsbyqRT-PCRofCEFtreatedwith721
recombinantchickenIFN1(1000u,6h).(B)KineticsofexpressionofselectedISGs722
assayedbyqRT-PCRfollowingtreatmentofCEFwithrecombinantchickenIFN1723
GiotisetalChISGVRsub_manuscript_R4.docx Page28of33
(1000u)for45,90,180or360min.ISGsshowingsimilarkineticexpressionprofiles724
arepairedvertically.725
726
Fig.4.Gene-levelvisualisationofRNA-seqreadsmappedtothechickengenome.727
AnnotatedCLCBioGenomicWorkbenchviewsofchickenchromosomeZ(A)and4(B)728
showingthelociaroundhomologuesofCCL19(ENSGALG00000028256)(A)and729
PYURF(ENSGALG00000026229)(B).Eachpanelshowstracksforgenes(labeledwith730
Galgal4–annotatednames),untranslatedregions(UTR),codingsequences(CDS)and731
mRNAtranscripts.LocationsofunannotatedNCBIGalgal5RefseqgenesLOC732
1008571891(A)andLOC422513(B)areindicated.RNA-seqreadsfromuntreatedand733
IFN-treatedCEF(6h,1000u)areshownmappedtothegenomeintheuppermostand734
lowesttracks,respectively,ineachpanel(totalsmappedtothechromosomeare735
indicatedtotheleftofthesetracks).ThelevelsofbasalandpeakRNA-readmappings736
areshowntotherightofthetracksunder“Scale”.Comparisonofthesefiguresin737
conjunctionwiththesizeofthepeaksallowsvisualestimatesofthelevelsof738
differentialexpressionforindividualexons(whichcanbecomparedwiththeformal739
numericalanalyses).Forinstance,IL11RA(A)aswellasCCGN2andHERC3(B)show740
nosignificantregulationbyIFN.Incontrast,CCL19andunannotatedLOC100857191in741
(A)showsignificantupregulation(96fold-butwithanFDRof0.031itfelloutsidethe742
cut-offforKal’sanalysisand,becauseofitsverylowbasalexpression,wasnot743
returnedbyBaggerly’s).In(B)PYURFshows24-foldsuppressionbyIFNbutthe744
sequencesurroundingPYURFshows87-foldinductionfromtheright-handendofthe745
unannotated,antisenseLOC422513andconsiderablyhigherupregulationfromthe746
left-handend(duetoitsloweruninducedlevels),consistentwiththeserepresenting747
homologuesofIFN-induciblehumangenesHERC6andHERC5.748
GiotisetalChISGVRsub_manuscript_R4.docx Page29of33
749
750
751
Additionalfile1.752
MicrosoftWordTable(.doc)753
TableofcuratedChISGsidentifiedbyindividualormultipletechnologies.754
755
1AnasteriskindicatesaGeneIDnotannotatedinENSEMBL.756
2TechnologiesidentifyingsignificantIRGsarelistedas‘1’RNA-seq(usingKal’sZ-test);757
‘2’Affymetrix32KGeneChipChickenGenomeArrayand‘3’ChickenGene1.0STArray’.758
ChISGssignificantbyoneorbothmicroarraysandRNA-sequsingKal’sZ-testunder759
relaxedcriteria(FC>2.5orFDR<0.05)areindicatedby‘(1)’.Aplusafterthe760
technologyidentifierindicatesthatIFN-inducedRNA-seqreaddensitywasobservedat761
thelocationoftheunannotatedgene.762
3IndicateswhetherhomologuesoftheChISGidentifiedarelistedintheInterferome763
websiteasinducedbyinterferoninHomosapiens(Hs),Musmusculus(Mm),both764
(Hs/Mm),orneither(***).765
766
Additionalfile2.767
MicrosoftExcelWorkbook(.xlsx)768
DetailedinformationonChISGsidentifiedbyRNA-seq,andmicroarray769
technologies770
[1]TechnologiesidentifyingsignificantIRGsarelistedas“1”RNA-seq(usingKal’sZ-771
test);“2”Affymetrix32KGeneChipChickenGenomeArrayand“3”ChickenGene1.0ST772
GiotisetalChISGVRsub_manuscript_R4.docx Page30of33
Array’.ChISGssignificantbyoneorbothmicroarraysandRNA-sequsingKal’sZ-test773
underrelaxedcriteria(FC>2.5orFDR<0.05)areindicatedby“(1)”.“+”afterthe774
technologyidentifierindicatesthatIFN-inducedRNA-seqreaddensitywasobservedat775
thelocationoftheunannotatedgene.776
[2]Interferomestatushttp://www.interferome.org/interferome777
[3]Humanhomologuedata(HUGO)http://www.genenames.org778
[4]Mouseorthologuedata(MGI)http://www.informatics.jax.org/marker/779
780
GiotisetalChISGVRsub_manuscript_R4.docx Page31of33
Table1.PrimersusedtoquantifygeneexpressioninmockorIFN-treatedCEF781
usingreal-timeqRT-PCR.782
783Gene Accession
number Forwardprimer(5’-3’) Reverseprimer(5’-3’)
GAPDH NM_204305.1 GGCACTGTCAAGGCTGAGAA TGCATCTGCCCATTTGATGTIFNβ NM_001024836.1 CAGTCTCCAGGGATGCACAG GAGAAGGTGGTGGTGAGAGCMX1 NM_204609.1 CACACCCAACTGTCAGCGAT ATGTCCGAAACTCTCTGCGGIFIT5 XM_421662.4 TGCTTCACCAGCTAGGACTCTGC TGGCTTTTGCTCTGTCACCACTTTG
ZC3HAV1 NM_001012938.1 TCGGCGCCTCTCTACGCCAT TCAGTCCACTGGCCGTGGTCAIRF8 NM_205416.1 ACAAGCAGGGCATCTTCATC TGTTCCCACTCCAGAAGACCSOCS1 NM_001137648.1 CTGCTGGATGCCTGCGGCTT GGGCCCGGTCGCGGTTTTAAIL15 NM_204571.1 CACTGTAAGTGGTCAGACGTTCTGA GGTTCCTGGCATTCTATATCCTCGT
RSAD2 XM_426208.4 GGACAAGGACGAGACAGTTCC TCCCGCCTCCTTAAGCATTGTRIM25 XM_415653.5 TCAAGAGTCCCACCCTTCCA AGCAGCTCAATGGACAGCATLGP2 HQ845773.1 ATCTCGCGGCATTGTCTTCA CTGCTGCTCATTCTGGGTCA
784
785
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Table2.Matrixshowingsignificanthitsfrommicroarray(Affymetrix32K786
GeneChipChickenGenomeArray)analysisofchickenembryofibroblasts(CEF)787
treatedwithrecombinantchickenIFN1(1000u,6h).788
789
Fold Change FDR All <0.05 <0.02 <0.01 <0.005 <0.001
All 38285 945 414 250 150 24 >1.1 17636 942 414 250 150 24 >1.5 1965 676 363 232 146 23 >2.0 677 458 296 206 135 22 >3.0 354 306 235 181 123 22
Expected by chance 47 8 2 0 0 790
Numbersofsignificantgenesareindicatedforfoldchangeinexpressionfrom>1.1to>791
3andfromfalsediscoveryrate(FDR)<0.001to<0.05(UnpairedTtestwith792
Asymptoticp-valuecomputationandBenjamini-HochbergMultipleTesting793
correction).794
795
GiotisetalChISGVRsub_manuscript_R4.docx Page33of33
Table3.ChISGsidentifiedbyRNA-sequsingBaggerly’stestbutnotKal’stest796
usingstandardcriteria(FC>3orFDR<0.01).797
798FeatureID Baggerley's
Weightedproportions
FC
Baggerley'sFDRp-valuecorrection
Kal’sRelaxedFDR<0.051
FAM26F 148 0.0000 YesTHEMIS2 118 0.0004 ENSGALG00000026152 55 0.0000 ENSGALG00000005148 54 0.0043 ENSGALG00000003110 52 0.0050 IL4I1 26 0.0040 C1orf168 24 0.0001 SPIRE2 18 0.0041 HRH1 17 0.0001 AZIN2 14 0.0000 ENSGALG00000029181 14 0.0000 ENSGALG00000001629 12 0.0001 B3GNT4 9 0.0000 GDPD4 8 0.0000 ATP6V1G3 7 0.0002 DUSP15 7 0.0002 IKBKE 7 0.0001 ANGPTL7 6 0.0000 ARHGEF28 6 0.0004 KCNJ5 6 0.0000 CHRD 5 0.0000 YesENSGALG00000002823 5 0.0005 ENSGALG00000004772 5 0.0000 ENSGALG00000006325 5 0.0000 ENSGALG00000027955 5 0.0000 FUT10 5 0.0000 TOR4A 5 0.0001 C1QTNF1 4 0.0000 CYBRD1 4 0.0000 ENSGALG00000000819 4 0.0028 ENSGALG00000020899 4 0.0000 ISLR 4 0.0000 JAM2 4 0.0000 KIAA0226 4 0.0028 MALL 4 0.0002 MAOA 4 0.0000 RBM43 4 0.0015 799
1ChISGssignificantbyKal’sZ-testunderrelaxedcriteria(FDR<0.05).800
801
RNA-seq Kals FC>3 FDR<0.01
n=138
3’ microarray FC>3 FDR<0.01
n=181 (129 recognised)
ST array FC>3 FDR<0.01
n=157 (125 recognised)
233 unique IRGs
36
25
3415
51
17
55
23
9
2319
70
20
29
17
7
1923
72
26
29
193 unique IRGs 193 unique IRGs
A B C
Figure 1 Click here to download Figure Giotis et al ChISG Fig1.pdf
Figure 2 Click here to download Figure Giotis et al ChISG Fig2.tiff
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Figure 3 Click here to download Figure Giotis et alChISG Fig3.pdf
A
8,200,000 8,210,000 8,220,0008,190,000 8,230,000
IL11RACCL19
RNA-seqCEF-MOCK6.2M reads
Gene
UTR
CDS
mRNA
RNA-seqCEF-IFN
6.2M reads
Scale
35
551
33
1793
LOC100857191
B
34,475,000 34,485,000 34,495,00034,465,000 34,505,000
CCGN2 PYURFHERC3
RNA-seqCEF-MOCK9.0M reads
Gene
UTR
CDS
mRNA
RNA-seqCEF-IFN
8.6M reads
Scale
37
1042
45
3355
LOC422513
Figure 4 Click here to download Figure Giotis et alChISG Fig4.pdf
Additional file 1
Click here to access/downloadSupplementary Material
Additional file 1.doc
Additional file 2
Click here to access/downloadSupplementary Material
Additional file 2.xlsx