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Giotis et al ChISG VR sub_manuscript_R4.docx Page 1 of 33 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. Giotis 1‡ , Rebecca C. Robey 1‡ , Natalie G. Skinner 2 , Christopher D. Tomlinson 3 , 4 Stephen Goodbourn 4 , Michael A. Skinner 1 * 5 6 1 Section 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 4 Institute 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 Genes 17 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 [email protected] 25

Transcript of Chicken interferome - avian interferon-stimulated genes ... et... · 1 Chicken interferome - avian...

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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

[email protected]

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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

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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

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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

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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

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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

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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

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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

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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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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

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(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

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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

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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

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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

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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

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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

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!"##$

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B

Figure 3 Click here to download Figure Giotis et alChISG Fig3.pdf

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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

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Additional file 1

Click here to access/downloadSupplementary Material

Additional file 1.doc

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Additional file 2

Click here to access/downloadSupplementary Material

Additional file 2.xlsx