De Novo Assembly And Characterization Of The Trichuris Trichiura Adult Worm Transcriptome Using Ion Torrent Sequencing

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ContentslistsavailableatScienceDirect

Acta

Tropica

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a

De

novo

assembly

and

characterization

of

the

Trichuris

trichiura

adult

worm

transcriptome

using

Ion

Torrent

sequencing

Leonardo

N. Santos

a

_,

_Eduardo

_S.

_Silva

a

_,

_André

_S.

_Santos

a

_,

_Pablo

_H.

_De

_Sá

b

_,

Rommel

T. Ramos

b

_,

_Artur

_Silva

b

_,

_Philip

_J.

_Cooper

c,d

_,

_Maurício

_L.

_Barreto

e,f

_,

Sebastião

Loureiro

e

_,

_Carina

_S.

_Pinheiro

a

_,

_Neuza

_M.

_{Alcantara-Neves}

a,∗

_,

Luis

G.C.

Pacheco

a,∗

a_Institute_of_Health_Sciences,_Federal_University_of_Bahia,_Salvador,_BA,_Brazil b_Institute_of_Biological_Sciences,_Federal_University_of_Pará,_Belém,_PA,_Brazil c_Institute_of_Infection_and_Immunity,_St._George’s_University_of_London,_London,_UK

d_Centro_de_{Investigacion}_en_Enfermedades_Infecciosas_y_Cronicas,_Pontiﬁcia_Universidad_Catolica_del_Ecuador,_Quito,_Ecuador e_Institute_of_Public_Health,_Federal_University_of_Bahia,_Salvador,_BA,_Brazil

f_Centro_de_Pesquisas_Gonc¸_alo_Muniz,_FIOCRUZ-BA,_Salvador,_BA,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received10July2015

Receivedinrevisedform23March2016 Accepted30March2016

Availableonline31March2016 Keywords: Trichuristrichiura Transcriptome Next-generationsequencing Functionalgenomics

a

b

s

t

r

a

c

t

Infectionwithhelminthicparasites,includingthesoil-transmittedhelminthTrichuristrichiura(human whipworm),hasbeenshowntomodulatehostimmuneresponsesand,consequently,tohaveanimpact onthedevelopmentandmanifestationofchronichumaninflammatorydiseases.Denovoderivationof helminthproteomesfromsequencingoftranscriptomeswillprovidevaluabledatatoaididentification ofparasiteproteinsthatcouldbeevaluatedaspotentialimmunotherapeuticmoleculesinnearfuture. Herein,wecharacterizedthetranscriptomeoftheadultstageofthehumanwhipwormT.trichiura,using next-generationsequencingtechnologyandadenovoassemblystrategy.Nearly17.6millionhigh-quality cleanreadswereassembledinto6414contiguoussequences,withanN50of1606bp.Intotal,5673 protein-encodingsequenceswereconfidentiallyidentifiedintheT.trichiuraadultwormtranscriptome; ofthese,1013sequencesrepresentpotentialnewlydiscoveredproteinsforthespecies,mostofwhich presentingorthologsalreadyannotatedintherelatedspeciesT.suis.Anumberoftranscriptsrepresenting probablenovelnon-codingtranscriptsforthespeciesT.trichiurawerealsoidentified.Amongthemost abundanttranscripts,wefoundsequencesthatcodeforproteinsinvolvedinlipidtransport,suchas vitellogenins,andseveralchitin-bindingproteins.Throughacross-speciesexpressionanalysisofgene orthologssharedbyT.trichiuraandthecloselyrelatedparasitesT.suisandT.murisitwaspossibletofind twenty-sixprotein-encodinggenesthatareconsistentlyhighlyexpressedintheadultstagesofthethree helminthspecies.Additionally,twentytranscriptscouldbeidentifiedthatcodeforproteinspreviously detectedbymassspectrometryanalysisofproteinfractionsofthewhipwormsomaticextractthatpresent immunomodulatoryactivities.Fiveofthesetranscriptswereamongstthemosthighlyexpressed protein-encodingsequencesintheT.trichiuraadultworm.Besides,orthologsofproteinsdemonstratedtohave potentimmunomodulatorypropertiesinrelatedparasitichelminthswerealsopredictedfromtheT. trichiuradenovoassembledtranscriptome.

Abbreviations: BLAST,BasicLocalAlignmentSearchTool;cDNA, complemen-taryDNA;DHPLC,denaturinghigh-performanceliquidchromatography;GO,Gene Ontology;KEGG,KyotoEncyclopediaofGenesandGenomes;NCBI,NationalCenter forBiotechnologyInformation;NR,non-redundant;rRNA,ribosomalRNA.

∗ Correspondingauthors.

E-mailaddresses:neuza@ufba.br(N.M.Alcantara-Neves),luis.pacheco@ufba.br (L.G.C.Pacheco).

1. Introduction

Trichuris trichiura (human whipworm) is a soil-transmitted helminthofhighpublichealthrelevanceandanestimated5 bil-lionhumansareatriskofstabletransmissionwiththisparasite, of whom nearly 1 billionare school-aged children (Pullan and Brooker,2012).Adultwhipwormmeasuresbetween30and50mm inlengthandcolonizesthehumanlargeintestine,whereitmay

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liveforuptotwo years,withfemaleslayingupto5000eggsa day(Bethonyetal.,2006).Clinicalpresentationoftrichuriasis typi-callyincludesintestinalmanifestations(diarrhea,abdominalpain), generalmalaise,weaknessand impairedcognitiveand physical development(WHO,2014).

Infectionwithhelminthicparasites,includingwhipworms,has been shown to modulate host immune responses and, conse-quently,tohaveanimpactonthedevelopmentandmanifestation ofchronichumaninflammatorydiseases(Bashietal.,2015;Maizels etal.,2014;Mishraetal.,2014).Specifically,epidemiological stud-ieshaveclearlydemonstratedaninverseassociationbetweenT. trichiurainfectionandType1skinhypersensivitytoaeroallergens inchildren(Rodriguesetal.,2008).Besides,thetherapeutic poten-tialofTrichurissuis(pigwhipworm)ovahasbeendemonstrated in some groups of patients withinflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, and in patients withrelapsing-remittingmultiplesclerosis(Flemingetal.,2011; Summersetal.,2003,2005a,2005b).

In an effort to better characterize the immunomodulatory potentialofhumanwhipwormproteins,ourgroupconducteda previousstudyinwhichseveralfractionsoftheT.trichiuraadult wormsomaticextract (hereafterrefereedasTtEFs)were evalu-atedfortheirimmunomodulatoryeffects oncytokineresponses byhumanperipheralbloodmononuclearcells(PBMCs)cultivated inthepresenceoftheseantigens(Santosetal.,2013).SixTtEFs wereidentifiedthatwereabletopromotegreaterproductionof theimmuneregulatorycytokineinterleukin(IL)-10,whencultured withPBMCs,andwerealsoabletoinhibithelperTcell1(TH1)and TH2cytokineproductionbyPBMCswhenco-culturedwith opti-malstimuli(e.g.phytohaemagglutininforTh2cytokines)(Santos etal.,2013).Additionalcharacterizationoftheseproteinfractions (TtEFs)bynano-liquidchromatography/massspectrometry iden-tifiedseveralproteins,mostofwhichhadorthologsintherelated parasiteTrichinellaspiralisandwereconsideredtobepromising candidatesforfutureevaluationasimmunomodulatorymolecules (Santosetal.,2013).Totakethisresearchastepfurthertoallow ustoengineertheseparasitemoleculesasrecombinantproteins, wedidatranscriptomicstudyofT.trichiuraadultwormtoidentify thegenesthatcodefortheseimmunomodulatoryproteins, tak-ingadvantageoftherecentlypublisheddraftgenomeassembly forT.trichiuraand thereferencegenomeand transcriptomefor themouseparasiteT.muris (Fothetal., 2014).We reporthere, toourknowledge,thefirsttranscriptomicanalysisofthespecies T.trichiura,whichwillcontributetothefunctionalannotationof therecentlyreleaseddraftgenomicsequence(Fothetal.,2014) andwillaiddiscoveryofT.trichiuramoleculesthatmaypossess immunomodulatoryproperties.

2. Materialsandmethods

2.1. CollectionofparasitesandtotalRNAextraction

AdultT.trichiurawormswereobtainedbytreatmentofinfected childrenfromtheprovinceofEsmeraldas–Ecuadorwithpyrantel pamoateandcollectionofwormsfromstoolsamplesfor1–2days after treatment, as described (Meekums et al., 2015). Ethical approvalwasprovidedbytheEthicalCommitteeofthe Universi-dadSanFranciscodeQuito–Ecuadorandwritteninformedconsent wasprovidedbyparentsorguardians.Adultwormswerewashed carefullyin0.15Mphosphate-bufferedsaline(pH7.4)and total RNAwasextractedusingastandardTrizol(Lifetechnologies) pro-tocolwiththeaidofzirconium/silicabeads(BioSpecProducts,Inc.). RNAqualitywasveriﬁedspectrophotometrically(260nm/280nm ratio=1.95)andquantitationwasdoneﬂuorometricallyusingthe Qubit®RNAAssaykit(LifeTechnologies).

2.2. DepletionofrRNAbyDHPLCandpreparationofthecDNA library

OnehundredmicrogramsoftotalRNAweresubjectedto deple-tionofribosomalRNAbydenaturinghigh-performanceliquid chro-matography(DHPLC)usingaRNASePTM_Cartridge_{(Transgenomic}®₎ column ina Wave® System4500 (Transgenomic®)machine,as describedindetailselsewhere(Castroetal.,2013).rRNA-depleted totalRNAwasthenusedforpreparationofthecDNAlibrarywith theIonTotalRNA-SeqKitv2(LifeTechnologies),accordingtothe manufacturer’srecommendations.

2.3. IonTorrentsequencinganddenovoassemblyofthe transcriptome

Transcriptome sequencing was achieved using the Ion Tor-rentPersonalGenomeMachineTM_(PGM)_and_the_Ion₃₁₈TM_chip, accordingtothemanufacturer’srecommendations(Life Technolo-gies).Atotalofthreereplicatesequencingrunswereperformed andtherawreadsweremergedintoasingleFASTQﬁle;shortreads (<50nt)wereremovedandtheremainingsequencesweretrimmed atboth5and3endswiththeFastX-Toolkit(http://hannonlab.cshl. edu/fastxtoolkit/index.html).Qualitycheckingof thesequences wasdonewiththeFastQCtoolthroughtheRNA-Rocketplatform (Warrenetal.,2015).

Denovoassemblyofthecleanreadswasgeneratedwiththe MIRA4.0assembler(Chevreuxetal.,2004).TheT.trichiura tran-scriptomeshortreadsdatasetispubliclyaccessible throughthe EuropeanNucleotideArchive(ENA),underaccessionPRJEB12315. ThedenovoassembledtranscriptsareavailableasaSupplementary material(SupplementaryﬁleS1).

Trimmedreadswerealsomappedagainsttherecentlyreleased draftgenomesequenceofT.trichiurav2.1(Fothetal.,2014)using theIonTorrentmapper (TMAP)strategyand theCLCGenomics Workbench.Then,weassociatedthelocustagswiththeir respec-tivegeneproductsaccordingtoT.trichiurageneannotationv.2.2 (Fothetal.,2014).

2.4. Functionalannotationofthetranscriptome

The Blast2GO tool v.3.1 (http://www.blast2go.org) and the TRAPID (Rapid Analysis of Transcriptome Data) tool (Van Bel et al., 2013) wereemployed to retrieve functional annotations for theT. trichiura assembled transcripts.Firstly, BLASTx simi-larity searches wereperformed againstthe non-redundant (nr) database of NCBI (E-value: <1E−05) using the de novo assem-bled transcripts as queries; conserved protein domains of the predictedproteinsandassignmentofthesequencestogene fam-ilieswereperformedthroughsearchesagainsttheOrthoMCLDB 5.0datasource(VanBelet al.,2013).Then,allassembled tran-scriptsweremanuallyannotatedusingtheTranslatetool(http:// web.expasy.org/translate/)followedbyBLASTpsimilaritysearches againstNCBI’snr(E-value:<1E−05)ortheHelmDBdatabase(http:// gasser-research.vet.unimelb.edu.au/helmdb/).WeobtainedGene ontology(GO)functionalclassiﬁcations(Ashburneretal.,2000)and KyotoEncyclopediaofGenesandGenomes(KEGG)pathway anno-tations(Kanehisaetal.,2014)fortheentiresetofproteinspredicted fromthemanuallyannotatedtranscriptome,throughtheBlast2GO v.3.1suiteusingdefaultparametersandthemostrecentreleases oftheGOandKEGGdatabasesasperDecember2015.Allsequences coding foras-yet-uncharacterizedproteinswerefurther charac-terizedthroughsearchesagainstthePROSITEcollectionofmotifs, usingScanProsite(http://prosite.expasy.org/scanprosite/). Predic-tionofprobablesignalpeptideswasperformedwiththeSignalP 4.1server(http://www.cbs.dtu.dk/services/SignalP/).

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2.5. Identiﬁcationofcandidatenovelprotein-encodingsequences andnon-codingtranscripts

Twodifferentstrategieswereusedtoidentifycandidatenovel transcriptsforthespeciesT.trichiura:(i)ﬁrstly,alldenovo assem-bledtranscriptsthatremainedunannotatedafterBLASTxsearches againstNCBI’sNRProteindatabasewerethenscannedforORFs withtheAUGUSTUSgenepredictionprogram,asdescribed(Stanke etal.,2006;Chenetal.,2015).SequencesforwhichnoORFscouldbe detectedwereconsideredaspotentialnon-codingtranscripts;all thesetranscriptswereusedinBLASTnsearchesagainstthe com-pletegenomesequenceofT.trichiurav.2.1throughtherecently releasedWormBaseParasitePlatform(Howeetal.,2016)inorder toconﬁrmthatthesenoveltranscriptsdoalignwiththereference genomebutdonotfallintoknowngenemodelsforthespecies. (ii)Inasecondstrategy,followingmanualannotationofprotein encodingtranscripts,allproteinspredictedfromtheT.trichiura transcriptomeinthisstudywerethencomparedwiththeentire setofproteinspredictedforthespeciesingenomeannotationv.2.2, usingtheCD-HITprogram(Fuetal.,2012)witha70%protein iden-titycut-off.Sequencesthatdidnotmatchproteinspredictedfrom knownT. trichiuragene productswerethencompared withthe entireproteinsetfortherelatedspeciesT.suis(Jexetal.,2014),in ordertodetectpotentialproteinorthologs.

2.6. AnalysisofT.trichiuratranscriptsthatcodeforproteinswith immunomodulatoryactivities

tBLASTnsearches(E-value:<1E−05)wereperformedagainstthe T.trichiuratranscriptomedatasetusingtheaminoacidsequences of the proteins that were identiﬁed previously through mass spectrometryanalysisofsixchromatographicfractionsofthe whip-wormsomaticextract(TtEF6,TtEF8,TtEF9,TtEF10,TtEF11and TtEF12)andwhichshowedinvitroimmunomodulatoryactivities (Santosetal.,2013).Theseproteinshadbeenidentiﬁedinthe previ-ousstudyusingProteinLynxGlobalServer(PLGS)searchesagainst proteindatabasesofrelatedparasiticworms,inparticularT.spiralis (Santosetal.,2013).

Three-dimensionalproteinstructuresweremodeledusingthe SWISS-MODEL server (Biasini et al., 2014) and validation was performedusing Prosa-web(Wiederstein and Sippl, 2007) and theSTINGMilleniumSuite(Higaetal.,2004).Multiplestructural alignmentwasobtainedwithMUSTANG(MultipleStructural Align-mentAlgorithm)(Konagurthuetal.,2006)andvisualizationofthe proteinstructureswasperformedusingUCSFChimerasoftware, version1.6.1(Pettersenetal.,2004).

2.7. Transcriptabundanceestimationandcross-speciesgene expressionanalysis

TheexpressionlevelsoftheT.trichiuratranscriptswere esti-matedbothwiththeCLCGenomicsWorkbench(CLCbio)andthe NextGENe®softwarev2.4(SoftGenetics),followingmappingofthe entiresetoftrimmedreadsagainsttheca.75Mbpdraftgenome assemblyofT.trichiurav.2.1andgeneannotationsv.2.2(GenBank: GCA000613005.1).Thenumbersofreadsmappingtotheexisting genemodelsforthespecieswerecalculatedandthennormalized toRPKM(ReadsPerKilobaseperMillionmappedreads)values.A RPKMthresholdvalueof0.3wassettoconﬁdentiallydetectthe presenceofatranscriptforaspeciﬁcproteinencodinggene.

A cross-speciesgene expression analysis wasperformed for comparingthetranscriptabundanceofgeneorthologssharedby T.trichiuraand theclosely relatedparasitesT.suisandT. muris. Forthis,theRNA-seqshortreadﬁlesfromT.suisandT.murisadult wormtranscriptomeswereretrievedfromArrayExpress(accession number:E-ERAD-125)andtheSequenceReadArchive(accession

Fig.1.AutomaticannotationoftheT.trichiuradenovoassembledtranscripts.All transcriptsequenceswereautomaticallyannotatedusingBLASTxsearchesagainst NCBI’snon-redundantproteindatabase.SequencesforwhichnoBLASThitscouldbe automaticallyretrieved(unannotated)werethenscannedforthepresenceofORFs, usingAUGUSTUS.AllthesetranscriptswerealsousedinBLASTnsearchesagainstthe T.trichiuradraftgenomeassemblyv.2.1,throughtheWormBaseParasiteplatform.

numbers: SRR1041654 and SRR1041655). Then, CLC Genomics Workbench(CLCbio)wasusedtomapthereadsagainst a ref-erence composedonly of T. trichiura predictedgene sequences (Fothetal.,2014).Thealignmentparametersusedforthese anal-yseswereasfollows:(i)mismatchcost=2;(ii)insertioncost=3; (iii)deletioncost=3;(iv)lengthfraction=0.7;and(v)similarity fraction=0.8.ResultswerenormalizedtoRPKMvalues.For confir-mationofexpressionprofilesobservedforspecifictranscripts,theT. trichiuragenesweremappedbacktotheircorrespondingorthologs intheothertwospecies,andthespecifictranscriptexpression lev-elswereobtainedfromtheworksbyJexandcollaborators(2014) andFothetal.(2014).

3. Resultsanddiscussion

3.1. SequencinganddenovoassemblyoftheT.trichiura transcriptome

Nearly 19 million raw reads (accumulated length of 2,642,638,062bp)wereobtainedbysequencingoftheT.trichiura cDNA library. Trimming and quality assessment resulted in approximately17.6million(93.6%)high-qualitycleansequences, withmostofthereadsabovetheQ20level(Table1;Fig.S1).De novotranscriptomeassemblyyielded6414contigswithanN50of 1606bp.Theaveragecontigsizeofthetranscriptomeassembled ab initiowas1404bp,and contiglengthrangedfrom183bpto 9499bp(Table1).Trimmedreadswerealsomappedagainstthe recentlyreleaseddraftgenomesequenceofT.trichiurav2.1(Foth etal., 2014)and92.08% of thereadsmapped tothereference; 81.64%ofthereadsmappedtopredictedproteincodingsequences (Table1),whichdemonstratesagoodqualityoftheRNAextraction protocolandofthecDNAlibrarypreparation(Sultanetal.,2014). Positional analysis ofspeciﬁc transcriptswas obtainedthrough theWormBaseParasite Platform(Howe etal., 2016), following alignmenttotheT.trichiurareferencegenomev.2.1;thisanalysis showedgoodconcordanceofthedenovoassembledtranscriptome withtheexistinggenemodelsforthespecies(Fig.S2A–E). 3.2. Proteinencodingsequencesandcandidatenoveltranscripts

Thegreat majorityofthe6414T.trichiuradenovoassembled transcriptsrepresented sequences for which it was possibleto retrieveautomaticannotationsfollowingBLASTxsearchesagainst knownproteinsequencesinpublicdatabases(Fig.1).Amongthe remaining440transcriptswithnoBLASTxhits,itwaspossibleto

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Table1

SummaryofresultsofT.trichiuraadulttranscriptomesequencingandassembly.

Statistics T.trichiuratranscriptomedataset

Numberofrawreads 18,838,891

Numberofﬁlteredreads 17,640,541

Totalbasepairs(bp)(rawdata) 2,642,638,062

Totalbasepairs(bp)(ﬁltereddata) 2,360,442,157

Sequencelengthrangeinnucleotides 50–362

AverageGCcontent 49.0%

Numberofcontigsassembledabinitio 6414

Contiglengthrange 183bp–9499bp

Averagecontiglength 1404bp

N50contigsize 1606bp

PercentofreadsaboveQ20level 97.6%

Readsmappedtoproteincodingregionsa _81.64%

Readsmappedtonon-codingregionsa _10.44%

Numberoftranscriptsassignedtospeciﬁcgenefamiliesb ₄₉₃₅_(76.9%) Numberoftranscriptscodingforrecognizableproteindomainsb ₄₁₅₄_(64.7%) Numberofproteinspredictedfromthemanuallycuratedtranscriptome 5673 NumberoftrancriptscodingforproteinsidentiﬁedinimmunomodulatoryfractionsoftheT.trichiuraproteinextractc ₂₀ a_Reads_were_mapped_against_the_draft_genome_assembly_of_T._trichiura_v2.1,_in_which₉₆₅₀_genes_were_predicted_for_this_species_(Foth_et_al.,_2014). b_As_predicted_by_the_TRAPID_tool_(Van_Bel_et_al.,_2013),_using_a_OrthoMCLDB_5.0_data_source.

c _Based_on_the_{immunomodulatory}_protein_fractions_identiﬁed_in_the_work_by_Santos_et_al._(2013).

Fig.2.ProteinspredictedfromtheT.trichiuratranscriptomeandcandidatenovel protein-encodinggenes(PEGs).Thedenovoassembledtranscriptsweremanually inspectedforthepresenceofproteincodingsequencesusingtheExpasy’s Trans-latetool,followedbyBLASTpsimilaritysearchesagainstNCBI’sNRortheHelmDB database.Allpredictedproteinswerealsomatchedwithcorrespondentproteins predictedfromtheT.trichiuraannotatedgenomicsequence.Unmatchedproteins werethenanalyzedagainstproteinspredictedfortherelatedparasiteT.suis.

predictnovelopenreadingframes(ORFs)for249sequences(Fig.1; TableS1).Onehundredand ninety-onetranscriptsrendered no hitswithknownproteinsequencesbut alignedwithhigh iden-tities(mean=97.0%)withtheT.trichiuradraftgenomeassembly, representingprobablenovelnon-codingtranscripts(Fig.1;Table S2).Ofthese,onlysevenwereintheneighborhoodsofknown pro-teinencodinggenes,consideringa100-nucleotideswindowsize (TableS2).Fivesequencesproducedsigniﬁcantalignmentswith non-codingRNAspresent intheNONCODE2016 database(Zhao etal., 2016)(TableS2). One hundredand seventy-oneofthese transcriptsarelongerthan200nucleotides,representingthen can-didatelongnon-codingRNAsofthespecies(TableS2).

Aftermanualcurationofthedenovoassembledtranscripts,5673 proteinencodingsequenceswereidentifiedintheT.trichiura tran-scriptome.Ofthese,4660matchedknownproteinspredictedfrom theT.trichiuragenomev.2.1(Fig.2).Oftheremaining1013protein encodingsequencesidentifiedinthemanuallycurated transcrip-tome,631matchedproteinspredictedfromthe genomeofthe relatedspeciesT.suis(Fig.2)(SupplementaryfileS2).

Thediscoveryofpotentialnovelproteinencodinggenes(PEGs) from the T. trichiura transcriptome, when compared to closely

relatedspecies,is notunexpectedgiventhefactthat only9650 genes have been predicted from the T. trichiura draft genome assemblyv.2.1,whilst14,261and11,004PEGswhere predicted forT.suisandT.muris,respectively(Jexetal.,2014;Fothetal., 2014).Atotalof7431geneorthologsaresharedbythelattertwo speciesaspredictedinthecurrentgenemodelsavailablein Worm-BaseParasiterelease5,whereasonlyca.5700PEGsofthecurrent T.trichiurageneannotationaresharedwiththeothertwospecies. Importantly,transcriptomicstudieswerealready availableforT. suisandT.muris(Cantacessietal.,2011;Jexetal.,2014;Fothetal., 2014),andthenaidedpredictionofPEGsinthenewlygenerated genomicsequencesofthesespecies.

3.3. Functionalannotationandclassiﬁcation

Mostofthe6414T.trichiuraassembledtranscriptsrepresented sequences that code for proteins predicted from the recently available genomic sequences for the species T. trichiura and T. suis(Fig.3A),withvariousorthologs sharedbytherelated par-asite T. spiralis and by ﬁlarial nematodes (Loa loa and Brugia malayi)(Fig.3B).Amongthese,4935transcriptscouldbeassigned to 2683 unique gene families (Table 1). A total of 22,799 GO functional annotations wereobtained for thepredictedprotein coding sequences;in thecategories representing biological pro-cesses(73.0%) and molecularfunctions (27.0%) (Fig.4Aand B). Amongthesequencesannotatedatthebiologicalprocesseslevel, the most highly scoredcategories were cellularand metabolic processes(3001and2885sequences,respectively),whereas114 predictedproteinswereclassiﬁedinimmunesystemprocessesand 194sequencesinreproductiveprocesses(Fig.4A).Atthemolecular functionslevel,theT.trichiurasequencesweredistributedineleven categories,with378predictedproteinsinvolvedwithtransporter activitiesandmostoftheremainingproteins(2842)involvedin bindingfunctions(Fig.4B).

T.trichiurapredictedproteinswerealsosubmittedtobiological pathwayanalysisbymappingagainstuniqueKEGGorthologs.We couldidentifyseveraltranscriptsthatcodeforenzymesinvolved inmetabolicmodulesthatareconservedamongparasitic nema-todes and that have been demonstrated tobe highly activated in adultworms (Tyagiet al., 2015), suchas reactionsinvolved in purinemetabolism. In total, thepredictedproteinscould be assigned to 88 different biochemical pathways(Table S3). The predominantpathwayswere:“Purinemetabolism”(ko00230,162 sequences), “Pyrimidine metabolism” (ko00240, 65 sequences),

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Fig.3.DistributionoftopBLASThitsfoundforproteinspredictedfromtheT.trichiuratranscriptome.(A)SpeciesdistributionofthetopBLASThitsfoundforeachT.trichiura protein,asdeterminedthroughBLASTpsimilaritysearchesofthetranslatedtranscriptomesequencesagainstthenon-redundantdatabaseofNCBI(E-valuecut-off:<1E−05₎

(B)distributionofthetotalhitsfoundforT.trichiuraproteins.

Fig.4. GeneOntology(GO)functionalannotationsofproteinspredictedfromtheT.trichiuratranscriptome.GOclassiﬁcationsofthepredictedproteinsaresummarizedin twomaincategories,atsecondlevel:(A)biologicalprocessesand(B)molecularfunctions.

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“Aminoacyl-tRNA biosynthesis” (ko00970, 58 sequences), and “Phosphatidylinositolsignalingsystem”(ko04070,41sequences) (TableS3).

3.4. Annotationoftranscriptsthatcodeforproteinsof uncharacterizedfunctions

Intotal, 2096(ca.35.0%)of theprotein-encodingtranscripts identifiedintheT.trichiuratranscriptomecodefor uncharacter-izedproteins(annotatedaseither‘unknown-functionproteins’or ‘hypotheticalproteins’).Thisproportionofuncharacterized pro-teins reflects the overall numbers predicted form the recently available genomic sequences of T. trichiura and T. muris (Foth etal.,2014).Eighty-fouroftheseproteinswereseentopossess signalpeptides. By searching for PROSITE signatures, usingthe ScanProsite approach (deCastro et al., 2006), we were ableto obtainadditionalfunctionalannotationsfor607ofthepredicted T. trichiurauncharacterizedproteins. Itwaspossible toidentify 1522PROSITEhits intheseproteins, belongingto337different domains,familiesandfunctionalsites.Twenty-sevenofthesehits werepresentinmorethan5uncharacterizedproteins,asshown inFig.S3A.Thezinc-fingerdomainwasthemostprevalentand wasfoundin48differentuncharacterizedproteins(Fig.S3A). Zinc-fingerdomainsarenucleicacid-bindingproteinstructuresmostly involvedintranscriptionregulation.Manyclassesofzinc-fingers arecharacterizedaccordingtothenumberand positionsofthe histidineandcysteineresiduesinvolvedinthezincatom coordi-nation(RosenfeldandMargalit,1993).Thezinc-fingerC2H2-type domain(PDOC00028),withDNAorRNAbindingproperty,andthe zincfingerRING-typesignature(PDOC00449),whichcanplayakey roleintheubiquitinationpathway(Itoetal.,2001),werethemost commonlyfoundintheT.trichiuraproteins(Fig.S3A).

Theproteinkinasedomain(PDOC00100)wasidentifiedin34 T. trichiura uncharacterizedproteins; proteinswiththe highest probabilitiesofbeingtrueproteinkinaseswillcommonlypresent twospecificsignatures:(i)serine/threonineresidues,involvedin ATPbinding,and(ii)tyrosineproteinkinases,withcatalytic activ-ity (Hanksand Hunter, 1995; Knightonet al.,1991).Six ofthe predictedT.trichiuraproteins,outof34proteinsidentifiedwith proteinkinasedomains,displayedbothproteinssignatures(Fig. S3B).

3.5. ExpressionlevelsofT.trichiuratranscriptsandcross-species comparisons

TheabundanceoftheT.trichiuratranscriptswasestimatedby normalizedRPKMvalues,followingmappingofthetranscriptome readsagainsttherecentlyreleaseddraftgenomeassemblyforthe species(Fothetal.,2014).AstringentRPKMthresholdof0.3was deﬁnedtoconﬁdentiallydetectthepresenceofatranscriptfora particulargene.

Thereis a highnumber of transcripts coding for unknown-functionproteins(hypotheticalproteins)amongthetop40most abundantprotein-encodingtranscriptsidentifiedinthe transcrip-tomeoftheT.trichiuraadultworm(Table2),similarlytowhathas beendemonstratedforthetranscriptionallymosthighlyexpressed genesofT.muris(Fothetal.,2014).Severalofthesehighlyexpressed transcriptsthat code for hypotheticalproteinsare predictedto bepartofagenefamilythatcontains8paralogsannotatedinT. trichiura(Fig.5);there are6 orthologs identifiedin therelated parasitesT.suisandT.muris,accordingtotheWormBaseParasite database(Fig.5).Fig.S4showsananalysisofuniquereadsidentified foreach of theparalogsin theT.trichiuratranscriptome. Note-worthy,thegeneorthologsfoundinT.suisandT.muriswerealso seenamongstthemosthighlyexpressedprotein-encodinggenes

accordingtoprevioustranscriptomicstudiesofthesespecies(Jex etal.,2014;Fothetal.,2014).

Consideringthatadultfemalewhipwormscanlaythousands of eggs per day, it is not unexpected to find a high tran-scriptional activity of genes that code for proteins involved in reproductiveprocesses. Accordingly, amongthemost abundant transcriptsidentifiedintheT.trichiuraadultwormtranscriptome wefoundsequencesthatcodeforproteinsinvolvedinlipid trans-port,suchasvitellogenins(whicharemajorcomponentsofyolk), andseveralchitin-bindingproteins(Table2).Interestingly,some oftheseprotein-encodingtranscriptswerealsoseentobemore expressedin femaleadult worms (particularly in theposterior body)intheT.muristranscriptome(Fothetal.,2014).Besides, tran-scriptscodingfortheseproteinswerealsoidentifiedamongstthe 20mosthighlyexpressedprotein-encodingtranscriptsinseparate T. suistranscriptomicstudies (Cantacessiet al.,2011;Jex etal., 2014).

Previousstudieshavedemonstratedthatitmightbepossible to achieve successful measurements of mRNA expression lev-elsthrough mappingof RNA-seqreadsto thepredictedcoding sequencesfromareference genomeof aclosely relatedspecies (HornettandWheat,2012).Thehumanandporcinewhipworms areverycloselyrelated;infact,thesespecieshaveonlyrecently beendemonstratedtobeseparatespeciesandunarguably distin-guishedatthegeneticslevel(Cutillasetal.,2009;Liuetal.,2012). TherelatednessbetweenT.trichiuraandthemouseparasiteT.muris hasalsobeenrecentlydemonstratedintheworkbyFothetal. (2014),wheretheyshowthatthesetwospeciesshareaveryhigh numberofgeneorthologs,withanaverageaminoacididentityof 79.0%betweenproteinorthologs(Fothetal.,2014).

Therefore,wehavehypothesizedinthisstudythatitmightbe possibletousetherecentlyreleasedT.trichiuraannotatedgenome asaclosereferencegenomeformappingT.suisandT.muris RNA-seqshortreads,inordertodiscovergeneorthologsthatarehighly expressedacrossallthethreespecies(fordetails;pleasereferto item2.7).Fig.6Ashowsthenumbersofgeneorthologsfoundas highlyexpressed(calculatedRPKM>200)foreachspecies.Itwas possibletoidentify26protein-encodinggenesthatareconsistently highlyexpressedintheadultstagesofthethreehelminthparasite species(Fig.6AandB);thesegenesarehighlyconservedamongthe Trichurisspecies,andtheirhighexpressionlevelshavebeen con-ﬁrmedfromprevioustranscriptomicstudiesofT.suisandT.muris (TableS4).Severalofthese26geneshadbeenidentiﬁedinthis studyamongthe40mosthighlyexpressedprotein-encodinggenes ofT.trichiura(Fig.6BandTable2).Noteworthy,genesbelongingto thegenefamilyshowninFig.5wereseenashighlyexpressedin allthreespecies(Fig.6B).Besides,wehavenoticedthattranscripts codingforproteinsinvolvedinreproductivefunctionsinfemale parasitesarealsoparticularlyhighlyrepresentedinthe transcrip-tomesofadultworms(Fig.6B).

3.6. T.trichiuratranscriptsencodingproteinswith immunomodulatoryactivities

In the present analysis, it waspossible to identify20 tran-scriptsthatcodeforproteinspreviouslydetectedintheT.trichiura immunomodulatory protein fractions (TtEFs) described in the studybySantosetal.(2013)(pleaserefertoitem2.6;Table3).Five ofthesetranscriptswereamongstthe200mosthighlyexpressed, asmeasuredbynormalizedtranscriptabundancevalues(RPKM) (Table3).

TtEF9 was the protein fraction presenting the greatest immunoregulatoryresponseinducingahighlevelofIL-10when culturedwithPBMCsandshowingstronginhibitoryeffectsagainst the production of TH1 and TH2 cytokines when co-cultured withoptimalimmunestimuli(Santosetal.,2013).Theproteins

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Table2

The40mosthighlytranscribedprotein-encodingsequencesidentiﬁedintheT.trichiuraadultwormtranscriptome.

Accessionnumbera_Descriptionb _Orthologsc _RPKMd

(Log2)

GeneOntology (GO) Annotationse

BiologicalProcess MolecularFunction CellularComponent CDW58692.1 Hypotheticalprotein TTRE0000701701 TMUEs005200110014.31 CDW52980.1 Hypotheticalprotein TTRE0000124301 Tsui7105291 14.23 CDW52979.1 Hypotheticalprotein TTRE0000124101 M51304084 14.06 CDW58325.1 Hypotheticalprotein TTRE0000663201 M51419266 14.03 CDW56655.1 Hypotheticalprotein TTRE 0000493701 TMUE s0052001100 13.83 Membrane CDW54233.1 Hypotheticalprotein TTRE0000250301 TMUEs002400290013.48 CDW58129.1 VitellogeninNandVWDand

DUF1943domaincontaining protein

Tsui7113118 13.32 Lipidtransport Lipidtransporteractivity

CDW54610.1 Spindle-polebodyprotein(Pcp1) M513 09656 13.11 CDW61263.1 Hypotheticalprotein TTRE0000971101 TMUEs005200100013.06 CDW61299.1 Hypotheticalprotein TTRE0000974801 Tsui7323554 12.92

CDW61002.1 Cellwall-associatedhydrolase Asuu8446313 12.66 Metabolicprocess Hydrolaseactivity CDW61247.1 Cellwall-associatedhydrolase Asuu8446313 12.55 Metabolicprocess Hydrolaseactivity CDW61305.1 Hypotheticalprotein TTRE0000975601 Tsui7125196 12.47 CDW60883.1 Hypotheticalprotein TTRE0000928701 Tsui7105291 12.00 CDW61059.1 Hypotheticalprotein TTRE0000948201

Tsui7121137 11.62 Lipidbinding

CDW60789.1 Cellwall-associatedhydrolase Asuu8446313 10.70 Metabolicprocess Hydrolaseactivity CDW54015.1 Poly-cysteineandhistidinetailed

proteinisoform2

Tsui7117112 10.46

CDW54307.1 Heatshockprotein90 Tsui7117373 10.20 Proteinfolding;ResponsetostressATPbinding;Unfoldedprotein binding

CDW59881.1 Hypotheticalprotein TTRE0000822501

Tsui7115052 10.08

CDW54937.1 Polyadenylatebindingprotein1 Tsui7108405 9.87 Nucleotidebinding;RNAbinding Cytoplasm CDW61304.1 hypotheticalprotein

TTRE0000975501

Asuu8446313 9.80

CDW56365.1 Polyubiquitin Tsui7109600 9.74 Cytoplasm

CDW58714.1 TILandCBM14domain containingprotein

Tsui7119446 9.49 Chitinmetabolicprocess Chitinbinding Extracellularregion CDW57445.1 CBM14domaincontaining

protein

Tsui7137747 9.22 Chitinmetabolicprocess Chitinbinding Extracellularregion CDW61148.1 Hypotheticalprotein

TTRE0000958101

Tsui7242031 9.20 Proteinfolding Calciumionbinding;Unfolded proteinbinding Endoplasmicreticulum CDW53632.1 Hypotheticalprotein TTRE0000189701 Tsui7129116 9.03 CDW52289.1 Hypotheticalprotein TTRE 0000054801 Tsui7134747 8.98

CDW53617.1 Angiotensinconvertingenzyme Tsui7118907 8.97 Proteolysis Peptidaseactivity; Metallopeptidaseactivity

Membrane CDW56602.1 Tyrosinasedomaincontaining

protein

Tsui7146002 8.94 Oxidation-reductionprocess Oxidoreductaseactivity CDW54940.1 CBM14domaincontaining

protein

Tsui7110776 8.88 Chitinmetabolicprocess Chitinbinding Extracellularregion CDW59986.1 NADHdehydrogenasesubunit5 Tsui7107151 8.79 Oxidation-reductionprocess Oxidoreductaseactivity Membrane CDW53952.1 Fattyacidsynthase Tsui7130170 8.79 Biosyntheticprocess Fattyacidsynthaseactivity

CDW55082.1 TSP1andCBM14domain containingprotein

Tsui7387368 8.67 Chitinmetabolicprocess Chitinbinding Extracellularregion

CDW57937.1 Calreticulin Tsui7272154 8.65 Proteinfolding Unfoldedproteinbinding Endoplasmicreticulum

CDW57439.1 CBM14domaincontaining protein

Tsui7118549 8.65 Chitinmetabolicprocess Chitinbinding Extracellularregion CDW56497.1 Phosphoenolpyruvate

carboxykinaseGTP

Tsui7136493 8.59 Gluconeogenesis Kinaseactivity

CDW61309.1 Piwidomainprotein Tsui7129624 8.57 Nucleicacidbinding

CDW60206.1 TrypsindomaincontainingproteinTsui7201621 8.55 Proteolysis Serine-typeendopeptidase activity

CDW56764.1 Bifunctional3_{phosphoadenosine} _Tsui7201551 _8.52 _{Phosphorylation} _{Adenylylsulfate}_kinase_activity

CDW59236.1 Hypotheticalprotein TTRE0000756701

Tsui7167102 8.44

Tsui=Trichurissuis;Asuu=Ascarissuum;TMUE=Trichurismuris.

a_NCBI’s_PROTEIN_Database._Several_of_the_most_highly_expressed_sequences_are_part_of_a_gene_family_with₈_paralogs_and₆_orthologs_annotated_in_WormBase_Parasite_database

(Howeetal.,2016).Thesearemarkedbold.

b _According_to_version_2.2_of_the_T._trichiura_genome_annotation.

c _Orthologs_retrieved_from_the_HelmDB_database_(Mangiola_et_al.,₂₀₁₃₎_or_the_WormBase_Parasite_database_(Howe_et_al.,_2016),_using_BLAST.

d _Normalized_transcript_abundances_obtained_using_CLC_Genomics_Workbench_(CLC_bio)._Results_are_expressed_in_Log2_of_Reads_Per_Kilobase_per_Million_mapped_reads

(RPKM).AllthesemeasurementswerealsocorroboratedbyanalysiswithNextGENe®_software_v2.4_{(SoftGenetics).}

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Fig.5. Agenefamilycodingforunknown-functionproteinswithhighexpressionlevelsintheT.trichiuratranscriptome.GenetreeshowingeightT.trichiuraparalogsand sixorthologsfoundinrelatedspecies,accordingtogenemodelspresentinWormBaseParasite.

Fig.6. Cross-speciescomparisonsoftranscriptabundances.RNA-seqshortreadsoftheadultstagesofthethreeTrichurisspeciesweremappedagainsttheT.trichiura annotatedgenes(fordetails,pleaserefertothemethodssection).(A)Venndiagramshowingthedistributionofthemosthighlyexpressed(RPKM>200)transcriptsacross thethreespecies.(B)Expressionlevelsof26geneorthologsconsistentlyhighlyexpressedinthethreeTrichurisspecies.HeatmapwasgeneratedwithLog2transformed RPKMvalues,usingtheMatrix2pngtool(PavlidisandNoble,2003).Anasterisk(*)indicatessequencesthatcodeforribosomalRNAs,asshowninFig.S6.Thearrowsindicate proteinencodinggeneswhoseorthologshavebeenshowntobehighlyexpressedinfemalewormsinprevioustranscriptomicstudiesofthespeciesT.murisandT.suis(Table S4),includinggenesinFig.5.

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Table3

ExpressionanalysisoftranscriptsencodingT.trichiuraproteinswithimmunoregulatoryactivities.

ProteinIDa _Description _Transcript

abundance (RPKM)b

TtEF6 TtEF8 TtEF9 TtEF10 TtEF11 TtEF12

CDW56935.1 Actin 179.54 X

CDW53601.1 Actindepolymerizingfactor2,isoformc 7.00 X

CDW53275.1 ATPsynthasesubunitbeta 89.03 X X X

CDW53384.1 Betahexosaminidase 22.47 X

CDW57885.1 Gutspeciﬁccysteineproteinase 6.85 X X X

CDW60307.1 NADHdependentfumaratereductase 12.15 X CDW53142.1 Eukaryotictranslationelongationfactor1A 249.53 X

CDW54435.1 FructosebisphosphatealdolaseclassI 76.75 X X X X X

CDW53185.1 Glutamatedehydrogenase 10.49 X

CDW56034.1 Heatshockprotein70 196.20 X X

CDW53003.1 Malatedehydrogenase 64.28 X

CDW54347.1 ProteinretinaldegenerationB 5.46 X

CDW58553.1 ProteinkinaseC,brainisozyme 3.68 X

CDW56947.1 LamininG2andCadherinandEGFCAdomaincontainingprotein 10.01 X CDW57784.1 Ufd2PcoreandU-boxdomaincontainingprotein 17.06 X CDW56963.1 NebulinandLIMandSH31domaincontainingprotein 4.87 X

CDW52208.1 Girdin 36.50 X

CDW54989.1 tRNA(cytosine(34)C(5))methyltransferase 11.39 X CDW56497.1 PhosphoenolpyruvatecarboxykinaseGTP 363.89 X X

CDW54395.1 Intermediateﬁlamentproteinifa1 281.45 X

a_Proteins_identiﬁed_previously_through_mass_spectrometry_analysis_of_six_{chromatographic}_fractions_of_the_whipworm_somatic_extract_(TtEF_6,_TtEF_8,_TtEF_9,_TtEF_10,

TtEF11andTtEF12),whichshowedinvitroimmunomodulatoryactivities(Santosetal.,2013).NCBI’sPROTEINDatabase.

b _Normalized_transcript_abundances_in_Reads_Per_Kilobase_per_Million_mapped_reads_(RPKM),_as_determined_by_analysis_with_the_NextGENe®_software_v2.4_{(SoftGenetics).}

TranscriptsthatareamongstthemosthighlyexpressedintheT.trichiuratranscriptomearemarkedbold.

predictedinthisstudyfromthetranslatedT.trichiura transcrip-tomewhicharefoundinTtEF9include:CDW56935.1(Actin Frag-ment);CDW53275.1(ATPsynthasesubunit␤);CDW57885.1(Gut speciﬁccysteineproteinase);CDW54435.1(Fructosebisphosphate aldolase); CDW53003.1 (Malate dehydrogenase); CDW58553.1 (ProteinkinaseC,brainisozyme);andCDW52208.1(Girdin).

The protein ‘Fructose bisphosphate aldolase’ (CDW54435.1) wasidentiﬁedinallbutone(TtEF10)oftheimmunomodulatory fractionspreviouslydescribed(Santosetal.,2013)(Table3). Like-wise,‘Gutspeciﬁccysteineproteinase’(CDW57885.1)and‘Heat shock protein 70 (HSP70)’ (CDW56034.1) were alsopresent in morethanoneoftheTtEFimmunomodulatoryfractions(Table3). Therefore,thesesequencesrepresentsuitablecandidatesforfuture expressionusingrecombinantDNAtechnology.Three-dimensional modelingoftheseT.trichiuraproteinsdemonstratedhigh struc-turalsimilaritywiththeircharacterizedorthologsfromT.spiralis (Fig.S5).

In addition to these proteins, we investigated sequences in theT.trichiuratranscriptomethatencodeproteinsdemonstrated to have potent immunomodulatory properties in related para-sitic helminths, including a serine-type endopeptidase from T. suisfirststagelarvae(Tsui7304731)(Ebneretal.,2014)andthe CathepsinL1fromFasciolahepatica(Dowlingetal.,2010).The pro-teinTsui7304731,alongwiththeproteinsofunknownfunction, Tsui7583957andTsui7234544,havebeenpreviouslyidentifiedby massspectrometryintheexcretory/secretoryextractfromL1 T. suislarvae,andhavedemonstratedimmunomodulatoryeffectsin amurinemodelofallergy(Ebneretal.,2014).tBLASTnsearches againsttheT.trichiuratranscriptomeusingtheaminoacidsequence ofTsui7304731identifiedthecodingtranscriptTrichurisc27 (E-value:2.8E−136).Structuralalignmentofthetwopredictedproteins also demonstrated high similarity (Fig. S5). Transcripts encod-ingproteins withsimilarities toTsui7583957 and Tsui7234544 werealsofoundintheT.trichiuratranscriptome:Trichurisc4299 (E-value:3E−092)andTrichurisc2345(E-value:2E−037), respec-tively.TheCathepsinL1ofF.hepaticaisanotherhelminthicprotein withimmunoregulatoryactivity(Dowlingetal.,2010).AtBLASTn searchagainsttheT.trichiuratranscriptomeidentifiedthecoding

transcriptTrichuris c3964,whichcodesforaprobableproteinwith highsimilaritytotheF.hepaticacounterpart(Fig.S5).

4. Conclusions

Inconclusion,wepresentthefirsttranscriptomicexploration oftheadultstageofthehumanwhipwormT.trichiura,using next-generationsequencingtechnologyandadenovoassemblystrategy. We wereabletoidentifyanumber oftranscriptsthat codefor potentialnewlydiscoveredproteinsforthespeciesT.trichiuraand alsofor previouslyunannotatednon-codingtranscripts.Besides, weidentifiedtranscriptsthatcodeforproteinspreviouslyreported topossessimmunomodulatory activities.Ourfindings willnow allowustoproducerecombinantT.trichiuraproteinsencodedby thesetranscriptsthatcouldbeevaluatedaspotentialtherapeutic moleculesforthetreatmentofchronicinflammatoryconditionsin humans.

Acknowledgements

Thisstudy wassupported by thefollowing research grants: INCT/MCT/CNPq Programme – Contract no. 5737862008; MCTI/CNPq/FNDCT Ac¸ão Transversal N◦ 79/2013 (RENORBIO); FAPESB (PET0005/2010 and JCB017/2013); and CAPES (project n◦0772012). LNS was recipient of a scholarship from FAPESB. NMAN and MLB were recipients of research fellowships from CNPq.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.actatropica.2016. 03.036.

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