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UseofMultipleSequencingTechnologiesToProduceaHigh-Quality GenomeoftheFungus Pseudogymnoascusdestructans ,theCausative AgentofBatWhite-NoseSyndrome KevinP.Drees,a JonathanM.Palmer,bRobertSebra,cJeffreyM.Lorch,dCynthiaChen,eCheng-CangWu,eJinWooBok,fNancyP.Keller,fDavidS.Blehert,d ChristinaA.Cuomo,gDanielL.Lindner,b JeffreyT.FosteraDepartmentofMolecular,CellularandBiomedicalSciences,UniversityofNewHampshire,Durham,NewHampshire,USAa;CenterforForestMycologyResearch, NorthernResearchStation,U.S.ForestService,Madison,Wisconsin,USAb;DepartmentofGeneticsandGenomicSciencesandIcahnInstituteforGenomicsandMultiscale Biology,IcahnSchoolofMedicineatMountSinai,NewYork,USAc;U.S.GeologicalSurvey,NationalWildlifeHealthCenter,Madison,Wisconsin,USAd;IntactGenomics, SaintLouis,Missouri,USAe;DepartmentofBacteriologyandDepartmentofMedicalMicrobiologyandImmunology,UniversityofWisconsin,Madison,Wisconsin,USAf; GenomeSequencingandAnalysisProgram,BroadInstituteofMITandHarvard,Cambridge,Massachusetts,USAgWhite-nosesyndromehasrecentlyemergedasoneofthemostdevastatingwildlifediseasesrecorded,causingwidespreadmortalityinnumerousbatspeciesthroughouteasternNorthAmerica.Here,wepresentanimprovedreferencegenomeofthefungal pathogen Pseudogymnoascusdestructans foruseincomparativegenomicstudies. Received 11April2016 Accepted 18May2016 Published 30June2016 Citation DreesKP,PalmerJM,SebraR,LorchJM,ChenC,WuC-C,BokJW,KellerNP,BlehertDS,CuomoCA,LindnerDL,FosterJT.2016.Useofmultiplesequencing technologiestoproduceahigh-qualitygenomeofthefungus Pseudogymnoascusdestructans ,thecausativeagentofbatwhite-nosesyndrome.GenomeAnnounc4(3): e00445-16.doi:10.1128/genomeA.00445-16. Copyright 2016Dreesetal.Thisisanopen-accessarticledistributedunderthetermsofthe CreativeCommonsAttribution4.0Internationallicense AddresscorrespondencetoJeffreyT.Foster,jeff.foster@unh.edu. S inceemergingin2006,white-nosesyndromehasrapidly spreadacrosseasternNorthAmericafromanindexsitein NewYork,causingthemortalityofmillionsofbatsofnumerous species( 1 ).Thepathogenwasidentiedas Pseudogymnoascusdestructans ( 2 3 ),apsychrophilicfungusthatinfectsbatsduring hibernation,whenbatbodytemperaturesdecreasetotheambient temperatureoftheirhibernacula( 4 ).Thegrowthofthisfunguson batsandsubsequentinvasionofepidermaltissuescancausea cascadeofdeleteriousphysiologicalchangesresultinginhigh mortalityrates( 5 6 ).ThefungusiswidespreadinEurasia( 79 ), andinitialgeneticandexperimentalresultssuggestthatitwas recentlyintroducedtoNorthAmerica( 10 11 ).Criticallymissing, however,aredetailedgenomicanalysesof P.destructans toshed lightontheorigins,evolution,globaldispersal,andpathogenicity ofthisfungus. Initialgenomesequencingeffortsfor P.destructans used454and Illuminaplatforms;however,unresolvedrepeatregionsinthegenomeresultedinfragmenteddraftassemblies( 12 ).Therefore,togenerateahigh-qualitycontiguousassemblyofthe P.destructans genome,weutilizedacombinationofdatafromPacBioSMRTreads, IlluminaMiSeq250-bppaired-endreads(SRR1952982),8-kb454 "jumping"libraries(SRP001346),andendsequencesfromanunbiasedrandomshearbacterialarticialchromosome(BAC)library withanaverageinsertsizeof100kb( 13 ). ThePacBioreadswereassembledwiththePacBioSMRTversion2.2analysispipeline( 14 )andyielded153contigs.UsingIllumina250-bppaired-endMiSeqreadsquality-trimmedtoQ30 withea-utilsversion1.1.2( 15 )anderror-correctedwithHammer ( 16 ),4548-kbjumpingreads,and 100-kbBAClibraryendsequences,thecontigswereextendedandscaffoldedwithSSPACE version3.0( 17 );gapswereremovedwithGapFillerversion1.10 ( 18 ).TwentyiterationsofSSPACE/GapFillerwerefollowedby Pilonversion1.8( 19 )tocorrectlocalmisalignmentsandindels fromtheassembly,resultingin96scaffolds.Aself-queryofthe improvedscaffoldswiththeNUCmerapplicationofMUMmer version3.23( 20 )indicatedthatthesmallerscaffolds( 42,633bp) andafewlarger scaffoldswerecompleteornearlycompleteduplicationsofthesequenceinotherscaffolds.Thirteenscaffolds,which hada 95%identityover95%oftheirlengthwithalargerscaffoldin theassembly,wereremoved.Thenalassemblycontains83scaffolds, is35.818201Mbinsize,hasan N50valueof1.168637Mb,andcontainsonly7,812bpofgaps.Thisisasignicantimprovementcomparedtopreviousdraftassembliesof P.destructans (AEFC01:1,847 scaffolds,30.6849Mb, N50105.158kb,2,328,879bpNs;AYKP01: 5,304scaffolds,30.2827Mb, N5017.914kb,0Ns). Thenew P.destructans genomecontains38.17%repetitivesequenceelements(RepeatModelerversion1.08,RepeatMasker version4.05, http://www.repeatmasker.org ),whichrequireda combinationoflong-readandlarge-insertsequencingtechnologiestoscaffold.Thenearlycompleteassemblypresentedhere shouldserveasavaluableresourcetofacilitatefungaldiseaseresearch. Nucleotidesequenceaccessionnumbers. Thiswhole-genome shotgunprojecthasbeendepositedinDDBJ/ENA/GenBankundertheaccessionnumber LAJJ00000000 .Theversiondescribedin thispaperistherstversion,LAJJ01000000.ACKNOWLEDGMENTSWewouldliketothankKatyPariseforpreparingtheDNAsequencedin thisstudy.Useoftrade,product,orrmnamesisfordescriptivepurposes onlyanddoesnotimplyendorsementbytheU.S.Government. crossmark GenomeAnnouncements May/June2016Volume4Issue3e00445-16 genomea.asm.org 1
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FUNDINGINFORMATIONThiswork,includingtheeffortsofJeffreyT.Foster,wasfundedby NationalScienceFoundation(NSF)(DEB-1336290).Thiswork, includingtheeffortsofJeffreyT.Foster,wasfundedbyDOI|U.S. GeologicalSurvey(USGS).Thiswork,includingtheeffortsofKevin P.Drees,JonathanM.Palmer,DanielLindner,andJeffreyT.Foster, wasfundedbyDOI|U.S.FishandWildlifeService(USFWS).REFERENCES1. TurnerGG,ReederDM,ColemanJH .2011.Ave-yearassessmentof mortalityandgeographicspreadofwhite-nosesyndromeinNorthAmericanbatsandalooktothefuture.BatResNews 52: 1327. 2. LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,HicksAC, BallmannAE,ColemanJTH,RedellDN,ReederDM,BlehertDS .2011. Experimentalinfectionofbatswith Geomycesdestructans causeswhitenosesyndrome.Nature 480: 376378. http://dx.doi.org/10.1038/ nature10590 3. BlehertDS,HicksAC,BehrM,MeteyerCU,Berlowski-ZierBM, BucklesEL,ColemanJTH,DarlingSR,GargasA,NiverR,Okoniewski JC,RuddRJ,StoneWB .2009.Batwhite-nosesyndrome:anemerging fungalpathogen?Science 323: 227. http://dx.doi.org/10.1126/ science.1163874 4. VerantML,BoylesJG,WaldrepW,WibbeltG,BlehertDS .2012. Temperature-dependentgrowthof Geomycesdestructans ,thefungusthat causesbatwhite-nosesyndrome.PLoSOne 7: e46280. http://dx.doi.org/ 10.1371/journal.pone.0046280 5. CryanPM,MeteyerCU,BoylesJG,BlehertDS .2010.Wingpathologyof white-nosesyndromeinbatssuggestslife-threateningdisruptionofphysiology.BMCBiol 8: 135. http://dx.doi.org/10.1186/1741-7007-8-135 6. VerantML,MeteyerCU,SpeakmanJR,CryanPM,LorchJM,Blehert DS .2014.White-nosesyndromeinitiatesacascadeofphysiologicdisturbancesinthehibernatingbathost.BMCPhysiol 14: 10. http://dx.doi.org/ 10.1186/s12899-014-0010-4 7. HoytJR,SunK,PariseKL,LuG,LangwigKE,JiangT,YangS,Frick WF,KilpatrickAM,FosterJT,FengJ .2015.Widespreadbatwhite-nose syndromefungus,northeasternChina.EmergInfectDis 22: 140142. http://dx.doi.org/10.3201/eid2201.151314 8. PuechmailleSJ,FrickWF,KunzTH,RaceyPA,VoigtCC,WibbeltG, TeelingEC .2011.White-nosesyndrome:isthisemergingdiseaseathreat toEuropeanbats?TrendsEcolEvol 26: 570576. http://dx.doi.org/ 10.1016/j.tree.2011.06.013 9. ZukalJ,BandouchovaH,BrichtaJ,CmokovaA,JaronKS,KolarikM, KovacovaV,KubtovA,NovkovA,OrlovO,PikulaJ,PresetnikP, ŠubaJ,ZahradnkovA,Jr,MartnkovN .2016.White-nosesyndrome withoutborders: Pseudogymnoascusdestructans infectiontoleratedinEuropeandpalearcticAsiabutnotinNorthAmerica.SciRep 6: 19829. http:// dx.doi.org/10.1038/srep19829 10. LeopardiS,BlakeD,PuechmailleSJ .2015.White-nosesyndromefungus introducedfromEuropetoNorthAmerica.CurrBiol 25: R217R219. http://dx.doi.org/10.1016/j.cub.2015.01.047 11. WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,CryanPM, WibbeltG,BlehertDS,WillisCKR .2012.Inoculationofbatswith European Geomycesdestructans supportsthenovelpathogenhypothesis fortheoriginofwhite-nosesyndrome.ProcNatlAcadSciUSA 109: 69997003. http://dx.doi.org/10.1073/pnas.1200374109 12. ChibucosMC,CrabtreeJ,NagarajS,ChaturvediS,ChaturvediV .2013. Draftgenomesequencesofhumanpathogenicfungus Geomycespannorumsensulato andbatwhitenosesyndromepathogen Geomyces ( Pseudogymnoascus ) destructans .GenomeAnnounc 1 (6):e01045-13. http:// dx.doi.org/10.1128/genomeA.01045-13 13. BokJW,YeR,ClevengerKD,MeadD,WagnerM,KrerowiczA, AlbrightJC,GoeringAW,ThomasPM,KelleherNL,KellerNP,Wu CC .2015.Fungalarticialchromosomesforminingofthefungalsecondarymetabolome.BMCGenomics 16: 343. http://dx.doi.org/10.1186/ s12864-015-1561-x 14. ChinCS,AlexanderDH,MarksP,KlammerAA,DrakeJ,HeinerC, ClumA,CopelandA,HuddlestonJ,EichlerEE,TurnerSW,KorlachJ 2013.Nonhybrid,nishedmicrobialgenomeassembliesfromlong-read SMRTsequencingdata.NatMethods 10: 563569. http://dx.doi.org/ 10.1038/nmeth.2474 15. AronestyE .2013.Comparisonofsequencingutilityprograms.Open BioinformaJ 7: 18. http://dx.doi.org/10.2174/1875036201307010001 16. MedvedevP,ScottE,KakaradovB,PevznerP .2011.Errorcorrectionof high-throughputsequencingdatasetswithnon-uniformcoverage.Bioinformatics 27: i137i141. http://dx.doi.org/10.1093/bioinformatics/btr208 17. BoetzerM,HenkelCV,JansenHJ,ButlerD,PirovanoW .2011.Scaffoldingpre-assembledcontigsusingSSPACE.Bioinformatics 27: 578579. http://dx.doi.org/10.1093/bioinformatics/btq683 18. NadalinF,VezziF,PolicritiA .2012.GapFiller:adenovoassembly approachtollthegapwithinpairedreads.BMCBioinformatics 13 (suppl 1):S8. http://dx.doi.org/10.1186/1471-2105-13-S14-S8 19. WalkerBJ,AbeelT,SheaT,PriestM,AbouellielA,SakthikumarS, CuomoCA,ZengQ,WortmanJ,YoungSK,EarlAM .2014.Pilon:an integratedtoolforcomprehensivemicrobialvariantdetectionandgenomeassemblyimprovement.PLoSOne 9: e112963. http://dx.doi.org/ 10.1371/journal.pone.0112963 20. KurtzS,PhillippyA,DelcherAL,SmootM,ShumwayM,AntonescuC, SalzbergSL .2004.Versatileandopensoftwareforcomparinglargegenomes.GenomeBiol 5: R12. http://dx.doi.org/10.1186/gb-2004-5-2-r12 .Dreesetal. GenomeAnnouncements 2 genomea.asm.org May/June2016Volume4Issue3e00445-16
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