Use of multiple sequencing technologies to produce a high-quality genome of the fungus pseudogymnoascus destructans, the causative agent of bat white-nose syndrome

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Use of multiple sequencing technologies to produce a high-quality genome of the fungus pseudogymnoascus destructans, the causative agent of bat white-nose syndrome

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Title:
Use of multiple sequencing technologies to produce a high-quality genome of the fungus pseudogymnoascus destructans, the causative agent of bat white-nose syndrome
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Genome Announcements
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Drees, Kevin P.
Palmer, Jonathan, M.
Sebra, Robert
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White-nose syndrome ( lcsh )
Genomics ( lcsh )
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serial ( sobekcm )

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Abstract:
White-nose syndrome has recently emerged as one of the most devastating wildlife diseases recorded, causing widespread mortality in numerous bat species throughout eastern North America. Here, we present an improved reference genome of the fungal pathogen Pseudogymnoascus destructans for use in comparative genomic studies.

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K26-05266 ( USFLDC DOI )
k26.5266 ( USFLDC handle )

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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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