eResearch Australasia Conference | Melbourne – Australia | 10 - 14 October - 2016 A Plant Biosecurity Virtual Laboratory Kathryn R. Napier Roberto A. Barrero1, Kathryn R. Napier1, James Cunnington2, Lia Liefting3, Sandi Keenan4, Rebekah Frampton4, Tamas Szabo1, Simon Bulman4, Adam Hunter1, Lisa Ward3, Mark Whattam2, Matthew I. Bellgard1,5 1 Centre for Comparative Genomics Murdoch University, Murdoch, Australia, [email protected]2Department of Agriculture and Water Resources, Knoxfield, Australia, [email protected]3Ministry for Primary Industries, Auckland, New Zealand, [email protected]4Plant Food Research, Lincoln, New Zealand, [email protected]5Australian Bioinformatics Facility, Bioplatforms Australia, [email protected]INTRODUCTION Historically, the geographical isolation of Australia and New Zealand, coupled with stringent quarantine screening measures, has provided protection from the introduction of exotic pests and pathogens that have the potential to harm human health, agriculture, the environment and the economy. However, increases in global trade and movement are placing significant pressure on these quarantine systems, with an increase in the frequency of incursions of pathogens causing the emergence of diseases and pests that are both difficult and costly to eradicate and control [1]. The challenge of maximising the benefits of global trade whilst minimising the negative impacts of biosecurity threats is one faced by most nations [2]. THE PROBLEM The diagnosis of viral pathogens is a crucial component of plant biosecurity surveillance, required to prevent the potential introduction of exotic plant viruses and viroids. Classical post entry quarantine (PEQ) detection and diagnostic protocols can be expensive and time and resource consuming, and can only screen against selected known viruses. The screening of imported plants into Australia using existing methods may result in plants spending up to two years in quarantine. This leads to significant losses in terms of time to access markets and money in terms of international competiveness. In 2011, the Department of Agriculture and Water Resources were invited to the Plant Biosecurity Cooperative Research Centre Science Exchange to present an ‘End user R&D needs perspective’. The vision of this ‘blue sky dreaming’ was to develop a reliable, accurate, sensitive, cost effective and easy to use diagnostic platform to detect all viruses in a single test. This led to a collaborative end user driven project funded by the Plant Biosecurity Cooperative Research Centre involving the Department of Agriculture and Water Resources, the Ministry for Primary Industries New Zealand, Plant and Food Research New Zealand, and the Centre for Comparative Genomics at Murdoch University, commencing in 2013. Recent studies have demonstrated both the detection of viral pathogens and the identification of novel viruses by the deep sequencing of small RNAs (small RNA-Seq) of plant species [3, 4]. RNA silencing is a natural anti-viral defence system present in plants, insects and invertebrates that recognises double stranded RNA (dsRNA) viral genomes and/or viral intermediate dsRNA sequences, and cleaves them into small interfering RNAs (siRNA) of 21-24nt in length [5]. These virus-derived siRNAs (viRNAs) can be abundant in plants, which allows the identification of viruses infecting a host through next generation sequencing (NGS) [6]. This offers an unprecedented paradigm shift to detect all known and novel plant viruses/viroids in a single small RNA next generation sequencing experiment that is also more low cost and time-effective than current PEQ detection methods. However, an effective eResearch solution is required to engage closely with end-users and key government stakeholders to analyse, manage, and store large amounts of sequencing data. THE AIM To develop a virtual laboratory for plant biosecurity for the detection of viruses and viroids, to be adopted by quarantine agencies without established bioinformatics expertise.
INTRODUCTIONHistorically, the geographical isolation of Australia and New Zealand, coupled with stringent quarantine screeningmeasures,hasprovidedprotectionfromtheintroductionofexoticpestsandpathogensthathavethepotentialtoharmhumanhealth,agriculture,theenvironmentandtheeconomy.However, increasesinglobaltradeandmovementareplacingsignificantpressureonthesequarantinesystems,withanincreaseinthefrequencyofincursionsofpathogenscausing the emergence of diseases and pests that are both difficult and costly to eradicate and control [1]. Thechallengeofmaximisingthebenefitsofglobaltradewhilstminimisingthenegativeimpactsofbiosecuritythreatsisonefacedbymostnations[2].
THEPROBLEMThe diagnosis of viral pathogens is a crucial component of plant biosecurity surveillance, required to prevent thepotentialintroductionofexoticplantvirusesandviroids.Classicalpostentryquarantine(PEQ)detectionanddiagnosticprotocolscanbeexpensiveandtimeandresourceconsuming,andcanonlyscreenagainstselectedknownviruses.Thescreeningof importedplants intoAustralia using existingmethodsmay result in plants spendingup to two years inquarantine. This leads to significant losses in terms of time to accessmarkets andmoney in terms of internationalcompetiveness.In 2011, the Department of Agriculture and Water Resources were invited to the Plant Biosecurity CooperativeResearch Centre Science Exchange to present an ‘End user R&D needs perspective’. The vision of this ‘blue skydreaming’wastodevelopareliable,accurate,sensitive,costeffectiveandeasytousediagnosticplatformtodetectallvirusesinasingletest.ThisledtoacollaborativeenduserdrivenprojectfundedbythePlantBiosecurityCooperativeResearchCentreinvolvingtheDepartmentofAgricultureandWaterResources,theMinistryforPrimaryIndustriesNewZealand, Plant and Food Research New Zealand, and the Centre for Comparative Genomics atMurdoch University,commencingin2013.Recentstudieshavedemonstratedboththedetectionofviralpathogensandtheidentificationofnovelvirusesbythedeep sequencing of small RNAs (small RNA-Seq) of plant species [3, 4]. RNA silencing is a natural anti-viral defencesystempresentinplants,insectsandinvertebratesthatrecognisesdoublestrandedRNA(dsRNA)viralgenomesand/orviral intermediate dsRNA sequences, and cleaves them into small interfering RNAs (siRNA) of 21-24nt in length [5].These virus-derived siRNAs (viRNAs) canbe abundant inplants,which allows the identificationof viruses infecting ahost throughnextgenerationsequencing (NGS) [6].Thisoffersanunprecedentedparadigmshift todetectallknownandnovelplantviruses/viroidsinasinglesmallRNAnextgenerationsequencingexperimentthatisalsomorelowcostand time-effective than current PEQ detection methods. However, an effective eResearch solution is required toengage closely with end-users and key government stakeholders to analyse, manage, and store large amounts ofsequencingdata.
THEAIMTo develop a virtual laboratory for plant biosecurity for the detection of viruses and viroids, to be adopted byquarantineagencieswithoutestablishedbioinformaticsexpertise.
METHODSSampleCollectionImported plants were grown within quarantine glasshouse facilities in Australia and New Zealand until samplecollection.TotalRNAand/orsmallRNAenrichedfractionwereextractedfromtissue,andstoredat -80oCwithinPEQfacilities until shipped on dry ice to a NGS service provider. Seven known positive control samples were initiallysequenced,followedbyanadditional35samples. DevelopmentofthevirtuallaboratoryThe plant biosecurity virtual laboratory for the detection of viruses and viroids was developed utilising Yabi(https://github.com/muccg/yabi) [7], an open source online intuitive analytical environment, that allows for thecustomisationoftoolsandscriptsthatruninacommandlineenvironmentinto‘draganddrop’toolsthatcanbeeasilyincorporated into analysis workflows. Yabi provides end users with the ability to run powerful, high performancecomputinganalysisworkflows,withouttheneedforextensivebioinformaticsexpertise.Yabialsoprovidesamechanismtomanage large amounts of raw and processed data in a secure and flexible environment, and files can be easilymanagedacrossdifferenthighperformancecomputingorcloudstorage infrastructures [7].Workflowscanbesaved,re-usedand sharedamongstusers, and importantly, comprehensiveprovenance for eachworkflow is kept includinginputfiles,theparametersusedforeachtool,andresultfiles.Thisenablesresearcherstoeasilytrackpreviousanalysesperformed.Yabicanbedeployedinexistinghighperformancecomputingcentresand/orasacloudinstance.
RESULTSWedevelopedanddesignedavirtuallaboratoryforthedetectionofvirusesandviroidsinplantquarantinesamplesforusebyquarantineagencies.Thiscloudbasedanalyticsenvironmentsimplifiescomplexbioinformaticsanalysisanddataprocessingworkflows(consistingofupto16differentoptimisedtools) forNGSdata intofoursimplesteps: i)uploadyourNGS data; ii) select desired analysisworkflow; iii) press ‘run’, and iv) download your results. This environmentallowsenduserstosecurelyreuseandshareworkflows,customisetoolparameters,viewresults,anddownloadresultfilesforfurtheranalysis.ThevirtuallaboratoryisabletoreliablydetectssRNA(+),dsDNAandssDNAvirusesandviroidsinplantquarantinesamples.Inaddition,thevirtuallaboratorywasalsoabletoidentifythecompletegenomesequenceofapossiblenovelpotyvirusinaquarantinedornamentalgrass.
CONCLUSIONSThedevelopmentofthevirtual laboratorywasachievedthroughcloseenduserengagementwiththeDepartmentofAgricultureandWaterResources,PlantandFoodResearchNewZealand,andtheMinistryforPrimaryIndustriesNewZealand.Over80enduserssuchaspolicyregulators,labmanagers,diagnosticians,researchersandgraduatestudentsinAustraliaandNewZealandhavetestedthisPlantBiosecurityCooperativeResearchCentrefundedvirtuallaboratorythrough several training workshops held in 2015 and 2016. The re-usable analysis and data processing workflowsminimises the hands-on requirements of end users, enabling them to rapidly process a large number of plantquarantine samples. The virtual laboratory also improves screening efficiency and diagnostic accuracy.We envisagethat thisvirtual laboratorywilldramatically reducethe timeof importedplants inPEQfacilities,andmake importofnewgeneticmaterialmorecosteffectiveforimporters,comparedtocurrentdiagnosticprotocols.
Biosecurity,G.GordhandS.McKirdy,Editors.2014,SpringerNetherlands.p.189-206.3. Kreuze, J.F., et al.,Complete viral genome sequence and discovery of novel viruses by deep sequencing of small
DRSIMONBULMAN,SANDIKEENAN,ANDREBEKAHFRAMPTONTheresearchteamcontributingtothisPBCRCfundedprojectatPlantandFoodResearch is ledbyDrSimonBulman.PlantandFoodResearchprovidesresearchanddevelopmentthataddsvaluetofruit,vegetable,cropandmarine-basedfoodproducts.
THEDEPARTMENTOFAGRICULTUREANDWATERRESOURCES
DRMARKWHATTAMANDDRJAMESCUNNINGTONDrMarkWhattam leads the team contributing to this PBCRC funded project at the Department of Agriculture andWater Resources. DrWhattam is the Director of theOperational Science Program,with expertise in plant pest anddiseasediagnostics,includingtestingofhigh-risknurserystockplantsinpostentryquarantine,andoperationaladvice.
MINISTRYFORPRIMARYINDUSTRIESNEWZEALAND
DRLISAWARDANDDRLIALIEFTINGDr LisaWard andDr Lia Liefting comprise the team contributing to this PBCRC funded project from theMinistry ofPrimaryIndustries.TheVirologyandPostEntryQuarantineteamismanagedbyDrLisaWard,whohasexpertiseintheidentificationofsuspectedexoticviral,viroidandphytoplasma-likediseasesinplantsamplescollectedduringtargetedsurveillance,passivesurveillanceandincursionresponse.
