APlantBiosecurityVirtualLaboratory
KathrynR.Napier
RobertoA.Barrero1,KathrynR.Napier1,JamesCunnington2,LiaLiefting3,
SandiKeenan4,RebekahFrampton4,TamasSzabo1,SimonBulman4,
AdamHunter1,LisaWard3,MarkWhattam2,MatthewI.Bellgard1,5
1CentreforComparativeGenomicsMurdochUniversity,Murdoch,Australia,[email protected]
2DepartmentofAgricultureandWaterResources,Knoxfield,Australia,[email protected]
3MinistryforPrimaryIndustries,Auckland,NewZealand,[email protected]
4PlantFoodResearch,Lincoln,NewZealand,[email protected]
5AustralianBioinformaticsFacility,BioplatformsAustralia,[email protected]
INTRODUCTION
Historically, the geographical isolation of Australia and New Zealand, coupled with stringent quarantine screening
measures,hasprovidedprotectionfromtheintroductionofexoticpestsandpathogensthathavethepotentialtoharm
humanhealth,agriculture,theenvironmentandtheeconomy.However,increasesinglobaltradeandmovementare
placingsignificantpressureonthesequarantinesystems,withanincreaseinthefrequencyofincursionsofpathogens
causing the emergence of diseases and pests that are both difficult and costly to eradicate and control [1]. The
challengeofmaximisingthebenefitsofglobaltradewhilstminimisingthenegativeimpactsofbiosecuritythreatsisone
facedbymostnations[2].
THEPROBLEM
The diagnosis of viral pathogens is a crucial component of plant biosecurity surveillance, required to prevent the
potentialintroductionofexoticplantvirusesandviroids.Classicalpostentryquarantine(PEQ)detectionanddiagnostic
protocolscanbeexpensiveandtimeandresourceconsuming,andcanonlyscreenagainstselectedknownviruses.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’wastodevelopareliable,accurate,sensitive,costeffectiveandeasytousediagnosticplatformtodetectall
virusesinasingletest.ThisledtoacollaborativeenduserdrivenprojectfundedbythePlantBiosecurityCooperative
ResearchCentreinvolvingtheDepartmentofAgricultureandWaterResources,theMinistryforPrimaryIndustriesNew
Zealand, Plant and Food Research New Zealand, and the Centre for Comparative Genomics at Murdoch University,
commencingin2013.
Recentstudieshavedemonstratedboththedetectionofviralpathogensandtheidentificationofnovelvirusesbythe
deep sequencing of small RNAs (small RNA-Seq) of plant species [3, 4]. RNA silencing is a natural anti-viral defence
systempresentinplants,insectsandinvertebratesthatrecognisesdoublestrandedRNA(dsRNA)viralgenomesand/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
hostthroughnextgenerationsequencing(NGS)[6].Thisoffersanunprecedentedparadigmshifttodetectallknown
andnovelplantviruses/viroidsinasinglesmallRNAnextgenerationsequencingexperimentthatisalsomorelowcost
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
sequencingdata.
THEAIM
To develop a virtual laboratory for plant biosecurity for the detection of viruses and viroids, to be adopted by
quarantineagencieswithoutestablishedbioinformaticsexpertise.
eResearchAustralasiaConference|Melbourne–Australia|10-14October-2016
METHODS
SampleCollection
Imported plants were grown within quarantine glasshouse facilities in Australia and New Zealand until sample
o
collection.TotalRNAand/orsmallRNAenrichedfractionwereextractedfromtissue,andstoredat-80 CwithinPEQ
facilities until shipped on dry ice to a NGS service provider. Seven known positive control samples were initially
sequenced,followedbyanadditional35samples. Developmentofthevirtuallaboratory
The 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 the
customisationoftoolsandscriptsthatruninacommandlineenvironmentinto‘draganddrop’toolsthatcanbeeasily
incorporated into analysis workflows. Yabi provides end users with the ability to run powerful, high performance
computinganalysisworkflows,withouttheneedforextensivebioinformaticsexpertise.Yabialsoprovidesamechanism
to manage large amounts of raw and processed data in a secure and flexible environment, and files can be easily
managed across different high performance computing or cloud storage infrastructures [7]. Workflows can be saved,
re-used and shared amongst users, and importantly, comprehensive provenance for each workflow is kept including
inputfiles,theparametersusedforeachtool,andresultfiles.Thisenablesresearcherstoeasilytrackpreviousanalyses
performed.Yabicanbedeployedinexistinghighperformancecomputingcentresand/orasacloudinstance.
RESULTS
Wedevelopedanddesignedavirtuallaboratoryforthedetectionofvirusesandviroidsinplantquarantinesamplesfor
usebyquarantineagencies.Thiscloudbasedanalyticsenvironmentsimplifiescomplexbioinformaticsanalysisanddata
processingworkflows(consistingofupto16differentoptimisedtools)forNGSdataintofoursimplesteps:i)upload
your NGS data; ii) select desired analysis workflow; iii) press ‘run’, and iv) download your results. This environment
allowsenduserstosecurelyreuseandshareworkflows,customisetoolparameters,viewresults,anddownloadresult
filesforfurtheranalysis.ThevirtuallaboratoryisabletoreliablydetectssRNA(+),dsDNAandssDNAvirusesandviroids
inplantquarantinesamples.Inaddition,thevirtuallaboratorywasalsoabletoidentifythecompletegenomesequence
ofapossiblenovelpotyvirusinaquarantinedornamentalgrass.
CONCLUSIONS
ThedevelopmentofthevirtuallaboratorywasachievedthroughcloseenduserengagementwiththeDepartmentof
AgricultureandWaterResources,PlantandFoodResearchNewZealand,andtheMinistryforPrimaryIndustriesNew
Zealand.Over80enduserssuchaspolicyregulators,labmanagers,diagnosticians,researchersandgraduatestudents
inAustraliaandNewZealandhavetestedthisPlantBiosecurityCooperativeResearchCentrefundedvirtuallaboratory
through several training workshops held in 2015 and 2016. The re-usable analysis and data processing workflows
minimises the hands-on requirements of end users, enabling them to rapidly process a large number of plant
quarantine samples. The virtual laboratory also improves screening efficiency and diagnostic accuracy. We envisage
that this virtual laboratory will dramatically reduce the time of imported plants in PEQ facilities, and make import of
newgeneticmaterialmorecosteffectiveforimporters,comparedtocurrentdiagnosticprotocols.
REFERENCES
1. Rodoni,B.,Theroleofplantbiosecurityinpreventingandcontrollingemergingplantvirusdiseaseepidemics.Virus
Res,2009.141(2):p.150-157.
2. Sharma,S.,etal.,TheBiosecurityContinuumandTrade:ToolsforPost-borderBiosecurity,inTheHandbookofPlant
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
RNAs:agenericmethodfordiagnosis,discoveryandsequencingofviruses.Virology,2009.388(1):p.1-7.
4. Candresse,T.,etal.,AppearancesCanBeDeceptive:RevealingaHiddenViralInfectionwithDeepSequencingina
PlantQuarantineContext.PLoSONE,2014.9(7):p.e102945.
5. Mlotshwa,S.,etal.,SmallRNAsinviralinfectionandhostdefense.TrendsPlantSci,2008.13(7):p.375-382.
6. Kreuze,J.,siRNADeepSequencingandAssembly:PiecingTogetherViralInfections,inDetectionandDiagnosticsof
PlantPathogens.2014,Springer.p.21-38.
7. Hunter,A.A.,etal.,Yabi:Anonlineresearchenvironmentforgrid,highperformanceandcloudcomputing.Source
CodeBiolMed,2012.7(1):p.1.
eResearchAustralasiaConference|Melbourne–Australia|10-14October-2016
THECENTREFORCOMPARATIVEGENOMICS
KATHRYNRNAPIER
Dr Kathryn Napier is a Research Associate at the Centre for Comparative Genomics (CCG) at Murdoch University. Dr
NapiergraduatedfromMurdochUniversitywithaPhDfocusingonEcologicalPhysiologyin2014,andalsohasaBSc.
(hons)inBiomedicalScienceandaBSc.inMathematicsandStatistics.DrNapierhasexpertiseinbioinformaticsandthe
deploymentofweb-basedeResearchsolutions.SincejoiningtheCCGin2013,shehasbeeninvolvedseveralprojects
includingthePlantBiosecurityCooperativeResearchCentre(PBCRC)fundedprojectdetailedinthispresentation.
ROBERTOBARRERO
DrRobertoBarreroisaSeniorResearchFellowattheCCGatMurdochUniversitywithexpertiseinbioinformatics,Next
GenerationSequencing(NGS)andsmallRNA’s.DrBarreroistheprojectleaderforthisPBCRCfundedproject.
MATTHEWIBELLGARD
ProfessorMatthewBellgardistheDirectorattheCCGatMurdochUniversity,theWestAustralianStateGovernment
CentreofExcellence.TheCCGundertakesuniquebiomedicalandagriculturalresearchanddevelopmentbypromoting
a collaborative understanding within and across fields of study. He is also Convenor of the Australian Bioinformatics
Facility.
ADAMAHUNTER
AdamHunteristheAssociateDirectorattheCCGatMurdochUniversity.HeleadstheCCGsoftwaredevelopmentand
infrastructureteam,andhasextensiveexperienceininformationandcommunicationstechnology.Hiscurrentareasof
focusincludehighperformanceandcloudcomputing,continuousintegrationandagileprogramming.
TAMASSZABO
TamasSzaboisaSeniorSoftwareDeveloperattheCCGatMurdochUniversity.Hehasexperienceinmanysectors,with
expertiseinprogramminglanguages,developmentmethodologies,opensourcedevelopment,operatingsystemsand
networks.
PLANTANDFOODRESEARCHNEWZEALAND
DRSIMONBULMAN,SANDIKEENAN,ANDREBEKAHFRAMPTON
TheresearchteamcontributingtothisPBCRCfundedprojectatPlantandFoodResearchisledbyDrSimonBulman.
PlantandFoodResearchprovidesresearchanddevelopmentthataddsvaluetofruit,vegetable,cropandmarine-based
foodproducts.
THEDEPARTMENTOFAGRICULTUREANDWATERRESOURCES
DRMARKWHATTAMANDDRJAMESCUNNINGTON
Dr Mark Whattam leads the team contributing to this PBCRC funded project at the Department of Agriculture and
Water Resources. Dr Whattam is the Director of the Operational Science Program, with expertise in plant pest and
diseasediagnostics,includingtestingofhigh-risknurserystockplantsinpostentryquarantine,andoperationaladvice.
MINISTRYFORPRIMARYINDUSTRIESNEWZEALAND
DRLISAWARDANDDRLIALIEFTING
Dr Lisa Ward and Dr Lia Liefting comprise the team contributing to this PBCRC funded project from the Ministry of
PrimaryIndustries.TheVirologyandPostEntryQuarantineteamismanagedbyDrLisaWard,whohasexpertiseinthe
identificationofsuspectedexoticviral,viroidandphytoplasma-likediseasesinplantsamplescollectedduringtargeted
surveillance,passivesurveillanceandincursionresponse.
eResearchAustralasiaConference|Melbourne–Australia|10-14October-2016