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SOFTWAREOpenAccessNanoDJ:aDockerizedJupyternotebookforinteractiveOxfordNanoporeMinIONsequencemanipulationandgenomeassemblyHéctorRodríguez-Pérez1,TamaraHernández-Beeftink1,JoséM.
Lorenzo-Salazar2,JoséL.
Roda-García3,CarlosJ.
Pérez-González4,MarcosColebrook3*andCarlosFlores1,2,5*AbstractBackground:TheOxfordNanoporeTechnologies(ONT)MinIONportablesequencermakesitpossibletousecutting-edgegenomictechnologiesinthefieldandtheacademicclassroom.
Results:WepresentNanoDJ,aJupyternotebookintegrationoftoolsforsimplifiedmanipulationandassemblyofDNAsequencesproducedbyONTdevices.
Itintegratesbasecalling,readtrimmingandqualitycontrol,simulationandplottingroutineswithavarietyofwidelyusedalignersandassemblers,includingproceduresforhybridassembly.
Conclusions:WiththeuseofJupyter-facilitatedaccesstoself-explanatorycontentsofapplicationsandtheinteractivevisualizationofresults,aswellasbyitsdistributionintoaDockersoftwarecontainer,NanoDJisaimedtosimplifyandmakemorereproducibleONTDNAsequenceanalysis.
TheNanoDJpackagecode,documentationandinstallationinstructionsarefreelyavailableathttps://github.
com/genomicsITER/NanoDJ.
Keywords:Genomeanalysis,Nanoporesequencing,Jupyter,DockerBackgroundIthasneverbeenbeforesoeasyandaffordabletoaccessandutilizegeneticvariationofanyorganismandpurpose.
Thishasbeenmotivatedbythecontinuousdevelopmentofhigh-throughputDNAsequencingtechnologies,mostcommonlyknownasNextGenerationSequencing(NGS).
Akeyimprovementisthepossibilityofobtaininglongsin-glemoleculesequenceswiththefastandcost-efficiencytechnologyreleasedbyOxfordNanoporeTechnologies(ONT)andthemarketingin2014oftheMinION,aport-able,pocket-size,nanopore-basedNGSplatform[1].
Sincethen,severalalgorithmsandsoftwaretoolshaveflourishedspecificallyforONTsequencedata.
Despiteitssize,itpro-videsmulti-kilobasereadswithathroughputcomparabletootherbenchtopsequencersinthemarket(1–10Gbasesby2017),thereforestillnecessitatingofefficientandinte-gratedbioinformaticstoolstofacilitatethewidespreaduseofthetechnology.
WhileMinIONhasshownpromiseindistinctapplica-tions[2],becauseofthelowcost,laptopoperability,andtheUSB-poweredcompactdesignofMinION,cutting-edgeNGStechnologyisnotanymorenecessarilylinkedtotheestablishedideaofalargemachinewithhighcostthatmustbelocatedincentralizedsequencingcentersorinalabora-torybench.
Asaconsequence,theutilityofMinIONinfieldexperimentstomovefromsample-to-answersonsitehavebeendemonstratedwithinfectiousdiseasestudies[3,4],off-Earthgenomesequencing[5],andspeciesidentifica-tioninextremeenvironments[6–8],amongothers.
Lever-agingofMinIONcapabilitiesintheacademicclassroomisanaturalextensionofthesefieldstudiestofacilitateTheAuthor(s).
2019OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
*Correspondence:mcolesan@ull.
edu.
es;cflores@ull.
edu.
esHéctorRodríguez-PérezandTamaraHernández-Beeftinkcontributedequallytothiswork.
3DepartamentodeIngenieríaInformáticaydeSistemas,UniversidaddeLaLaguna,SantaCruzdeTenerife,Spain1ResearchUnit,HospitalUniversitarioNuestraSeoradeCandelaria,UniversidaddeLaLaguna,SantaCruzdeTenerife,SpainFulllistofauthorinformationisavailableattheendofthearticleRodríguez-Pérezetal.
BMCBioinformatics(2019)20:234https://doi.
org/10.
1186/s12859-019-2860-zeducationofgenomicsinundergraduateandgraduatestu-dents[9].
Todate,thereisnospecificsoftwaresolutionaimedtofacilitateONTsequenceanalysesbyintegratingcapabilitiesfordatamanipulation,sequencecomparisonandassemblyinfieldexperimentsorforeducationalpurposestohelpfacilitatelearningofgenomics[9].
WehavedevelopedNanoDJ,aninteractivecollectionofJupyternotebookstointegrateavarietyofsoftware,advancedcomputercode,andplaincontextualexplanations.
Inaddition,NanoDJisdistributedasaDockersoftwarecontainertosimplifyin-stallationofdependenciesandimprovethereproducibilityofresults.
ImplementationNanoDJisdistributedasaDockercontainerbuiltunder-neathJupyternotebooks,whichisincreasinglypopularinlifesciencestosignificantlyfacilitatetheinteractiveex-plorationofdata[10],andhasbeenrecentlyintegratedinthewidelyusedGalaxyportal[11].
TheDockercontainerallowsNanoDJtoruninanisolated,self-containedpack-age,thatcanbeexecutedseamlesslyacrossawiderangeofcomputingplatforms[12],havinganegligibleimpactontheexecutionperformance[13].
NanoDJintegratesdi-verseapplications(Additionalfile1:TableS1)organizedinto12notebooksgroupedonthreesections(Fig.
1;Table1).
Mainresultsarepresentedasembeddedobjects.
Inaddition,oneofthenotebookswasconceivedforedu-cationalpurposesbysettingaparticularlysimpleproblemandtheinclusionoflow-levelexplanations.
Tofacilitatetheuseoftheeducationalnotebookandbypassingthein-stallationofDockerandNanoDJ,alightweightversionofthisnotebookandsmallsetsofONTreadscanbeutilizedfromaweb-browserusingBinder(https://mybinder.
org)intheNanoDJGitHubrepository.
Inaddition,aspartoftheCyVerseproject(https://www.
cyverse.
org/),NanoDJhasbeenincorporatedintoVICE,avisualandinteractivecomputingenvironmentthatfacilitatestrainingofONTdataanalysis.
WeillustratetheversatilityofNanoDJindistinctscenariosbyprovidingresultsfromfourcasestud-ies(Additionalfile1:TextS1).
Input,basecalling,andsimulationsInputdatacanbealistofFAST5filesfrompreviousbase-calledruns(e.
g.
aMetrichoroutput)orevent-levelsignaldatatobebasecalledusingthelatestONTcaller.
TheusercanalsosimulatereadswithNanoSimandpre-computedmodelparameters.
Thispossibilityisimportantindifferentscenariosastohelpdesigninganexperiment,ortobypasstechnicaldifficultiesinacademicsetups[9].
Summary,qualitycontrolandfilteringEitherforasimulatedoranempiricalrun,theuserwillobtainsummarydataandplotsinformingofreadlengthdistribution,GCcontentvs.
length,andreadlengthvs.
qualityscore(whenavailable).
Ifbarcodeswereusedintheexperiment,Porechopcanbeusedfordemultiplexing,barcodetrimmingandtofilteroutreads.
GenomeassemblyandcomparisonDependingontheapplication,sequencedatacanbealignedagainstreferencesequencesorusedforgenomeas-semblyusingdiversemethods.
Alignmentisperformedei-theragainstone(BWAandRebaler)ormultiple(BLAST)referencesequences,providingthegenerationofBAMfilesfordownstreamapplications(e.
g.
,variantidentification)orinformationofspeciescomposition.
Alternatively,theusermayoptforadenovoassembly.
NanoDJallowstheuseofsomeofthebest-performingalgorithms(Canu,Flye,andMiniasm),ortocombineONTreadswithothersobtainedwithsecond-generationNGSplatformsforahybridassem-bly(UnicyclerandMaSuRCA).
ThelatterprovidesmoreFig.
1SimplifiedschemeofallNanoDJfunctionalitiesRodríguez-Pérezetal.
BMCBioinformatics(2019)20:234Page2of4effectiveassembliesandreducederrorratecomparedtoas-sembliesbasedonlyonONTreads[14].
NanoDJincludesthepossibilityofcontigcorrection(Racon,Nanopolish,andPilon).
Assembliescanbeevaluatedwiththeembed-dedversionofQUAST,andrepresentedwithBandage.
LimitationsandfuturedirectionsFornon-expertusers,itwouldhavebeenbetterifNanoDJwasenvisagedasanon-lineapplicationtofacilitateitsuse.
However,ourmainobjectivewastointegratemajortoolsfortheanalysisofONTsequencesinaninteractivesoft-wareenvironmenttofacilitatelearningthebasicsbehindONTsequenceanalysiswhileprovidingausefultoolforprofessionals.
ProvidingitasaDockerizedsolutionsimplybolstersthefocusontheuseofthetool,reducingthebur-denofinstallingalldependenciesbytheuser.
Atthemo-ment,NanoDJissetfortheanalysisofsmallgenomesandtargetedNGSstudies,althoughfocusingonprimaryandsecondaryanalysisofDNAsequences.
Theintegrationoftoolsforvariantidentificationandtertiaryanalysis(anno-tationofvariantsorsequenceelements,interpretation,etc.
)[15,16],aswellasforepigenetics[17]anddirectRNAsequencing[18]willbethefocusoffurtherdevelop-mentsofNanoDJ.
ConclusionsWepresentNanoDJasanintegratedJupyter-basedtool-boxdistributedasaDockersoftwarecontainertofacili-tateONTsequenceanalysis.
NanoDJisbestsuitedfortheanalysesofsmallgenomesandtargetedNGSstudies.
WeanticipatethattheJupyternotebook-basedstructurewillsimplifyfurtherdevelopmentsinotherapplications.
AvailabilityandrequirementsProjectname:NanoDJProjecthomepage:https://github.
com/genomicsITER/NanoDJOperatingsystem(s):Windows,Linux,MacOSProgramminglanguage:Bash/PythonOtherrequirements:DockerinstallationLicense:GPLAnyrestrictionstousebynon-academics:NoneAdditionalfileAdditionalfile1:TableS1.
ApplicationsintegratedinNanoDJ.
TextS1.
Testingoncasestudydatasets.
TableS2.
DatasetsforillustrativeusesofNanoDJ.
TableS3.
Comparisonofdenovoassembliesusingdifferentinputsorwithanassemblycorrector.
TableS4.
Comparisonofthreedenovoassemblersinahigh-coverageONTdataset.
TableS5.
Comparisonofresultsfromtwohybriddenovoassemblers.
FigureS1.
Humanmito-chondrialDNAvariantrepresentationagainstthereferencesequence.
TableS6.
SourceofmitochondrialDNAgenomes,simulationsandclassi-ficationresults.
(DOCX1544kb)AcknowledgementsNotapplicableFundingThisresearchwasfundedbytheInstitutodeSaludCarlosIII(grantsPI14/00844andPI17/00610),theSpanishMinistryofScience,InnovationandUniversities(grantRTC-2017-6471-1;MINECO/AEI/FEDER,UE),theSpanishMinistryofEconomyandCompetitiveness(grantMTM2016–74877-P),whichwereco-financedbytheEuropeanRegionalDevelopmentFunds'AwayofmakingEurope'fromtheEuropeanUnion,AreaTenerife2030fromCabildodeTenerife(CGIEU0000219140),andbytheagreementOA17/008withInsti-tutoTecnológicoydeEnergíasRenovables(ITER)tostrengthenscientificandtechnologicaleducation,training,research,developmentandinnovationinGenomics,PersonalizedMedicineandBiotechnology.
Thefoundingen-titieshadnoroleinthedesignofthestudy,analysis,interpretationofdataorinmanuscriptwriting.
AvailabilityofdataandmaterialsAlldatageneratedoranalysedduringthisstudyareincludedinthispublishedarticleanditssupplementaryinformationfiles.
RawreadsfromMinIONandIlluminaareavailablefromtheSRAdatabase(accessionnumber(s)PRJNA451111,PRJNA451107).
Authors'contributionsHRPscriptedandtestedthesoftware,andcontributedtodataanalysis;THBwasinvolvedindataanalysisandinterpretation;JLSwasinvolvedindataanalysis;JRGrevisedandtestedthesoftwareandrevisedthemanuscript;CPGwasinvolvedinvisualization,dataanalysisandrevisedthemanuscript;MCconceivedtheproject,revisedandtestedthesoftware,andrevisedthemanuscript;CFconceivedtheproject,designedthesoftware,interpretedthedata,andcriticallyrevisedthemanuscript.
Allauthorshavereadandapprovedthefinalmanuscript.
Table1SummaryofNanoDJnotebooksNameFunctionality0.
0_QualityControl.
ipynbEvaluatethequalitycontrolandsequencehandling1.
0_Basecalling.
ipynbTranslatestheeventsortherawelectricalsignalfromanONTsequencer(FAST5format)toaDNAsequencetoobtainaFASTAoraFASTQfile1.
1_Trim+Demux.
ipynbPerformsequencetrimminganddemultiplexing2.
0_DeNovo_Canu-Miniasm.
ipynbDenovoassemblywithCanuorMiniasm,andpolishwithRaconandPilon3.
0_DeNovo_Canu+polish.
ipynbNanopolishmodulestoimprovetheCanuassembly4.
0_DeNovo_Flye.
ipynbDenovoassemblywithFlyesoftware5.
0_DeNovo_Hybrid.
ipynbPerformdenovoassemblyofNanoporereadsinconjunctionwithIlluminareadsusingMaSuRCAand/orUnicyclersoftware6.
0_AssemblyCompare.
ipynbComparedistinctassemblyresultsbasedonQUASTsoftware7.
0_SimulateReads.
ipynbObtainsimulatedreadsmadewithNanosimsoftwareandtheNanosim-hforkwithprecomputedmodels8.
0_Alignment.
ipynbReference-basedassemblyusingeitherBWA,BLASTorRebalersoftware9.
0_AssemblyGraph.
ipynbAssemblygraphvisualizationEducational.
ipynbPerformsbasecalling(withAlbacore),qualitycontrolsteps,andaBLAST-basedclassificationofthereads(foreducationalpurposes)Rodríguez-Pérezetal.
BMCBioinformatics(2019)20:234Page3of4EthicsapprovalandconsenttoparticipateNotapplicable.
ConsentforpublicationNotapplicable.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Authordetails1ResearchUnit,HospitalUniversitarioNuestraSeoradeCandelaria,UniversidaddeLaLaguna,SantaCruzdeTenerife,Spain.
2GenomicsDivision,InstitutoTecnológicoydeEnergíasRenovables(ITER),SantaCruzdeTenerife,Spain.
3DepartamentodeIngenieríaInformáticaydeSistemas,UniversidaddeLaLaguna,SantaCruzdeTenerife,Spain.
4DepartamentodeMatemáticas,EstadísticaeInvestigaciónOperativa,UniversidaddeLaLaguna,SantaCruzdeTenerife,Spain.
5CIBERdeEnfermedadesRespiratorias,InstitutodeSaludCarlosIII,Madrid,Spain.
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