單分子測序特性詳解

SMRT? Sequencing:SMRT? Sequencing:FromfinishedgenomescomplexmicrobialcommunitiesZuweiQian,PacificBiosciencesForResearchUseOnly.Notforuseindiagnosticsprocedures.?Copyright2015byPacificBiosciencesofCalifornia,Inc.AllrightsJune21,SINGLESINGLEMOLECULE,REAL-TIME(SMRT)DNASMRTSEQUENCING單SMRTSEQUENCING單分子SMRT測序特LongReads長讀-Average>10,000平均讀長>10,000HighConsensusAccuracy一致性準確率-Uniform,UnbiasedCoverage均一,無偏好覆-LackofGC%orsequencecomplexityDNAModificationDetection堿基修飾檢-Epigenome描述表觀基因PACBIORSIIANDSEQUELSYSTEMSPacBioRSMachineNumberZMWPACBIORSIIANDSEQUELSYSTEMSPacBioRSMachineNumberZMWperOutputper0.5–1~5-8InputDNAper~250~250N50Read15–20~15ConsensusWHATWHATISNEEDEDFORAHIGH-QUALITY獲取高質(zhì)量基因組測序的至關重要因Longsequencereads（長讀取ResolveProvidescontiguousgenomeUniformityinsequencingcoverage（均衡的測序覆蓋度Lackofbias(GC%,sequenceAllowssequencingtheentireAbsenceofsystematicsequencingerrors（無測序系統(tǒng)偏差RandomerrorswashoutinfinalProvidesaccurategenomeWHATISNEEDEDFORWHATISNEEDEDFORAHIGH-QUALITYLongsequencereads（長讀取ResolveProvidescontiguousgenomeUniformityinsequencingcoverage（均衡的測序覆蓋度Lackofbias(GC%,sequenceAllowssequencingtheentireleastbiasofAbsenceofsystematicsequencingerrors（無測序系統(tǒng)偏差RandomerrorswashoutinfinalProvidesaccurategenome-1/wp-content/uploads/2015/09/Revolutionize-Genomics-with-SMRT-DRAFTvs.GAPLESSGENOMESDRAFTvs.GAPLESSGENOMES（基因組草圖和無洞基因組圖對于基因組二代測序技術的短板讀長二代測序技術的短板讀長導致組裝不完無法跨越重復序覆蓋度不均一（G/C或A/T富含區(qū)域是重災區(qū)）法達到真正擴增等因素導致系統(tǒng)錯誤（指測序的準確率只適用于做SNP檢測，不適用作大范圍的結構變異檢NGSPLAGUEDBYSEQUENCINGBIAS二代測序飽受序列偏好的困擾NGSPLAGUEDBYSEQUENCINGBIAS二代測序飽受序列偏好的困擾GC-AT-LowUniformcoverage&ofDNAsequencecontextRossetal.(2013)GenomeBiology14:RS1.Chaissonetal.(2015)NatureReviewsGenetics16:TECHNOLOGYCOMPARISON(SHANK3EXAMPLE)PacBiodataat/2014/Human54x/fast.htmlHiSeqdataat/sra/SRR642636TECHNOLOGYCOMPARISON(SHANK3EXAMPLE)PacBiodataat/2014/Human54x/fast.htmlHiSeqdataat/sra/SRR642636SHANK3–INVOLVEDINAUTISMSHANK3–INVOLVEDINAUTISM85%GC-二代測序和三代測序的覆蓋度對比二代測序和三代測序的覆蓋度對比PAPERSINENVIRONMENTALMICROBIALAPPLICATIONAREAS---IdentificationofRestriction-ModificationSystemsofBifidobacteriumanimalisPAPERSINENVIRONMENTALMICROBIALAPPLICATIONAREAS---IdentificationofRestriction-ModificationSystemsofBifidobacteriumanimalissubsp.lactisCNCMI-2494bySMRTSequencingandAssociatedMethylomeAnalysisCompleteGenomeSequenceofEnterococcusmundtiiQU25,anEfficientL-(+)-LacticAcid-ProducingBacterium------Comparisonofsingle-moleculesequencingandhybridapproachesforfinishingthegenomeofClostridiumautoethanogenumandanalysisofCRISPRsystemsinindustrialrelevantThegenomeoftheanaerobicfungusOrpinomycessp.strainC1Arevealstheuniqueevolutionaryhistoryofaremarkableplantbiomassdegrader/content/early/2013/05/20/AEM.00821-13.full.pdf(fungal)Localizedelectrontransferratesandmicroelectrode-basedenrichmentofmicrobialcommunitieswithinaphototrophicmicrobialmat/pmc/articles/PMC3902354/(16S)Long-termoperationofmicrobialelectrosynthesissystemsimprovesacetateproductionbyautotrophicmicrobiomes/doi/abs/10.1021/es400341bCapturingsinglecellgenomesofactivepolysaccharidedegraders:anunexpectedcontributionofVerrucomicrobia-----ExploringtherolesofDNAmethylationinthemetal-reducingbacteriumShewanellaoneidensisMR-1ThegenomesequenceofStreptomyceslividans66revealsanoveltRNA-dependentpeptidebiosyntheticsystemwithinametal-relatedgenomicislandhttp://intl-Acrowdsourcedfundedprojecttosequenceafern./projects/azolla-a-little-fern-with-massive-green-GenomesequenceofCandidatusMicrothrixparvicellaBio17-1,along-chain-fatty-acid-accumulatingfilamentousactinobacteriumfromabiologicalwastewatertreatmentplant-NewbacteriawithpotentialcommercialSinglecellgenomicstudyofDehalococcoidetesspeciesfromdeep-seasedimentsofthePeruvianMargin/wp-GenomeSequenceofBacilluspumilusMTCCB6033/content/2/2/e00327-DeNovoAssemblyoftheStreptomycessp.StrainMg1GenomeUsingPacBioSingle-MoleculeSequencing./content/1/4/e00535-Valueofanewlysequencedbacterialgenome./1949------Diverseantibioticresistancegenesindairycowmanure/content/5/2/e01017-GenomeSequenceandMethylomeofSoilBacteriumGemmatirosakalamazoonensisKBS708T,aMemberoftheRarelyCultivatedGemmatimonadetesPhylumGenomeSequenceofPseudomonassp.StrainP482,aTomatoRhizosphereIsolatewithBroad-SpectrumAntimicrobialActivity.NEAR--KorenS.,et.al.(2013)ReducingassemblyofialgenomeswithNEAR--KorenS.,et.al.(2013)ReducingassemblyofialgenomeswithRFECTCONSENSUS3代和2代微生物基因組組裝序列完成度對比(20133代和2代微生物基因組組裝序列完成度對比(2013年Innercircle:Outercircle:NEWGOLDSTANDARDMICROBIALGENOMESTanizawaNEWGOLDSTANDARDMICROBIALGENOMESTanizawaetal.BMCGenomics(2015)KINETICVARIATIONGENOMEACROSSTHEMITOCHONDRIALKINETICVARIATIONGENOMEACROSSTHEMITOCHONDRIALCollaborationwithN.Sondheimer(CHOP),A.Chen-Plotkin(UPenn),E.Schadt(Mt.Sinai)IMPORTANCEOFFINISHEDMICROBIOLOGYcapableoffermentingwastegases(CO,H2,CO2)intoethanol&commodityGENOMESIMPORTANCEOFFINISHEDMICROBIOLOGYcapableoffermentingwastegases(CO,H2,CO2)intoethanol&commodityGENOMESAPPLIED-Previousdraftgenomein~100-Closed,high-qualitygenomegeneratedfromPacBio-onlydatawithoutmanualBrownetal.(2014)Comparisonofsingle-moleculesequencingandhybridapproachesforfinishingthegenomeofClostridiumautoethanogenumandanalysisofCRISPRsystemsinindustrialrelevantClostridia.BiotechnologyforBiofuels7:40FINISHEDGENOMESAREESSENTIALFORMETABOLICRECONSTRUCTIONSGenesmissedby2nd-genassemblies,FINISHEDGENOMESAREESSENTIALFORMETABOLICRECONSTRUCTIONSGenesmissedby2nd-genassemblies,butpresentinPacBioBrownetal.(2014)Comparisonofsingle-moleculesequencingandhybridapproachesforfinishingthegenomeofClostridiumautoethanogenumandanalysisofCRISPRsystemsinindustrialrelevantClostridia.BiotechnologyforBiofuels7:40FOODBORNEFOODBORNEMICROBIALSEQUENCINGPUBLICATIONSFOODBORNEOUTBREAKS-AcomparativeanalysisofmethylomeprofilesFOODBORNEOUTBREAKS-AcomparativeanalysisofmethylomeprofilesofCampylobacterjejunisheepabortionisolateandgastroentericstrainsusingPacBiodata.AGapless,UnambiguousGenomeSequenceoftheEnterohemorrhagicEscherichiacoliO157:H7StrainEDL933.AnovelTn3-likecompositetransposonharboringblaVIM-1inKlebsiellapneumoniaespp.pneumoniaeisolatedfromriverAnevaluationofalternativemethodsforconstructingphylogeniesfromwholegenomesequencedata:acasestudywithSalmonella.AnalysisoftheCampylobacterjejuniGenomebySMRTDNASequencingIdentifiesRestriction-ModificationMotifs.BacteriophageP70:uniquemorphologyandunrelatednesstootherListeria-----predominatedbythenewallelicvariantsubAB2-SafetyofthesurrogatemicroorganismEnterococcusfaeciumNRRLB-2354foruseinthermalprocessvalidation.TracingOriginsoftheSalmonellaBareillyStrainCausingaFood-borneOutbreakintheUnitedStates.VerticaltransmissionofhighlysimilarblaCTX-M-1-harboringIncI1plasmidsinEscherichiacoliwithdifferentMLSTtypesinthepoultryproductionpyramid.Whole-GenomeSequencing:OpportunitiesandChallengesforPublicHealth,Food-borneOutbreakInvestigations,andtheGlobalFoodSupply.----Comparativegenomicanalysisandvirulencedifferencesincloselyrelatedsalmonellaentericaserotypeheidelbergisolatesfromhumans,retailmeats,andComparativegenomicsofenterohemorrhagicEscherichiacoliO145:H28demonstratesacommonevolutionarylineagewithEscherichiacoliComparativeGenomicsoftheCampylobacterlariCompleteandAssembledGenomeSequenceofStaphylococcusaureusRKI4,aFood-PoisoningStrainExhibitingaNovelS.aureusPathogenicityIslandCarryingCompleteGenomeSequenceofaMultidrug-ResistantSalmonellaentericaSerovarTyphimuriumvar.5-StrainIsolatedfromChickenCompleteGenomeSequenceofEnteroinvasiveEscherichiacoliO96:H19AssociatedwithaSevereFoodborneCompleteGenomeSequenceofListeriamonocytogenesLm60,aStrainwithanEnhancedColdAdaptationCompleteGenomeSequenceofPluralibactergergoviaeFB2,anN-AcylHomoserineLactone-DegradingStrainIsolatedfromPackedFishCompleteGenomeSequenceofSalmonellaentericasubsp.entericaSerovarAgona4600042-1,AssociatedwithaMultistateOutbreakintheUnitedCompleteGenomeSequenceoftheQualityControlStrainStaphylococcusaureussubsp.aureusATCCCompleteGenomeSequenceofVibrioparahaemolyticusEnvironmentalStrainUCM-CompleteGenomeSequencesofaClinicalIsolateandanEnvironmentalIsolateofVibrioCompleteGenomeSequencesofSalmonellaentericaSerovarHeidelbergStrainsAssociatedwithaMultistateFood-BorneIllnessCompleteGenomeSequencesofTwoEscherichiacoliO145:H28OutbreakStrainsofFoodCompleteSequencesofSixIncA/CPlasmidsofMultidrug-ResistantSalmonellaentericasubsp.entericaSerotypeDiverseantibioticresistancegenesindairycowDraftgenomesequenceofPantoeaagglomeransR190,aproducerofantibioticsagainstphytopathogensandfoodborneDraftWhole-GenomeSequencesofNineNon-O157ShigaToxin-ProducingEscherichiacoliEnterobacterasburiaestrainL1:completegenomeandwholegenomeopticalmappinganalysisofaquorumsensingExploringbacterialepigenomicsinthenext-generationsequencingera:anewapproachforanemergingFirstCompleteGenomeSequenceofClostridiumsporogenesDSM795T,aNontoxigenicSurrogateforClostridiumbotulinum,DeterminedUsingPacBioSingle-MoleculeReal-TimeFirstFullyClosedGenomeSequenceofSalmonellaentericasubsp.entericaSerovarCubanaAssociatedwithaFood-BorneFullyAssembledGenomeSequenceforSalmonellaentericasubsp.entericaSerovarJavianaGenomeSequenceofSalmonellabongoriStrainN268-08,aRareClinicalGenomeSequenceofSerratiaplymuthicaRVH1,IsolatedfromaRawVegetable-ProcessingGenomeSequencesof228ShigaToxin-ProducingEscherichiacoliIsolatesand12IsolatesRepresentingOtherDiarrheagenicE.coliGenomeSequencesofThreeFrequentlyUsedListeriamonocytogenesandListeriaivanoviiGenomesequencingandcomparativegenomicsprovidesinsightsontheevolutionarydynamicsandpathogenicpotentialofdifferentH-serotypesofShigatoxin-producingEscherichiacoliGenome-WideMethylationPatternsinSalmonellaentericaSubsp.entericaGenomicepidemiologyoftheEscherichiacoliO104:H4outbreaksinEurope,HamburgerHighresolutionassemblyandcharacterizationofgenomesofCanadianisolatesofSalmonellaIntegrativeanalysisofSalmonellosisinIsraelrevealsassociationofSalmonellaentericaSerovar9,12:l,v:-withextraintestinalinfections,disseminationofendemicS.entericaSerovarTyphimuriumDT104biotypes,andsevereunderreportingofoutbreaks.NoncontiguousFinishedGenomeSequenceofStaphylococcusaureusKLT6,aStaphylococcalEnterotoxinB-PositiveStrainInvolvedinaFoodPoisoningOutbreakinPrevalenceofsubtilasecytotoxin-encodingsubABvariantsamongShigatoxin-producingEscherichiacolistrainsisolatedfromwildruminantsandsheepdiffersfromthatofcattleandpigsandIDENTIFICATIONIDENTIFICATIONOFDMGFORBEERSPOILINGMICROBEDETECTIONDETECTIONOFSEQUENCINGDNABASEMODIFICATIONSSMRTFlusbergetal.DETECTIONOFSEQUENCINGDNABASEMODIFICATIONSSMRTFlusbergetal.(2010)NatureMethods7:461-SIGNATURESOFDIFFERENTDNASIGNATURESOFDIFFERENTDNAMODIFICATIONSDNADetectablebyotherSequencingBIFIDOBACTERIACHARACTERIZATION-PROBIOTICSStudyfundedbyDanoneNutriciaBIFIDOBACTERIACHARACTERIZATION-PROBIOTICSStudyfundedbyDanoneNutriciaMETHYLATIONINLACTOCOCCAL936-TYPEMETHYLATIONINLACTOCOCCAL936-TYPEPHAGES-AmongmostfrequentlyisolatedphagesindairyutilisingLactococcuslactisstarter-Continuetonegativelyimpactcheese-FirstfunctionalMTasesidentifiedinmembersof936-typeMurphyetal.(2014)Methyltransferasesacquiredbylactococcal936-typephageprovideprotectionagainstrestrictionendonucleaseactivity.BMCGenomics15:831-842.METHYLATIONINLACTOCOCCAL936-TYPEMETHYLATIONINLACTOCOCCAL936-TYPEPHAGES-AmongmostfrequentlyisolatedphagesindairyutilisingLactococcuslactisstarter-Continuetonegativelyimpactcheese-FirstfunctionalMTasesidentifiedinmembersof936-type-ProvideprotectionagainstHphIand-AdaptiveresponsetoincreasingfitnessinadynamicdairyMurphyetal.(2014)Methyltransferasesacquiredbylactococcal936-typephageprovideprotectionagainstrestrictionendonucleaseactivity.BMCGenomics15:831-842.METAGENOMICSAPPROACHES???METAGENOMICSAPPROACHES???Shot-gunPCRDECIDINGWHICHMETAGENOMICSMRTSEQUENCINGOPTIONTOUSEFORYOURPROJECTFull-Length2kbProjectMediumDECIDINGWHICHMETAGENOMICSMRTSEQUENCINGOPTIONTOUSEFORYOURPROJECTFull-Length2kbProjectMediumtoLow(forabundant<ngs,low100ng,good10ng,low16SreferenceleveldesiredGeneorprotein-16SrRNA-COMMUNITYANALYSISSTILLMAJOR16SrRNA-COMMUNITYANALYSISSTILLMAJORPrimerPrimerTRIAGEAHETEROGENEOUSATTRIBUTESPOPULATIONMULTIPLEByskindarklightTRIAGEAHETEROGENEOUSATTRIBUTESPOPULATIONMULTIPLEByskindarklight+TRIAGEAHETEROGENEOUSATTRIBUTESPOPULATIONMULTIPLEByskincolor,tall,short,short,TRIAGEAHETEROGENEOUSATTRIBUTESPOPULATIONMULTIPLEByskincolor,tall,short,short,tall,+++HIGH-QUALITYHIGH-QUALITYSINGLE-MOLECULEACCURACYSMRTbellDNAHIGH-QUALITYSINGLE-MOLECULEACCURACYSMRTbellDNACircularConsensusSequencing(CCS)SequencingHIGH-QUALITYSINGLE-MOLECULEACCURACYSMRTbellDNACircularConsensusSequencing(CCS)SequencingthesameDNAbasemultipletimes:CCSADVANTAGESOFFULLADVANTAGESOFFULLLENGTH16SFORMETAGENOMICANALYSIS-Onlyfull-length16SsequencehasbeenproventobeadequatefortreeconstructionnecessaryforprecisephylogeneticplacementExamplelayout:BulletSlidewithADVANTAGESOFADVANTAGESOFFULLLENGTH16SSEQUENCINGTFORMETAGENOMICANALYSIS-Multiplephylawereoftencompletelymissedbyshort-read-TheresolvingpowerofFL16Sdatasetswasmoreapparentinsampleswithcomplexmicrobialcommunitiesandwhenreferencesequencesindatabasewerescarce-NoamplificationrequiredduringFL16Ssequencingmeansplatform-specificbiasisgenerallyverylowcomparedtoshort-readtechnology.-FL16SsequencingoffershighestcontigaccuracywithoutdiscriminationagainstGC-richor-poorregions.-Longreadsresultinafinerphylogeneticresolutionthatmaynotbeachievedbyshorter16Sgenesequencing-ResurgenceofFLsequencesaffordedbylongreadsequenceasthegoldstandardhasthepotentialtotransformmicrobialcommunitystudiesExamplelayout:BulletSlidewithFULL-LENGTHFULL-LENGTH16SSEQUENCINGFULL-LENGTH16SSEQUENCING“UsingPhyloTagstoassessmicrobialcommunitydiversityinenvironmentalFULL-LENGTH16SSEQUENCING“UsingPhyloTagstoassessmicrobialcommunitydiversityinenvironmentalsamplesallowsustofillimportantgapsinthetreeoflifewhileimprovingclassificationandmicrobialcommunityprofilingaccuracywithimportantimplicationsforinferredmetabolicpotentialandbiogeochemicalrolesofuncultivatedmicroorganismsinnaturalandhumanengineeredecosystems.”FULL-LENGTH16SFULL-LENGTH16SSEQUENCINGFULL-LENGTH16SFULL-LENGTH16SSEQUENCINGFULL-LENGTH16SSEQUENCINGFULL-LENGTH16SSEQUENCINGChunLab-PacBioMicrobiomeCloudCLmicrobiomeBIOINFORMATICSMADEEASYCHUNLABChunLab-PacBioMicrobiomeCloudCLmicrobiomeBIOINFORMATICSMADEEASYCHUNLABSMRTsoftwarePACBIOSHOTGUNPACBIOSHOTGUNMETAGENOMICSPUBLICATIONS-ASingle-cellgenomeforThiovulumALE:agenericassemblylikelihoodevaluationframeworkforassessingtheaccuracyofgenomeandmetagenomeassemblies.Anenvironmentalbacterialtaxonwithalargeanddistinctmetabolicrepertoire.BacteroidesdoreidominatesgutmicrobiomepriortoautoimmunityinFinnishchildrenathighriskfortype1diabetes.Buzzoff,that'smybee!Capturingsinglecellgenomesofactivepolysaccharidedegraders:anunexpectedcontributionofCaughtinthemiddlewithmultipledisplacementamplification:themythofpoolingforavoidingmultipledisplacementamplificationbiasinametagenome.CSSSCL:apythonpackagethatusescombinedsequencesimilarityscoresforaccuratetaxonomicclassificationoflongandshortsequencereads.DetectingepigeneticmotifsinlowcoverageandmetagenomicsDiscoveryofadivergentHPIV4fromrespiratorysecretionsusingsecondandthirdgenerationmetagenomicsequencing.Diverseantibioticresistancegenesindairycowmanure.DraftgenomesequenceofSulfurospirillumsp.strainMES,reconstructedfromthemetagenomeofamicrobialelectrosynthesisGenomesequencedeterminationandmetagenomiccharacterizationofaDehalococcoidesmixedculturegrownoncis-1,2-GenomicandmetabolicdiversityofMarineGroupIThaumarchaeotainthemesopelagicoftwosubtropicalgyres.Genomicsandhostspecializationofhoneybeeandbumblebeegutsymbionts.Gillbacteriaenableanoveldigestivestrategyinawood-feedingImprovedmetagenomeassembliesandtaxonomicbinningusinglong-readcircularconsensussequenceNearlyfinishedgenomesproducedusinggelmicrodropletculturingrevealsubstantialintraspeciesgenomicdiversitywithinthehumanmicrobiome.Nextgenerationsequencingtechnology:Advancesandapplications.Next-generationsequencingforpathogendetectionandPhyloPythiaS+:Aself-trainingmethodfortherapidreconstructionoflow-rankingtaxonomicbinsfrommetagenomesThemethylomeofthegutmicrobiome:disparateDammethylationpatternsinintestinalBacteroidesdoreiTheSantaPolasalternasamodelforstudyingthemicrobiotaofhypersaline----------------------METAGENOMICASSEMBLYCOMMUNITYBMETAGENOMICASSEMBLYCOMMUNITYB–HUMANMICROBIOMEMOCKAcinetobacterbaumanniiATCC17978ActinomycesodontolyticusATCC17982BacilluscereusATCC10987BacteroidesvulgatusATCC8482ClostridiumbeijerinckiiNCIMB8052DeinococcusradioduransR1chromosome1DeinococcusradioduransR1chromosome2EnterococcusfaecalisOG1RFHelicobacterpylori26695LactobacillusgasseriATCC33323ListeriamonocytogenesEGD-eNeisseriameningitidisMC58PseudomonasaeruginosaPAO11Rhodobactersphaeroides2.4.1chromosome2StaphylococcusepidermidisATCC12228StreptococcusagalactiaeStreptococcuspneumoniaeHUMANMICROBIOMEMOCKHUMANMICROBIOMEMOCKCOMMUNITYAcinetobacterbaumanniiATCC17978BacilluscereusATCC10987BacteroidesvulgatusATCC8482ClostridiumbeijerinckiiNCIMB8052DeinococcusradioduransR1chromosome1DeinococcusradioduransR1chromosome2EnterococcusfaecalisOG1RFHelicobacterpylori26695LactobacillusgasseriATCC33323ListeriamonocytogenesEGD-eNeisseriameningitidisMC58PseudomonasaeruginosaPAO1Rhodobactersphaeroides2.4.1chromosomeStaphylococcusaureussubsp.aureusagalactiaemutansASSEMBLYCOMPARISONExample-Propionibacteriumacnes31210Technology-AssemblyAssemblySizeASSEMBLYCOMPARISONExample-Propionibacteriumacnes31210Technology-AssemblyAssemblySizeRESULTS-SUMMARYRESULTS-SUMMARYIMPROVEDMETAGENOMEASSEMBLIESiversalmarkerIMPROVEDMETAGENOMEASSEMBLIESiversalmarkerrganismsinhabitcomplexFranketal.(2016)ScientificReports|6:25373|DOI:“PacBioCCSdataprovidesadramatimprovementintheassemblyofungenesincomparisontoHiSeq2000“thisenabledenhancementsinreconstructionsofunculturedIMPROVEDMETAGENOMEASSEMBLIESfamilyandwasestimatedtorepresent~36%IMPROVEDMETAGENOMEASSEMBLIESfamilyandwasestimatedtorepresent~36%ofthetotalwhereasunFirm_1isadeeplybranchedunculturedrepresentativeaffiliatedtotheFirmicutes,accountingfor~5%.”“…unClos_1isanas-yetunculturedbacteriumaffiliatedtotheIMPROVEDMETAGENOMEASSEMBLIESclearpatternsthatareindicativeofseveralnumericallyIMPR