GLAPD: Whole Genome Based LAMP Primer Design for a ...

Therefore, LAMP primer design is more complex than PCR primers. Designing a LAMP primer set to specifically identify a group of genomes (group- ... DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Supplementaldata totalviews ViewArticleImpact SHAREON AbdEl-LatifHesham AssiutUniversity,Egypt DebmalyaBarh InstituteofIntegrativeOmicsandAppliedBiotechnology(IIOAB),India PallaviSingh NorthernIllinoisUniversity,UnitedStates Theeditorandreviewer'saffiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction MaterialsandMethods Results Discussion Conclusion DataAvailabilityStatement AuthorContributions Funding ConflictofInterest Acknowledgments SupplementaryMaterial Footnotes References Opensupplementaldata Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex Checkforupdates Peoplealsolookedat METHODSarticle Front.Microbiol.,13December2019Sec.FoodMicrobiology https://doi.org/10.3389/fmicb.2019.02860 GLAPD:WholeGenomeBasedLAMPPrimerDesignforaSetofTargetGenomes BenJia1†,XuelingLi2†,WeiLiu2,ChangdeLu2,XiaotingLu2,LiangxiaoMa2,Yuan-YuanLi2*andChaochunWei1,2* 1DepartmentofBioinformaticsandBiostatistics,SchoolofLifeSciencesandBiotechnology,ShanghaiJiaoTongUniversity,Shanghai,China 2ShanghaiCenterforBioinformationTechnology,Shanghai,China Loop-mediatedisothermalamplification(LAMP)technologyhasbeenappliedinawiderangeoffieldssuchasdetectionoffoodbornebacteriaandclinicalpathogensduetoitssimplicityandefficiency.However,existingLAMPprimerdesigningsystemsrequireaconservedgeneorashortgenomeregionasinput,andtheycan’tdesigngroup-specificprimers.Withthegrowingnumberofwholegenomesavailable,itispossibletodesignbetterprimerstotargetasetofgenomeswithhighspecificitybasedonwholegenomes.WepresenthereawholeGenomebasedLAMPprimerdesigner(GLAPD),anewsystemtodesignLAMPprimerforasetoftargetgenomesusingwholegenomes.CandidatesingleprimerregionsareidentifiedgenomewideandthencombinedintoLAMPprimersets.Foragivensetoftargetgenomes,onlyprimersetsamplifyingthemandonlythesegenomeswillbeoutput.Inordertoacceleratetheprimerdesigning,aGPUversionisprovidedaswell.TheeffectivenessofprimersdesignedbyGLAPDhasbeenassessedforawiderangeoffoodbornebacteria.GLAPDcanbeaccessedathttp://cgm.sjtu.edu.cn/GLAPD/orhttps://github.com/jiqingxiaoxi/GLAPD.git.AsimpleonlineversionisalsosuppliedtohelpuserstolearnandtestGLAPD:http://cgm.sjtu.edu.cn/GLAPD/online/. Introduction Loop-mediatedisothermalamplification(LAMP)isasimple-operating,effectiveandreliablemethodtoamplifyDNAsequence(Notomietal.,2000;Paridaetal.,2008;MoriandNotomi,2009).Theamplificationisunderaconstanttemperature(about62°C)andtherunningtimeisshort(within1h).Inmanyapplicationscenarios,LAMPisabetteroptionthanpolymerasechainreaction(PCR)becausethereactioncanbeinsmallandportabledevices(Curtisetal.,2012;Chaumpluketal.,2016).AbasicLAMPprimersetcontainsfoursyntheticprimersderivedfromsixprimerregions(SupplementaryFigureS1).Therefore,LAMPprimerdesignismorecomplexthanPCRprimers.DesigningaLAMPprimersettospecificallyidentifyagroupofgenomes(group-specific)atthesametimehasahighdemandinmanyapplicationfieldslikefoodborneharmfulbacteriadetection,clinicalpathogenidentification,agriculturalpathogenidentification,andsoon. Thegroup-specificprimersmeanthattheycanbeappliedtomanytargetgenomesbelongingtoagroup(theprimersarecommon)andatthesametimetheprimerscan’tamplifyanyothergenomesnotincludedinthisgroup(theprimersarespecific)(Jarman,2004;Kalendaretal.,2017).Forexample,thereare16strainsofwhitespotsyndromeviruswithcompletegenomesandmorethan100thousandothervirusesinNCBInucleotidedatabase(uptoOctober29th,2018).Agroup-specificprimerforwhitespotsyndromevirusshouldonlyamplifythe16strainsbutnootherviruses.Traditionally,thegroup-specificprimersaredesignedbasedonconservedgenes(Pengetal.,2015),genomeregions(Yaoetal.,2016)orthemultiplesequencealignment(MSA)ofthesegenesorgenomicregions(Kurosakietal.,2010).ButthismethodislimitedbythesmallnumberofsuitablegenesandthedifficultytogenerateMSAforalargenumberofsequences(ChenandTompa,2010).Inaddition,theprimersbasedontraditionalmethodsoftencan’tmeettherequirementsinpractice.Forexample,theLAMPprimersetfromWangetal.(2015)targetingStaphylococcusaureuswasnotabletoamplifysomeS.aureusstrainsandmightamplifysomeunexpectedgenomes(moredetailsinresultspart).Withmorewholegenomesavailable(O’Learyetal.,2016),it’sabettermethodtodesigngroup-specificprimersbasedonwholegenomes(Treven,2015;Demkinetal.,2017). TherearesomesystemsforLAMPprimerdesignnow.ThemostpopularoneisPrimerExplorerV51,anonlinesoftware.However,themaximumlengthofitsinputsequenceislimitedupto2,000bps.Therefore,designingprimersbasedonthewholegenomeisnotprovided.Inaddition,commonorspecificprimerscanbedesignedbyPrimerExplorerV5usingMSAresultsbyclickingthe“Common”or“Specific”buttonseparately.However,thesetwobuttonscan’tworkatthesametime,whichmakesitnotstraightforwardtodesigngroup-specificprimers.AnothersystemisLAVA(Torresetal.,2011),whichcandesigncommonprimersforagroupoftargetgenomes.However,itrequiresMSAresultsastheinput,whichlimitsthetargetregiontoageneorconservedgenomicregion.LAVAdoesn’tcheckthespecificityofprimers.FastPCR(Kalendaretal.,2017)isasystemthatcandesignLAMPprimersusingthewholegenome.SimilarasPrimerExplorer,ithasanonlineversion,anditdesignscommonprimersorspecificprimersseparately.Itcan’tdesigngroup-specificprimersinonerun.Accordingtotheauthors’bestknowledge,noexistingsystemcandesigngroup-specificLAMPprimersusingwholegenomes. HerewepresentGLAPD(wholegenomebasedLAMPprimerdesigner),anewsystemtodesigngroup-specificLAMPprimersets.Byusingthewholegenomesequencesasinputdata,GLAPDcanensurethespecificityoftheprimersandincreasethechancetodesignasuccessfulprimerset.Agraphicsprocessingunit(GPU)versionofGLAPDisalsoprovided. MaterialsandMethods TheGenomeBasedLAMPPrimerDesignSystemGLAPD ThesystemdiagramofGLAPDislistedinFigure1.GLAPDhasthreesteps:(I)identifyingcandidatesingleprimerregions;(II)combiningsingleprimersintoLAMPprimerset;and(III)checkingtheLAMPprimerset.Theinputs,outputsandthecomputationstepsarelistedbelowinmoredetails. FIGURE1 Figure1.ThesystemdiagramofGLAPD.TheinputsofGLAPDincludethetargetgroupofgenomes,thereferencegenomes,andthebackgroundgroupofgenomes.TherearethreestepstogenerateLAMPprimersets:(I)identifyingcandidatesingleprimerregions;(II)combiningsingleprimersintoLAMPprimersets;(III)checkingtheLAMPprimersets.Thecheckpartcontainscommonalitycheckbasedontargetgroup,specificitycheckbasedonthebackgroundgroupandthetendencycheckofbindingamongsingleprimers.PartsI,II,andIIIcouldbeacceleratedbyusingGPU. Inputs Inthisstep,twogroupsaredefinedfirst.ThetargetgroupisdefinedasagroupofgenomesorgenomeregionswhichareexpectedtobeamplifiedbytheLAMPprimerset.TheprimersgeneratedbyGLAPDareexpectedtoidentifyeachtargetgenome.Ifitfailedtogenerateaprimersetforthegroupoftargetgenomes,thesystemwilloutputprimersthatcanamplifythemaximumnumberofgenomes. Similartothetargetgroup,abackgroundgroupisdefinedasagroupofgenomesorgenomeregionswhicharenotexpectedtobeamplified.PrimersdesignedbyGLAPDshouldnotamplifyanygenomeinthebackgroundgroup. Onegenomefromthetargetgroupneedstobeselectedasthereferencegenomewhichwillbeusedasthetemperatetogenerateprimersequences.Thereferencegenomecanberandomlypickedfromthetargetgroup. IdentifyingSinglePrimerRegions AbasicLAMPprimersetcontainsfoursyntheticprimersfromsixprimerregions,namedF3,F2,F1c,B1c,B2,andB3.SequencesfromF1candF2aresynthesizedintooneprimerFIPandsequencesfromB1candB2aresynthesizedintoanotherprimerBIP.ThepositionsrelationshipamongthesesingleprimersareshowedinSupplementaryFigureS1.InordertodesignaLAMPprimerset,thosecandidateprimerregionsareidentifiedfirst.TheyarethencombinedintoLAMPprimersets. GenomebasedLAMPprimerdesigneridentifiesallcandidateprimerregionsinthereferencegenomeaccordingtoprimer’slength,GC-content,meltingtemperature(Tm),stabilityandsoon(SupplementaryTableS1).ThesecondarystructureofprimerischeckedbyGLAPDusingthethermodynamicalapproachsimilartoPrimer3program(Untergasseretal.,2012).Atthesametime,theendofeachprimerischeckedtoexcludesymmetricsequencesandhomopolymers.GLAPDusescustomizedparameterstoidentifyprimerregionsaccordingtotheGC-contentofthetargetregion(betweenF3andB3).IfGC-contentofthetargetregionishigh,theGC-contentandTmofprimersaresettobehigh,viceversa. CombiningSinglePrimersIntoPrimerSets PrimersfromsixregionsarecombinedintoonebasicLAMPprimerset.GLAPDusesthepositionalrelationship(SupplementaryFigureS1)amongthesixregions,GC-contentrelationshipbetweenprimersandwholeamplificationregion(SupplementaryTableS1),Tmrelationshipamongprimers(theTmsofF1candB1care3°Chigherthanotherprimers’)tocombinethem.ThenthecombinedLAMPprimersetwillbecheckedforcommonality,specificityandtendencyofbindingamongsingleprimers. CheckingCommonalityandSpecificityoftheLAMPPrimerSet AnidealLAMPprimersetshouldbeabletoamplifyallthetargetgenomesbutnotgenomesfromthebackgroundgroup.Inordertodothischeck,firstly,allsingleprimersarealignedtothetargetgenomesandthebackgroundgenomesusingBowtie(Langmeadetal.,2009).Bydefault,nomismatchisallowedwhenaprimerisalignedtothetargetgroup.IfGLAPDfailstodesignLAMPprimersetstoamplifyalltargetgenomes,asmallnumberofmismatchesareallowedwhenprimersarealignedtothetargetgroup.However,ifaprimercanbealignedtoabackgroundgenomewithintwomismatches(bydefault),thisprimerisconsideredasnotspecific.Themoremismatchesrequiredtoaligntheprimertothebackgroundgenomes,themorespecificistheprimer.Nomatterhowmanymismatchesinaprimer,nomismatchisallowedinthe5′ofF1candB1c,andthe3′ofF3,F2,B2,B3,LF,andLBprimer.Afterthealignment,primers’positions,strandinformation(plusorminus)andthenumberofmismatchesineachgenomearerecorded. Usingtheinformationgeneratedaboveandthepositionalrelationshipofthesixprimerregions(SupplementaryFigureS1),GLAPDcheckstheabilityofaLAMPprimersettoamplifygenomesinthebackgroundgroup.IfaLAMPprimersetcanamplifyanybackgroundgenome,thissetwillbediscarded.Therefore,moreflexiblethresholdsforpositionalrelationshipamongprimerscanbeusedtoimprovethespecificityinthisstep.AftertheLAMPprimersetpassesthisspecificitycheck,thenumberofgenomesorgenomeregionsinthetargetgroupamplifiedbytheprimersetiscalculatedusingthesamemethodinspecificitycheck. CheckingtheBindingTendencyofAnyTwoPrimers TheLAMPprimersetpassedthecommonalityandspecificitycheckwillbecheckedforeverysingleprimer’stendencyofbindingtoothersingleprimersinthisprimerset.ThischeckusesthethermodynamicalapproachsimilarasthePrimer3programdoes. OutputtingLAMPPrimerSets TheLAMPprimersetpassedallabovecheckstepswillbeoutput.WhenGLAPDhasdesigned10(bydefault)LAMPprimersetssuccessfully,orGLAPDhascheckedallcandidateLAMPprimersets,thesystemstopsautomatically.Theoutputscontainthesequences,positions,lengthsoftheprimersandgenomeswhichcanbeamplified.TheLAMPprimersetsarenotoverlappedwitheachother.Bydefault,theshortestdistancebetweentwoLAMPprimersets’F3regionsis300bps. LoopPrimers Inordertoacceleratetheamplification,twoadditionalloopprimers(LFandLB)canbeadded(SupplementaryFigureS1).GLAPDcanalsodesignLAMPprimersetwithloopprimers.Thecandidateprimerregionsareidentifiedforloopprimersfromthereferencegenomefirst.ThosecandidateregionsmustmeettherequirementslistedinSupplementaryTableS1.Then,GLAPDcombinesloopprimerswithothersingleprimersintoaLAMPprimerset.ALAMPprimersetcouldcontainoneortwoloopprimers.TheTmsofloopprimersaresettobe3°ChigherthanTmofF3,F2,B2,andB3.Atlast,thisLAMPprimersetischeckedforthetendencyofprimerannealinganditscommonality. GPUVersion GraphicsprocessingunitscanbeusedtoaccelerateGLAPDinthreesteps.Inthestepofidentifyingcandidatesingleprimerregions,GLAPDcanidentifiesthemfrommanypositionsofthereferencegenomesimultaneously.Manyprimers’GC-content,stability,TmandsecondarystructurecanbecalculatedinparallelinGPUs.Inthestepofcombiningsingleprimers,eachthreadofGPUisassignedwithadifferentcandidateF3primerthenGLAPDtriestodesignallLAMPprimersetscontainingthisF3primerinparallel.Inthecheckingstep,eachthreadcalculatesthenumberoftargetgenomesandbackgroundgenomesthatcanbeamplifiedbytheprimersetdesignedinthisthread,andeverysingleprimer’stendencyofbindingtoothersingleprimers.Ineachthread,onlytheLAMPprimersetamplifyingthemaximumnumberoftargetgenomeswillbereturnedtoCPUforoutput. Databases Threedatabasesweregenerated.Database-1wasthedatabaseofcompletegenomesequencesofbacteriaandarchaea,whichwasdownloadedfromNCBI’sFTPonAugust5th,2013.Itcontained4,902sequencefilesfrom2,599strains(about9GB). Database-2wasthedatabaseofallcompletemitochondrionsequencesofsuina,bovinae,andcaprinae,whichwasdownloadedfromNCBInucleotidedatabaseonJune14th,2016.Inthisdatabase,120,394,and209mitochondrionsequencesforsuina,bovinaeandcaprinae,respectivelywereselected. Database-3wasthedatabaseofcompletegenomesequencesofbacteriaandarchaeadownloadedfromNCBI’sNucleotidedatabaseonSeptember19th,2018.Itcontained15,728bacterialsequencefilesand494archaeasequencefiles(about60GB). Database-4wasthedatabaseofcompletegenomesequencesofvirusesdownloadedfromNCBI’sNucleotidedatabaseonAugust2th,2017.Itcontained163,576virusessequencefiles. DNAExtraction Theactivatedstrainswereculturedinappropriatemethod(SupplementaryTableS2)andcollectedwhentheculturesreachedanopticaldensityat600nm(OD600)between0.6∼1.0.ThegenomicDNAofthestrainswereextractedusingAxyPrepTMMultisourceGenomicDNAMiniprepKit(AxygenBioscientific,Inc.,UnitedStates).Thesamplesweregroundintopowderwithliquidnitrogenandhomogenizedwith400μlofcelllysisbuffer,andallotherstepsfollowedthemanufacturer’sinstructions. LAMPReaction Loop-mediatedisothermalamplificationreactionwasperformedina25μlreactionmixture.Themixturecontained1×ThermoPolBuffer(contained2mmol/LMgSO4),6mmol/LMgSO4(total8mmol/L),1.4mmol/LofeachdNTP,1.6μmol/Lofeachinnerprimers(FIPandBIP),0.2μmol/Lofeachouterprimers(F3andB3),8unitsofBstDNAPolymerase(LargeFragment). WhentesttheLAMPprimerdesignedforS.aureus,themixturecontained20ngoftemplateDNAofeach29bacterialstrains.WhentesttheLAMPprimer’sspecificitydesignedforVibriovulnificusandVibriocholerae,themixturealsocontained20ngoftemplateDNAofeach29bacterialstrainsandwhentestitscommonality,theamountoftemplateDNAwas50ng,5ng,500pg,50pg,5pg,500fg,50fg,5fgand0.5fg,respectively.InallNTC(notemplatecontrol)reaction,templateDNAwasreplacedbysterilizedwater. TheLAMPreactionwascarriedoutat62°Cfor60minusingaVeritiTMDxThermalCycler(ThermoFisher,UnitedStates),theninactivatedBstDNAPolymeraseat80°Cfor10min.Afterthereaction,1μl1000×SYBRGreenIwasaddedintothesolutiontoconfirmwhetherthereactionoccurred.Inapositivereaction,thecolorofthesolutionwasgreenandinanegativereaction,thecolorwasorange. Results ExperimentalValidationoftheGroup-SpecificPrimersDesignedbyGLAPD Group-SpecificLAMPPrimersofStaphylococcusaureus Staphylococcusaureusisonemaintypeoffoodbornepathogensaroundtheworld(Kadariyaetal.,2014;Paudyaletal.,2017).Traditionally,itsnuc,mecAgeneswereusedtodesigntheLAMPprimersets(Wangetal.,2015;Chenetal.,2017).ButthetwogenesarenotconservedamongallS.aureus(Hoeghetal.,2014;Karmakaretal.,2016)andmayexistinotherStaphylococcusspp.(Borjessonetal.,2015).Therefore,existingLAMPprimersets(Wangetal.,2015)forS.aureusareneithercommonnorspecificenough(SupplementaryTablesS3,S4). Indatabase-1theS.aureusspecieshad43strains(SupplementaryTableS3).GLAPDusedthose43strainsasthetargetgroupandtherestofthegenomesindatabase-1asthebackgroundgroupthendesignedseveralgroup-specificLAMPprimersets.Oneset(Table1)waslocatedinapredictedgenewhichcodes50SribosomalproteinL14.ThisLAMPprimersetwascommonforallS.aureusandnotforanyotherbacteria.Itwasvalidatedbyexperiments(SupplementaryFigureS2). TABLE1 Table1.TheLAMPprimersetdesignedbyGLAPDforS.aureus. VibrioGroup-SpecificLAMPPrimerSet Vibriospp.isoneofthemainpathogenicbacteriainseafood(Huehnetal.,2014;Mizanetal.,2015).MostLAMPprimersaredesignedonlyfortheidentificationofV.cholerae(Okadaetal.,2010)orV.vulnificus(Hanetal.,2011).InspitethattheV.vulnificusandVibrioparahaemolyticuscanbedetectedsimultaneouslyinonereaction,itrequiredtwoLAMPprimersetsinthereaction(Wangetal.,2016). Indatabase-1,therewereeightstrainsofV.choleraeandthreestrainsofV.vulnificus(SupplementaryTableS5).V.choleraeandV.vulnificusbelongedtoVibriogenus.GLAPDusedthose11strainsasthetargetgroupandtherestofthegenomesindatabase-1asthebackgroundgroup.Severalgroup-specificLAMPprimersetsweredesignedbyGLAPD.Oneset(Table2)waslocatedinagenewhichcoded30SribosomalproteinS20.ThisLAMPprimersetwascommonforallV.choleraeandV.vulnificus,andwasspecifictothesebacteriaonly.ItwasvalidatedbyexperimentslistedinSupplementaryFigureS3. TABLE2 Table2.TheLAMPprimersetdesignedbyGLAPDforV.choleraeandV.vulnificus. Besidesthetwogroup-specificLAMPprimersetsmentionedabove,wehaveappliedGLAPDtodesigngroup-specificprimersformorethantenotherfoodbornepathogens.GLADPcouldsuccessfullydesigngroup-specificprimersformostofthesefoodbornepathogensandmorethanhalfofthoseprimersworkedwellinrealexperiments(SupplementaryTableS6). Theflexiblesettingoftargetandbackgroundgroup. WhenGLAPDisusedtodesigngroup-specificprimers,thetargetgroupandbackgroundgroupcouldbedefinedflexiblybyusers.Someexamplesarelistedbelowtoshowthisflexibility. (1)Todesigngroup-specificprimerforallgenomesinagenus.Salmonellaisacommonbacterialpathogenresponsibleforsalmonellosis,acommondiseaseaffectstheintestinaltraceanditcancausesubstantialsocioeconomicburden(Liuetal.,2018).Indatabase-3,471completegenomesand1assemblygenomewith66contigswereSalmonella.GLAPDcoulddesignthegenus-levelsalmonella-specificLAMPprimersetsuccessfully.OneLAMPprimersetdesignedbyGLAPD(SupplementaryTableS7)wasinagenecoding(2E,6E)-farnesyldiphosphatesynthase.Itcouldamplifyall472genomeswithoutanymismatches,anditwasspecifictoalltargetgenomesconsideringallothergenomesindatabase-3asthebackgroundgenomes. (2)Todesigngroup-specificprimerforsomegenomesinagenus.BothV.choleraespeciesandV.vulnificusspeciesbelongtoVibriogenus.Indatabase-3,44completegenomeswereV.choleraeand19genomeswereV.vulnificus.TheprimersetlistedinTable2wasstillcommoninall63genomeswithoutanymismatches,andisspecifictothetargetgenomeconsideringtheothergenomesindatabase-3asthebackgroundgroup(don’tallowanymismatches). (3)Todesigngroup-speciesprimerforallgenomesinaspecies.Indatabase-3therewere351completegenomesofS.aureus,theprimersetinTable1wasneithercommonnorspecificinthenewdatabase.Onenewprimerset(SupplementaryTableS8)designedbasedondatabase-3wasoverlapwithtwogenescodingS4domain-containingproteinYaaAandDNAreplicationrepairproteinRecF.Itwascommonforall351genomeswithoutanymismatchesandspecifictothetargetgenomesaswell. (4)Todesigngroup-specificprimerforsomegenomesinaspecies.EnterohemorrhagicEscherichiacoliO157:H7wasamajorfoodbornepathogenanditcauseddiarrhea(Tarretal.,2005;Limetal.,2010).TheE.coliO157:H7wasonegroupofE.colispecies.Indatabase-3,therewere59completegenomesofE.coliandthreewereO157:H7(Inordertoexcludeambiguousgroup,onlytakecareofpartE.coligenomes,thoseaccessionnumbermuststartwith“NC”).OneLAMPprimerset(SupplementaryTableS9)designedbyGLAPDoverlapswithtwogenescodingrecombinaseandpeptidetransporter.Thisprimersetwascommonforallthreegenomeswithoutanymismatchandspecifictoallthesethreegenomesaswell(withallothergenomesindatabase-3asthebackgroundgroup). GLAPDCanDesignPrimersforOtherOrganisms GenomebasedLAMPprimerdesignerwasfirstdevelopedforrapidlydetectingfoodbornepathogensusingLAMPtechnology.Butgroup-specificprimersforotherorganismscanalsobedesignedbyGLAPD.Forexample,wehavetriedtodesigngroup-specificprimerstodetecthalalproductsusingGLAPD.Porkisnotallowedinhalalproducts(Nakyinsigeetal.,2012).ThepreviousLAMPprimersetsforporkidentificationweredesignedonDN1geneandcytbgenelocatedinmitochondria(Yangetal.,2014;Ranetal.,2016).ButtheLAMPprimerset(Yangetal.,2014)couldn’tcoverallmitochondriagenomes(SupplementaryTableS10).Indatabase-2,therewere120mitochondriasequencesofsuniawhichwereconsideredtobefromporkproducts.WeusedGLAPDtodesignseveralLAMPprimersetswiththe120suniamitochondrionsequencesasthetargetgroupandtherestindatabase-2asthebackgroundgroup.Oneoftheseprimersets(SupplementaryTableS11)wasina16SrRNAgene.ThisLAMPprimersetwascommonforallporkmitochondriaandwouldamplifytheporkmitochondriaonly. Wealsotriedtodesigngroup-specificprimersforseveraltypesofaquaticanimalvirusesindatabase-4.GLAPDcoulddesigngroup-specificprimersformostaquaticanimalvirusesusingdefaultparameters(SupplementaryTableS12).Thegroup-specificprimersofinfectioushematopoieticnecrosisvirusandspringviraemiaofcarpviruswerevalidatedbyexperiments.IfGLAPDcouldn’tdesigngroup-specificprimers,itwouldoutputthemostcommonprimers. ComparisonWithExistingSystems PrimerExplorerV5wasanonlineLAMPprimerdesignsoftware.Acandidategenomicregionwasrequiredastheinput.However,GLAPDdidnotrequirethepriorknowledgeaboutthecandidategeneorgenomicregion.UsingthesamesequencesfromthecandidateregionscontainingprimersetsdesignedbyGLAPD,theresultsofPrimerExplorerV5andGLAPDwereverysimilar.Mostprimersfromtwodesignersoverlappedandsomeofthemwereidentical(Figure2).Themainreasonofdifferenceswasthedifferentprimercombinationstrategies. FIGURE2 Figure2.ComparisonoftheprimersetsdesignedbyGLAPDandPrimerExplorerV5.ThesequencecontainingLAMPprimersetdesignedbyGLAPDwasinputtoPrimerExplorerV5todesignprimers.FourscenariosofLAMPprimersetswereshown:(A)LAMPprimersetforS.aureusbasedondatabase-3;(B)LAMPprimersetforV.choleraeandV.vulnificus;(C)LAMPprimersetforSalmonellabasedondatabase-3;(D)LAMPprimersetforE.coliO157:H7basedondatabase-3.TheprimersdesignedbyGLAPDwereshowningreensolidarrowsandprimersdesignedbyPrimerExplorerV5wereinredblankarrows.TheSalmonella’sB2primerdesignedbyPrimerExplorerwasthesameastheB3primerdesignedbyGLAPD. ExistingsystemssuchasPrimerExplorer,LAVAandFastPCRcandesigncommonprimersetforagroupofgenomeswithoutconsideringthespecificitycomparingtoabackgroundgroup(LAVAwasnotusedtodesignprimersbecauseitwasnotdownloadableanymore).Userscaninputasetofgenomicregionsconsideringthefirstregionasthetargetandtherestasthebackgroundgrouptodesignspecificprimers.However,itisnotstraightforwardtodesignprimersetsdirectlywithagiventargetgroupandagivenbackgroundgroupatthesametime.Forexample,themecAandnucgeneswereusedascandidateregions(Wangetal.,2015;Chenetal.,2017)todesignprimersforS.aureus.Indatabase-3,therewere351S.aureusgenomes,amongwhich235genomeshadmecAgeneandall351genomeshadnucgene.Thenon-redundant19mecAsequencesand44nucsequenceswereusedasinputsforPrimerExplorerandFastPCRtodesignprimers.FormecAgene,theMSAresultwasgeneratedbyclustalW(Thompsonetal.,1994)withdefaultparameter,andonly68.1%nucleotideswereidentical.PrimerExplorercouldn’tdesignanyLAMPprimersetsusingthisMSAresultandFastPCRalsocouldn’tdesignanyprimersusingthe19mecAsequences.Fornucgene,theMSAalignmentresultwasalsogeneratedbyclustalWandonly74.7%nucleotideswereidentical.FastPCRcouldn’tdesignanycommonprimersforthe44sequences.PrimerExplorercoulddesigntwoLAMPprimersets.However,becauseofthelowsimilarity,thereweremanymutationsinthedesignedprimerregionsindifferenttargetgenomes(Figure3,redunderline),whichindicatedthatthecommonalityofthisprimersetwasnotgood.Therefore,usingPrimerExplorerandFastPCRtodesigncommonprimersetforS.aureuswiththemecAandnucgeneregionsasinputwasnotsuccessful.Onthecontrary,GLAPDcouldusethewholegenometodesignLAMPprimersetsfortheS.aureusgenomessuccessfully.MoreexamplesareinSupplementaryMaterial(SupplementaryFigureS4andSupplementaryTableS14). FIGURE3 Figure3.ThecommonprimersetsofnucgenedesignedbyPrimerExplorer.ThetwoprimersetsaredesignbyPrimerExplorerusingMSAresultofnon-redundantnucgenesequences.Therednucleotidewithredunderlinemeansamutationamongthosegenesequences. MorecomparisonsbetweenGLAPDandthoseexistingsystemswerelistedinTable3. TABLE3 Table3.ThecomparisonwithexistingLAMPprimerdesigningsystems. Discussion WhyDoWeUseGroup-SpecificPrimers? Detectingmultiplefoodbornepathogenssimultaneouslycanhelpthefoodsafetybecauseitcanspeedthedetectionofpathogens.WhenuseLAMPorPCR,thetraditionalmethodforthisaimisaddingmultiplesetsofprimersintooneassay.Eachprimersetcanonlybeappliedtoonepathogen.Thismethodhassomedisadvantages:(1)alargenumberofdifferentprimersinoneassaymayincreasetheriskofgeneratingprimerdimer,andtheefficiencyofoneprimersetcouldbeinhibitedbyotherprimersets(Xuetal.,2012;Zhaoetal.,2014);(2)It’sachallengetasktofindafeasiblecombinationofmultipleprimersetsduetothehugenumberofdifferentcombinationsofcandidateprimersetsinadditiontothetestofeachprimersetforeachtargetgenome. GenomebasedLAMPprimerdesigner,ontheotherhand,candesigngroup-specificprimers,whichcanavoidthosedisadvantages.Inoneassay,onlyoneprimersetisneeded.Lessprimersinassaycandecreasetheriskofinteractionsamongprimersandreducethetestworkloadaswell. DesigningPrimersBasedontheWholeGenome Traditionally,primersaredesignedforsomeconservedgenesorasmallgenomeregion.Mostregionsofthewholegenomeareneglected.Inaddition,manyprimerdesignsystemshavealimitedlengthofinputsequences.IfnoprimerscouldbedesignedbyGLAPD,thechancetofindsuitableprimerswouldbelow. GenomebasedLAMPprimerdesignercanusewholegenomesasinputdirectly.Itscansallcandidatesingleprimersderivedfromthewholegenome,thenallcandidateprimersarecombinedintoprimersetsandtestedonebyone.Thiscanvastlyimprovethesuccessrateofprimerdesign. ANewStrategyIsUsedtoDesignGroup-SpecificPrimers Thegroup-specificprimerscanbedesignedbasedonconservedgenes,genomicregionsorMSAresults.However,existingmethodshavelimitations:(1)Therearelimitednumberofwell-knownconservedgenesineachorganism.Thisnumberwillbemuchsmallerforaslightlylargernumberofdifferenttargetorganisms.(2)Theconservedgenesmayexistinsomebackgroundorganisms.Forexample,the16SrRNAgenesareconservedinmanybacteria,thereforenon-targetorganismsmayalsobeamplifiediftheycontainverysimilar16SrRNAgenes;(3)ItisdifficulttogenerateaMSAresultsfromabignumberofinputsequences,anditisalmostimpossibletogenerateMSAformanygenomes. GenomebasedLAMPprimerdesignerusesadifferentstrategytoavoidthoseproblems.Firstly,GLAPDsearchesallcandidateprimersgenomewide,thenthecandidatesingleprimersarealignedwithtargetandbackgroundgenomes.Thealignmentinformationaboutpositionsandstrandsisusedtocheckprimers’commonalityandspecificity.Thedistancebetweentwosingleprimerscanbedifferentineachtargetgenomeaslongasthedistanceiswithintheallowedrange.Thesequencesbetweenprimerscanalsobedifferentindifferenttargetgenomesaslongastheprimerregionsareconservedamongtargetgenomes.Inotherwords,GLAPDcandesigngroup-specificprimersinvariablegenomeregionswithahighersuccessrate.Thisstrategycanalsobeusedindesigningprimersforotheramplifications,likePCR. TheParameterConfigurationinGLAPD ParametersandthresholdsusedinGLAPDweresimilarwithPrimerExplorerV5.GoodresultswerestillachievedifGLAPDusedloosethresholds.Forexample,whenthe5′stabilityofF1corB1cwassettobebiggerthan-4kcal/mol,theexperimentalresultsoftheprimerswerestillgood.Moreexperimentsmightbeneededtodecidebetterparametersorthresholdsforaspecificgroupoforganisms. GPUVersionofGLAPD InordertoacceleratetheLAMPprimerdesign,aGPUversionofGLAPDwasdeveloped.TheGPUversionwasverypromisingtoacceleratetheprimerdesignprocedureinidentifyingcandidatesingleprimerregions(GPUversionisthreetimefasterthanCPUversion)andcombiningsingleprimerstoaprimersetmaybeslowerthanCPUversioninsomescenarios(SupplementaryTableS13).WearecurrentlyworkingonittoimprovetheperformanceoftheGPUversion. Conclusion Designinggroup-specificprimersisadifficulttaskforamplificationslikePCR,anditisevenmorechallengingforLAMPduetothenumberofprimersinLAMPprimerset.HerewepresentanewLAMPprimerdesigner,GLAPD,todesignaLAMPprimersettargetingonagroupofgenomes.Insteadofusingwell-knowngeneregions,thewholegenomecouldbeuseddirectlyforprimerdesign,whichincreasedthesuccessrate. GenomebasedLAMPprimerdesignercouldbeappliedtodesignLAMPprimersfortheidentificationofanyorganismswithoutknownregionsasinput.TheresultsofGLAPDaresimilartoPrimerExplorerV5whenthesamesequencesareinput.TheeffectivenessofGLAPDwerevalidatedinexperiments.WithGLAPD,thechancetosuccessfullydesignaLAMPprimersettoidentifyagroupoforganismishigherthanbeforeanditcanbeagoodsystemtoacceleratingtheapplicationofLAMPtechnologyinmanyfieldssuchasfoodquarantine,epidemicdiseasesurveillanceandsoon.GLAPDcanbedownloadedfromhttp://cgm.sjtu.edu.cn/GLAPD/orhttps://github.com/jiqingxiaoxi/GLAPD.git.UserscanalsolearnandtestGLAPDusingthesimpleonlineversion:http://cgm.sjtu.edu.cn/GLAPD/online/. DataAvailabilityStatement Alldatasetsgeneratedforthisstudyareincludedinthearticle/SupplementaryMaterial. AuthorContributions CW:conceptualizationanddesignofthesystem.BJandCW:systemimplementation.BJandLM:systemtest.XLi,WL,CL,XLu,andY-YL:experimentalvalidationofdesignedLAMPprimers.BJ,XLi,Y-YL,andCW:writingthemanuscript.Allauthorshavereadandeditedthemanuscript. Funding ThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(61472246),theNationalBasicResearchProgramofChina(2013CB956103),theNationalHigh-TechR&DProgram(863)(2014AA021502),andCross-InstituteResearchFundingofShanghaiJiaoTongUniversity(YG2017ZD01andYG2015MS39). ConflictofInterest Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WethanktheHighPerformanceComputingCenter(HPCC)attheShanghaiJiaoTongUniversityforcomputing. 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PubMedAbstract|GoogleScholar Keywords:LAMP,group-specificprimer,wholegenomebasedprimerdesigner,foodbornepathogens,primerdesign Citation:JiaB,LiX,LiuW,LuC,LuX,MaL,LiY-YandWeiC(2019)GLAPD:WholeGenomeBasedLAMPPrimerDesignforaSetofTargetGenomes.Front.Microbiol.10:2860.doi:10.3389/fmicb.2019.02860 Received:17June2019;Accepted:26November2019;Published:13December2019. Editedby: AbdEl-LatifHesham,AssiutUniversity,Egypt Reviewedby: DebmalyaBarh,InstituteofIntegrativeOmicsandAppliedBiotechnology(IIOAB),India PallaviSingh,NorthernIllinoisUniversity,UnitedStates Copyright©2019Jia,Li,Liu,Lu,Lu,Ma,LiandWei.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:Yuan-YuanLi,[email protected];ChaochunWei,[email protected] †Theseauthorshavecontributedequallytothisworkandsharefirstauthorship Peoplealsolookedat Download