RDPManual.docx
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RDPManual.docx
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RDPManual
UnfinishedRDP3
InstructionManual
Contents
1Introduction1
2OpeningAlignmentFiles1
3SettingAnalysisOptions1
3.1GeneralSettings1
3.2RDPSettings2
3.3GENECONVSettings2
3.4BootscanSettings3
3.5MaxChiSettings4
3.6ChimaeraSettings4
3.7SiScanSettings5
3.8LARDSettings5
3.9PhylProSettings5
3.10DNADistancePlotSettings5
3.11TOPALSettings6
3.12Recombinationratesettings7
3.13MatrixSettings7
3.14TreeSettings6
4BeginningANAnalysis6
4.1AutomatedExploratoryRecombinationAnalysis6
4.2ManualAnalyses7
5ExaminingautomatedanalysisResults7
5.1TheSchematicSequenceDisplay7
5.2TheRecombinationInformationDisplay8
5.3ThePlotDisplay8
5.4TheSequenceDisplay8
5.5TheDendrogramDisplay9
5.6TheCompatibilityMatrixDisplay9
6SavingResults9
7SupplementaryAnalyses10
8RECOMBINATIONSIGNALDETECTIONmethods10
8.1TheRDPMethod10
8.2GENECONV11
8.3Bootscan11
8.4MaxChi12
8.5Chimaera13
8.6SiScan13
8.73SEQ14
8.8PhylPro14
8.9LARD14
8.10DNADistancePlots14
8.11TOPAL15
9SuPPLEMENTARYMETHODS15
9.1BreakpointDistributionPlot(HotspotDetection)15
9.2Matrices15
10ASTEP-BY-STEPGUIDETOUSINGRDP315
10.1CompilingaGoodDataset15
10.2MakingaGoodAlignment15
10.3SettingUpaPreliminaryScanforRecombination15
10.4TestingandRefiningPreliminaryHypotheses15
9.5SoftwareCrashes/FileIncompatibilities15
11RunningRDP3fromacommandline15
12PossibleproblemsWITHUSINGRDP15
12.1PoorAlignments15
12.2RecombinantReferenceSequences15
12.3AdjacentRegionsofDifferentOrigin15
12.4Degeneracies15
12.5SoftwareCrashes/FileIncompatibilities15
13Acknowledgements15
14Appendix15
14.1ProgramFiles15
14.2CitingRDPandOtherPrograms16
15REFERENCES16
1Introduction
RDP3(RecombinationDetectionProgramversion3)isaWindows95/98/NT/XPprogramfordetectingandanalysingrecombinationsignalsinasetofalignedDNAsequences.Whileanumberofotherprogramshavebeenwrittentocarryoutthesametask(seeDrouinetal.1999,PosadaandCrandall,2001,Posada,2002,andtheweb-sitehttp:
//www.bioinf.manchester.ac.uk/recombination/programs.shtmlandreferencestherein),mymotivationforwritingRDPhasbeentoproduceananalysistoolthatisbothaccessibletouserswhoareuncomfortablewiththeuseofUNIX/DOScommandlinesandpermitsamoreinteractiveroleintheanalysisofrecombination.
BesidesimplementingtheoriginalRDPmethoditalsoimplementsaBootscanningmethod(Salminienetal.,1995;Martinetal.,2005b),theGENECONVmethod(Padidametal.,1999),theMaximumChiSquaremethod(MaxChi;MaynardSmith,1992;PosadaandCrandall,2001),theChimaeramethod(PosadaandCrandall,2001),theSisterScanningMethod(SiScan;Gibbsetal.,2000),the3SEQmethod(Bonietal.,2007),theReticulatecompatibilitymatrixmethod(JakobsenandEasteal,1996)andtheTOPALDSSmethod(McGuireandWright,1998,2000).RDP3isalsoawindowsinterfaceforarangeofotherrecombinationdetectionandanalysisprogramsincludingLARD(LikelihoodAssistedRecombinationDetection;Holmesetal.,1995)andLDHAT(McVeanetal.,20022004).
Ifyouareimpatientandwanttostartanalysingyoursequenceswithoutreadingthemanualitisstronglyrecommendedthatyougostraighttothestep-by-stepguideinsection10.Thisguidewillhelpyouusetheprograminthewayitwasintendedtowork.
2OpeningAlignmentFiles
AnumberofdifferentalignmentfileformatsarerecognizedbyRDP3includingPHYLIP,GDE,FASTA,CLUSTAL,GCG,NEXUS,MEGAandDNAMAN.Toopenafilepressthe“Open”button(Fig1inthecommandbuttonpanel)andselectthefiletobeopened.Thedirectoryfromwhichthefileisselectedis“remembered”byRDP3whenitisshutdown.Onceloadedthealignedsequencesandtheirnamesaredisplayedinthe“sequencedisplaypanel”(Fig1).Alsodisplayedarethedegreesofnucleotideidentityindifferentregionsofthealignedsequencesinan“identitydisplaypanel”(Fig1).
Besidesalignmentfiles,RDP3projectfiles(witha“.rdp”extension)mayalsobeloaded.Inadditiontoalignedsequencesthesefilesalsocontaininformationonpossiblerecombinationeventsdetectedinpreviousanalysissessions.
AstillexperimentalfeaturethathasbeenimplementedinRDP3,istheabilitytoreadORFpositionsandnamesfromGenBankfiles.Theprogramrequires
(1)thataGenBankfilebeopenedafteranalignmentfileand
(2)thatoneofthesequenceswithinthemultiplealignmentisthesameasthesequenceintheGenBankfile.RDP3willautomaticallyscanthesequencesinthealignmenttocheckwhetheranymatchthesequenceintheGenBankfile.IfyouareunabletoloadaparticularGenBankfilesuccessfully,sendmethefiletogetherwithyouralignmentandI’llfixtheproblemforyou.
3SettingAnalysisOptions
Pressingthe“Options”buttoninthecommandbuttonpanelwillallowyoutomodifyRDP3ssettings.ForcasualusersofRDP3,theprogram’sdefaultsettingsshouldworkfineformostdatasets.Theonlysettingsthatyoushouldeverneedtochangeareitalicised.
3.1GeneralSettings
3.1.1Generalrecombinationdetectionoptions.Thevariousrecombinationdetectionmethodscanbesettoperceivesequencesasbeingeitherlinearorcircular.Notethatevenlinearsequencescanbeanalysedasthoughtheyarecircularandthiswillinnowayinvalidatetheanalysisresults.
ThehighestacceptableP-valuesettingisthehighestacceptableprobabilitythatsequencescouldsharehighidentitiesinpotentiallyrecombinantregionsduetochancealone(thecalculationofP-valuesdiffersforthedifferentmethodsandwillbediscussedinsection8).TheoptimalhighestP-valuesettingvariesdependingonthenumberofsequencesinthealignmentbeinganalysed,therecombinationmethodsbeingusedtoexaminethealignment,thesizeoftheslidingwindowsthatareused(forRDP,Bootstcan,MaxChi,ChimaeraandSiScan),andonwhetherthemultiplecomparisoncorrectionsettingisonoroff.
Thedefaultsettingformultiplecomparisoncorrectionis“on”asthismakesthecalculatedP-values“experiment-wide”(orglobal)ratherthan“currentlyselectedsequencetriplet/pairwide”(orlocal)estimatesofprobability.Notethattherearetwomultiplecomparisoncorrection“on”settings.Thedefaultis“Bonferronicorrection”butamodificationofthiscalled“step-downcorrection”isalsooffered.ThesecorrectionsactasP-valuemodifiersthatdecreasestheP-valuecutoffaccordingtothesizeofthedatasetbeingexamined.ForahighestacceptableP-valuesettingof0.05withmultiplecomparisoncorrection“off”youwouldexpectthatapproximately5%ofP-valuesthatarecalculatedwouldmaketheP-valuecutoffbychancealone.Foralargedatasetyouwouldthereforeexpectmanyfalsepositiveresults.ForthesameP-valuecutoffbutwithmultiplecomparisoncorrectionsetto“on”youwouldexpecttoonlyencounteronefalsepositivein~5%ofthedatasetsthatareexamined.Inmostsituations(<100sequenceswithanalysedsequencessharing>70%identity)ahighestacceptableP-valuesettingof0.05whenmultiplecomparisoncorrectionisonand0.0001whenitisoffshouldgivefewfalsepositivesbutstillenabletheidentificationofmostdetectablerecombinationevents.IfthecorrectionsettingisofftheP-valuecut-offmustbeverycarefullyselectedbasedonthenumberoffalsepositivesyouarepreparedtotolerate.Whenalargedatasetcontainingsequenceswithlowdiversity(e.g.100sequencesallsharing>95%identity)isanalyseditmayinfactbeimpossibletodetectanyrecombinationthatispresentifoneofthemultiplecomparisoncorrectionsettingsareon.Inthesecasesitmaybebesttoanalysethedatasetusingthepermutationtestsoffered(seesection3.1.2)withthemultiplecomparisoncorrectionsettingoffandaP-valuecut-offof0.001–thiswillgiveyousomeideaoftheexpectedfalsepositiverateforeachidentifiedrecombinationsignal.
3.1.2Permutationoptions:
Ifthe“numberofpermutationssetting”issettoanumberlargerthan0,RDP3willrunitsautomatedrecombinationdetectionanalysesinpermutationmode.Thisinvolvesgeneratingagroupofsimulatedrecombinationfreedatasets(thenumberthataresimulatedisspecifiedbyyouinthespaceprovided),whicharethenanalysedbytheprogramusingtheexactsamesettingsthatitusestoanalysearealdataset.Thereareseveralwaysinwhichtheresultsfromsuchananalysiscanbeinterpreted.Firstly,ifRDP3identifiesmorerecombinationeventsintherealdatasetthanitdoesin95%ormoreofthesimulateddatasetsthenthisisequivalenttoaP-value<=0.05oftheirbeingnoevidenceofrecombinationinthedataset–i.e.youcanbemore95%ormoresurethatthereissomeevidenceofrecombinationinthedataset.Thisresultdoesnot,howevertellyouwhichofthedetectedrecombinationeventsareactualrecombinationevents–theresultsimplytellsyouthatsomeofthemareprobablyreal.SecondlyifRDPdetectsasinglerecombinationsignalintherealdatasetthathasabetterassociatedP-valuethanthebestsignalsin95%ormoreofthesimulateddatasetsthenthisistheequivalentofsayingthatthissignalhasaP-value<=0.05–i.e.thatyoucanbe95%confidentthattherecombinationeventassociatedwiththisrecombinationsignalisarealeventandnotafalsepositive.
RDP3canusetwodifferentapproachestosimulatethesequencesusedinthepermutationtest.Thesimplestinvolvesshufflingalignmentcolumnstodestroymostoftherecombinationsignalsevidentinanalignment.Whilethishasthepleasingeffectofmaintai
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