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WhatdoIdoifmyblastsearchesseemtohaveallthetophitsfromthesamegenusorspecies?Ifthebacterialspeciesyouareusingtoannotateisclinicallysignificantorofgreatresearchinterest,youmayfindthatwhenyouperformblastsearches(particularlyinnr)thatyouseeminglyonlygethitsthataredifferentstrainsorisolatesofthesamespecies.Thisobviouslydoesn’tgiveyoumuchinformationabouthowwellconservedtheproteinonwhichyouareworkingiscomparedtoproteinsinothergenera.Thereisamethodtomodifyblasttoletyouexcludesuchhitsfromyoursearches.IwilluseagenefromClostridiumbotulinumasanexampletoillustratethisusingtheproteinsequenceofthegenewiththelocustagCLJ_B3418.Figure1showsthetopnrblasthitsforthisprotein.YoucaneasilyseethatallofthehitsbutonearefromClostridiumbotulinumwithveryhighlevelsofcoverageandidentities.TheyareessentiallyallthesameproteinfromdifferentisolatesofClostridiumbotulinum.

Figure1.Theblastresultsusinganon-filterednrblastsearchforCLJ_B3418.Theblastsearchcanbesetupslightlydifferentlytopreventthisproblemfromoccurring.Asnotedinfigure2,wecansetthesearchuptoexclude,inthiscase,thetaxid:1485(Clostridium).ThetaxidnumberstandsfortheNCBITaxonomyIDnumber.Byexcludingthetaxidnumber1485,allblasthitsinthattaxonomicclassificationwillnotbeincluded.TodothiswetypethegenusnameClostridiumintheOrganismtextboxbelowthesequenceinputbox.Asyoutypeapulldownmenuofoptionswillappearwhichyoucansubsequentlyjustclickonto

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select(seehighlightedmenuiteminFigure2).ThesimplyclicktheExcludecheckboxnexttotheorganismnameandthenselectblast.

Figure2.SettingupablastsearchtoexcludetheCostridiumtaxid:1485.Figure3showstheresultsoftheblastresultforthesameproteinAFTERexcludingtheClostridiumtaxid1485.Notethedifferentnamesappearinginthesearchresults.However,alsonotethatthetophitisnolongertheonethatmatchestheproteinunderinvestigationinthespeciesyouareworkingon.ThusyouwouldtaketheFIRSTnrhitasthetophitinthiscaseinsteadofskippingoverthefirstone.NotealsothatifyouusethisblastresulttoselectsequencesfortheT-CoffeealignmentthatyouwillsubsequentlydointheSequenceBasedSimilarityModule,thatyouwillneedtoaddtheFASTAformattedsequenceoftheproteinunderinvestigationtothetopofthelistbeforeconstructingthealignment.Studentsshouldalsoaddacommentintheirtextbookofwhichtaxidnumberwasexcludedfromtheirsearch.Experimentwithdifferentlevelsofexclusion(onlyonespecies)oraddmultipleoptionsforexclusion(i.e.,thegenus)orsomewhereinbetween(differentspecificspeciesexcludedbyaddingadditionalorganismboxesinwhichtoenterchoicesbyusingthe+optiontotherightoftheexcludecheckboxtoaddanother).

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Figure3.ThenrblastresultsforCLJ_B3418AFTERexcludingtheCostridiumtaxid:1485.Notethedifferentgenusandspeciesnamesofthetophits.YoucanalsousetheNCBITaxonomyBrowsertofinddifferentlevelsoftaxidtouseinyourexclusionsearches,especiallyifitisnotclearwhatyoushouldchoosefromthepulldownmenuinBLAST.TheuseoftheTaxonomyBrowserisdescribedingeneraltermsintheHorizontalGeneTransfersectionoftheprojectmanual.Briefly,goto:https://www.ncbi.nlm.nih.gov/taxonomyandenterthenameofyourorganism’sgenusinthesearchwindow(Clostridiumbotulinumistheexampleusedbelow)andclickonSearch.AresultsimilartoFigure4willdisplay.Clickontheorganismhyperlinkinblue,andyouwillbetakentothefulllineageoftheorganism(nextfigure).

Figure4.ResultsofsearchingforClostridiumbotulinumintheNCBITaxonomybrowser.

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Figure5belowshowsaportionoftheC.botulinumresults.Inthelineageline,thelastentryisthegenus(Clostridum),butyoucanhoverthecursoroveranyofthelevelsoftaxonomyandseethenameofthelevel(i.e.,family,orderetc.).Figure6showswhatwilldisplaywhentheClostridiumhyperlinkisselected.

Figure5.TheClostridiumbotulinumresultsfromNCBITaxonomybrowser(notcomplete).OfinterestintheresultsfromclickingontheClostridiumhyperlinkdisplayeddisplayedinFigure6istheTaxonomyIDof1485(exactlytheonewefoundbylimitingtheBLASTresultsfromwithintheBLASTtool).Youcouldusethisinformationtosimplytype“Clostridium(taxid:1485)”–donot,however,includethequotationmarks-intheorganismwindowoftheBLASTsearchandclickexcludeasbefore.

Figure6.TheClostridiumgenustaxidinformation.

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Wecanalsogofurther“up”intaxonomicwindowtoexcludemorethanonegenus(thoughyoushouldnothavetodothatroutinely).Forexample,Figure7showsthedisplaythatwouldcomeupifweclickedontheClostridiaceae(i.e.,thefamilytowhichthegenusClostridumbelongs)hyperlinkinsteadoftheClostridiumhyperlink.Differentgenerawillappearthatarepartofthisfamily.ClickingontheClostridiaceaelinkfromthispagewillresultintheinformationshownintheFigure8.

Figure7.ThedisplayresultingfromselectionoftheFamilyClostridiaceaeinthetaxonomybrowser.

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HereweseethattheClostridiaceaefamilyhastheTaxonomyIDof31979.ToexcludethisFamilyfromtheBLASTresults,wewouldsimplytypein“Clostridiaceae(taxid:31979)”intotheorganismboxintheBLASTsearchandclickexclude.Thenextimagewillshowhowtheautofilloptionwillhighlightoncewepasteinthetaxid.

Figure8.ThetaxonomyidentificationnumberoftheFamilyClostridiaceae.

Figure9.ABLASTsearchsetuptoexcludemembersoftheFamilyClostridiaceaefromthesearchresults.Finally,Figure10showstheBLASTresultsfromdoingtheexclusionatthislevel.

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Figure10.BLASTresultsafterexcludingtheFamilyClostridiaceaefromthesearch.