EBI web resources III: Web-based tools in Europe (EBI...

EBI web resources III: Web-based tools in Europe (EBI, ExPASy,

EMBOSS, DTU)

Yanbin Yin

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Homeworkassignment41. Downloadhttp://cys.bios.niu.edu/yyin/teach/PBB/purdue.cellwall.list.lignin.fa to

yourcomputer2. SelectaC3HproteinandaF5Hproteinfromtheabovefileandcalculatethe

sequence identitybetweenthemusingtheWaterserveratEBI.3. Performamultiple sequencealignmentusingMAFFTwithallFASTA

sequences inthefile4. Builtaphylogenywiththealignmentusingthe"AlaCarte"modeat

http://www.phylogeny.fr/5. Buildanotherphylogenystartingfromtheunalignedsequences usingthe

“one-click”modeathttp://www.phylogeny.fr/;ifyouencounteranyerrorreports,trytofigureoutwhyandhowtosolveit (hint:skiptheGblocks step).

Writeareport(inwordorppt)toincludealltheoperations,screenshotsandthefinalphylogeniesfromstep3and4.

Dueon10/11(sendbyemail)

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Officehour:Tue,ThuandFri2-4pm,MO325AOremail:yyin@niu.edu

Outline

• Handsonexercises!

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Pairwisealignment(includingdatabasesearch)tools

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FasterSlower

LessmatchesMorematchesBLASTFASTASSEARCH

BLATBWABowtiePSI-BLAST

PSI-SearchHMMER3RPS-BLAST

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http://www.ebi.ac.uk/

Tothebottomofthepage

Clickbynames(A-Z)

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Thisisaverylong listoftoolsScrolldowntofindFASTA

OrCtrl+F andtypefasta

Wearegonna tryFASTAtool

ClickonFASTA[nucleotide]

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ClickGenomesWe’regonna searchArabidopsisgenome

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Clickonthislittlearrow ChooseArabidopsis

Gotohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faandcopythefirstseq (CesA)andpastehere

Changeheretoprotein

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Tfastx:allowframeshiftbetweencodons

Tfasty:alsoallowframeshiftwithincodons

Toleratesequenceerrors

Good forfinding pseudogenes

http://www.ebi.ac.uk/Tools/sss/fasta/help/index-genomes.html

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Should befinished veryquickly

Rawoutput (plain text)Graphicalpresentationoftheoutput

ShowEMBLformatofthesubject(hit)Showalignment

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Inthealignment, lookfor/ frameshift\ frameshift* stopcodon

Weareattherawoutputview

Inorder toalignthequeryprotein tothesubjectgenomicDNA,reading frameshavetomove1or2baseahead(1baseinsertionor2baseinsertion)

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BLASTgivesshorteralignmentbecauseitsalignmentbreakswhereitseesframeshifts

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GobacktothetoolA-Zpage:http://www.ebi.ac.uk/services/all

Ctrl+F andtypessearch

SSEARCHisacommand intheFASTApackageimplementing Smith-Watermanalgorithm

Ssearch canonlydoprotein-protein ornucleotide-nucleotide searchesSlowerbutmostaccurate

Canonlydopr-prornt-nt search

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GobacktothetoolA-Zpage:http://www.ebi.ac.uk/services/all

Ctrl+F andtypeemboss

EMBOSS:EuropeanMolecularBiologyOpenSoftwareSuiteEMBOSS:TheEuropeanMolecular BiologyOpenSoftwareSuite(2000)Rice,P.Longden,I.andBleasby,A.TrendsinGenetics16,(6)pp276--277

EMBOSScontainhundredsofcomputerprogramsforsequenceanalysis

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Needleman-wunsch algorithmSmith-Watermanalgorithm

Equivalenttothebl2seqcommandoftheBLASTpackage

Let’stryneedlefirst

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Globalvs localalignment:• inalocalalignment, youtrytomatchyourquerywithasubstring (aportion)

ofyoursubject(reference)• inaglobalalignmentyouperformanendtoendalignmentwiththesubject

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CslA:539aaCesA:1089aa

Gotohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faCopy&pasteCesACopy&pasteCslA

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- gap.Negativescore:positivescore|identical

ThisisdifferentfromwhatBLASTshowsthealignment

Notadatabasesearch,sonoE-valueisreported

Thisisneedleoutput

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Thisiswateroutput

Thebestwaytofind theoptimallyaligned regionsandcalculatethesimilaritybetweentwosequences

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Selectheretotryblast2seq

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Gotohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faCopy&pasteCesACopy&pasteCslA

Chooseblastp

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FragmentedalignmentsThisblast2seqoutput

Multiplesequencealignmenttools

Foundationformanyotherfurtheranalyses:phylogeny,evolution,motif,

proteinfamilyetc.

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http://www.ebi.ac.uk/Tools/msa/TheMSApageshowsninetoolsandwe’regonna tryClustal Omega,MAFFTandMUSCLE

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Youcanalwayscheckthehelppage

Gohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faandcopypasteallthe9protein seq here

Thensubmit

ThisisClustal Omegapage

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Thisiscalledclustal formatofMSA

ColorAAbasedonchemicalproperties,e.g.acidicAAinblue

Checktheevolutionaryrelatedness

Gettextformatsummaryoftheresults

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Txtformattodescriberelatednessandcanbevisualizedgraphicallyasatreegraph

Matrixtellshowsimilareachpairofseqs is

Bothcanbecopypastetonotepadandsaveasplaintextfile

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ThisisMAFFTpageYoucanalwayscheckthehelppage

Gohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faandcopypasteallthe9protein seq here

ChangeheretoClustalW

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IDsweretruncated

ForcethefirstMresiduealigned

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ThisisMUSCLEpage

Gohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.faandcopypasteallthe9protein seq here

ChangeheretoClustalW

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MolecularSystemsBiology7:539,2011

accuracy speedMAFFT>Clustal Omega>MUSCLE>>ClustalW

http://mafft.cbrc.jp/alignment/software/about.html

SowhichMSAtoolshould Iuse?

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http://www.ebi.ac.uk/Tools/msa/Visualizealignment

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Youcanalwayscheckthehelppage

Gohttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa.alnandcopypastetheMSAbuiltabove

ThisisMview page

MView reformatstheresultsofasequencedatabasesearch(BLAST,FASTA,etc)oramultiplealignment(MSF,PIR,CLUSTAL,etc)addingoptionalHTMLmark-uptocontrolcolouring andwebpagelayout.MView isnotamultiplealignmentprogram,norisitageneralpurposealignmenteditor

37Consensuslettersexplainedathttp://bio-mview.sourceforge.net/manual/manual.html#ref-output-formats

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AnotherMSAvisualizationtool:ESPript http://espript.ibcp.fr/ESPript/ESPript/

Clickheretostart

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Copyhttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa.alnandpastetoatxtfileandsaveitonyourdesktop,andupload to

Afterthefileisuploaded

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Anewwindowpopped out,viewinPDF

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Wejusttriedtheverybasicfunction.ThiswebserverhasmanymoreusefulfunctionssuchasdisplayingsecondarystructuresalongwithMSA.Tolearnmore:http://espript.ibcp.fr/ESPript/ESPript/esp_tutorial.php

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ExPASy:ExpertProteinAnalysisSystematSIBCollectionofexternal/internaltools

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http://expasy.org/

Clickongenomics, thensequencealignment

Thiswebsitecollectandclassifyweblinkstohundreds ofbioinfo tools

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Thispageliststoolsforsequencealignment

We’regonna try

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http://weblogo.berkeley.edu/logo.cgi

Uploadthefilethatwedownloaded fromhttp://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa.aln

Toggle thistoallowlogoshown inmultiline

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Clicktoincrease

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Youcanalsocopypasteasegmentofthealignment toweblogoNoneedtousetheentirealignment

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Pastethecopiedsegmenthere

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WithMSAyoucanbuildaphylogeny todescribetherelatednessofseqs

Seqs

MSA

Phylogeny

Graph

Wearegonna trythiswebsite

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http://phylogeny.lirmm.fr/phylo_cgi/index.cgi

Threemodesofphylogenyreconstruction

Trytheoneclickmode

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Oneclickmodeusesthesetools

Givethisjobaname

http://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa

Gblocks isaprogramautomaticallyeditthealignment

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EMBOSS:EuropeanMolecularBiologyOpenSoftwareSuite

EMBOSS:TheEuropeanMolecularBiologyOpenSoftwareSuite(2000)Rice,P.Longden,I.andBleasby,A.TrendsinGenetics16,(6)pp276--277

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http://emboss.sourceforge.net/

EMBOSScontainhundredsofcomputerprogramswritteninClanguageforsequenceanalysis

ThebestwaytouseistoinstallitonaLinuxcomputer

Herewe’regonna trysomepublicwebserversthathaveEMBOSSpackageinstalled

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Manyothersarenotaccessible,butthisoneis

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ThisiscalledEMBOSSexplorer,whichisawebinterfacetosupportrunning EMBOSSprograms throughweb

350+programsputintodifferentgroups

Wewilltryafewprograms inthispackage

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Themostbasicone:translateanucleotideseq toanaminoacidseq (relatedtofinding theopenreadingframes)

Find theprogramtranseq inthenucleictranslationgroup

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Copyandpastetheseq inhttp://cys.bios.niu.edu/yyin/teach/PBB/nt-example.faIt’sanassembledtranscriptfromESTdataofsomealgalspeciesWedonotknowifitindeedencodeaproteinandifyeswhereistheORFRemembermRNAcontainsuntranslated region(UTR)

Chooseallsixframes

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Thisislikelytherightframe

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Ifthisisacorrectresult?Youcantakethent seq todoblastatNCBI

Puttheseq IDherebecauseitisinGenBank already

Chooseswiss-prot becauseitissmallerandhighquality

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ClickformattingoptionsChooseplaintextviewClickreformat

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Thisisthealignmentofourquerywiththebesthit,theframeis+2,sameasthetranseq result

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ThisisthelongestORF

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ATGCGCTA

TACGCGAT

TAGCGCATReverse

Complementhttp://cys.bios.niu.edu/yyin/teach/PBB/nt-example.fa

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ATGCGCTA

TGCGCTRegion2-7

http://cys.bios.niu.edu/yyin/teach/PBB/nt-example.fa

Region57-400

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CalculateGCcontent

ATGCGCTAGC%=50%

Changetoyestogetapic

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ATGCGCTA

16possibledinuc64possible trinuc256possibletetranuc

Defaultisdinuc

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Equaloccurrence:1/16

Application: scangenometolookforregionswithabnormalcompositions

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http://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa,copypastethe1st seq

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PopulartoolsdevelopedatTechnicalUniversityofDenmark

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Google:cbs dtu

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http://cys.bios.niu.edu/yyin/teach/PBB/nt-example.fa

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ThislistsalltheATGintheseq,eachwasscoredtoindicateitslikelihood tobeastartcodon

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http://cys.bios.niu.edu/yyin/teach/PBB/cesa-pr.fa,copypastethe1st seq

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Nextclass:ClustalX andMEGA

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