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1 Phylogeny Workshop By Eyal PrivmanEyal Privman The Bioinformatics Unit G.S. Wise Faculty of Life Science Tel Aviv University, Israel November 2009 http://ibis.tau.ac.il/twiki/bin/view/Bioinformatics/Phylogeny2009
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2 Why should we care about phylogeny? "Nothing in biology makes sense except in the light of evolution" (Theodosius Dobzhansky, 1973)
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33 Alignment and phylogeny are mutually dependant Inaccurate tree building MSA Sequence alignment Phylogeny reconstruction Unaligned sequences
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44 Alignment and phylogeny are both challenging 25% of residues are aligned wrong Based on BAliBASE: a large representative set of proteins
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55 Alignment and phylogeny are both challenging 5% of tree branches are wrong Based on simulations of 100 protein sequences
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66 Multiple sequence alignment (MSA) progressive alignment ABCDEABCDE Guide tree A D C B E MSA Pairwise distance table Iterative
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77 Multiple sequence alignment (MSA) Several advanced MSA programs are available. Today we will use two: MAFFT – fastest and one of the most accurate PRANK – distinct from all other MSA programs because of its correct treatment of insertions/deletions
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88 MAFFT Web server & download: http://align.bmr.kyushu-u.ac.jp/mafft/online/server/ http://align.bmr.kyushu-u.ac.jp/mafft/online/server/ Efficiency-tuned variants quick & dirty or slow but accurate Nucleic Acids Research, 2002, Vol. 30, No. 14 3059-3066 © 2002 Oxford University PressOxford University Press MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Kazutaka Katoh, Kazuharu Misawa 1, Kei-ichi Kuma and Takashi Miyata *
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99 Choosing a MAFFT strategy quick & dirty slow but accurate
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10 Choosing a MAFFT strategy quick & dirty slow but accurate
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11 Choosing a MAFFT strategy quick & dirty slow but accurate
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12 Choosing a MAFFT strategy L-INS-i ooooooooooooooooooooooooooooooooXXXXXXXXXXX-XXXXXXXXXXXXXXX------------------ --------------------------------XX-XXXXXXXXXXXXXXX-XXXXXXXXooooooooooo------- ------------------ooooooooooooooXXXXX----XXXXXXXX---XXXXXXXooooooooooo------- --------ooooooooooooooooooooooooXXXXX-XXXXXXXXXX----XXXXXXXoooooooooooooooooo --------------------------------XXXXXXXXXXXXXXXX----XXXXXXX------------------ G-INS-i XXXXXXXXXXX-XXXXXXXXXXXXXXX XX-XXXXXXXXXXXXXXX-XXXXXXXX XXXXX----XXXXXXXX---XXXXXXX XXXXX-XXXXXXXXXX----XXXXXXX XXXXXXXXXXXXXXXX----XXXXXXX E-INS-i oooooooooXXX------XXXX---------------------------------XXXXXXXXXXX-XXXXXXXXXXXXXXXooooooooooooo ---------XXXXXXXXXXXXXooo------------------------------XXXXXXXXXXXXXXXXXX-XXXXXXXX------------- -----ooooXXXXXX---XXXXooooooooooo----------------------XXXXX----XXXXXXXXXXXXXXXXXXooooooooooooo ---------XXXXX----XXXXoooooooooooooooooooooooooooooooooXXXXX-XXXXXXXXXXXX--XXXXXXX------------- ---------XXXXX----XXXX---------------------------------XXXXX---XXXXXXXXXX--XXXXXXXooooo-------- quick & dirty slow but accurate
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13 MAFFT output Saving the output Choose a format: Clustal, Fasta, or click "Reformat" to convert to a selection of other formats Save page as a text file A colored view of the alignment
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14 PRANK
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15 Classical alignment errors for HIV env
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16 PRANK Web server: http://www.ebi.ac.uk/goldman-srv/webPRANK/http://www.ebi.ac.uk/goldman-srv/webPRANK/
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17 PRANK output If you need a different format – copy the results to the READSEQ sequence converter: http://www-bimas.cit.nih.gov/molbio/readseq/ http://www-bimas.cit.nih.gov/molbio/readseq/
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18 Downloadable PRANK http://www.ebi.ac.uk/goldman-srv/prank/prank/ –PRANK: A command-line program interface –PRANKSTER: A program with graphical user interface
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19 1.Download and unzip the sequence files from my homepage (Google "Eyal Privman" and look for the workshop materials under "Teaching"). Open "fahA.fas" in Notepad – these are 65 protein sequences in FASTA format. 2.Run PRANKSTER, open the "fahA.fas" file, and run "Alignment" "Make alignment" 3.While you wait: Copy the sequences into the MAFFT web server and run the "automatic" "moderate" strategy – which strategy did MAFFT choose for you? Click "Reformat", choose "phylip|phylip4", and save as "fahA.mafft.phylip" 4.When PRANKSTER finishes click File Save, and save the MSA in Phylip format by the name "fahA.prank.phylip"
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20 Phylogeny reconstruction Different approaches (algorithms / programs): Distance based methods (e.g. neighbor-joining, as in ClustalW) Fast but inaccurate Maximum parsimony (e.g. MEGA)MEGA Maximum likelihood methods (e.g. phyML, RAxML) Accurate but slowerphyMLRAxML Bayesian methods (e.g. MrBayes) Most accurate but very slowMrBayes ABCDEABCDE Guide tree A D C B E MSA Pairwise distance table
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21 PhyML The most widely used maximum likelihood (ML) program Web server & download: http://www.atgc-montpellier.fr/phyml/http://www.atgc-montpellier.fr/phyml/ Accepts input MSA in PHYLIP format only: Interleaved: Sequencial:
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22 Downloadable PhyML Less user-friendly, but allows using local computer power Run "phyml.bat" Drag the file from Windows Explorer to the blue window Enter "d" to switch from DNA to AA Enter "y" to run
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23 1.Give "fahA.prank.phylip" or "fahA.mafft.phylip" as input to the phyML webserver (don't forget to choose "Amino-acids" and enter your email) 2.Run it with the local installation of "phyml.bat" You should end up with a file: "fahA.prank.phylip_phyml_tree.txt"
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24 RAxML Web server: http://phylobench.vital-it.ch/raxml-bb/http://phylobench.vital-it.ch/raxml-bb/ Similar maximum likelihood (ML) methodology as phyML, but much faster Faster results Better results in same run-time
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25 Downloadable RAxML A command-line program: http://icwww.epfl.ch/~stamatak/index-Dateien/Page443.htm (On that page you will also find instructions for running on Windows, and the RAxML manual) http://icwww.epfl.ch/~stamatak/index-Dateien/Page443.htminstructionsmanual easyRAx takes care of some of the RAxML options for you: http://projects.exeter.ac.uk/cee m/easyRAx.html but installation is a somewhat more complex http://projects.exeter.ac.uk/cee m/easyRAx.html
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26 1.Give "fahA.prank.phylip" or "fahA.mafft.phylip" as input to the RAxML webserver (don't forget to tick "Protein sequences" and enter your email) Save the resulting tree file as: "fahA.prank.phylip.raxml"
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27 FigTree: tree visualization and figure creation Manipulate a node Manipulate a clade Manipulate a taxon
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28 1.Open "fahA.prank.phylip_phyml_tree.txt" in FigTree 2.Play around with the different options and make a pretty figure! 1.Find out how to color specific clades, as below 2.Try each of the three options under "Layout" 3.Export a figure in PDF format (File Export Graphic … )
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29 Thanks for your attention and happy phylogeny …
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