From basic Concepts to Advanced applications Molecular Evolution & Phylogeny By Ofir Cohen The Bioinformatics Unit G.S. Wise Faculty of Life Science Tel Aviv University, Israel Feb

2 of 20 Part II: Tools

3 of 20 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 Tools - Alignments

4 of 20 MAFFT Web server (& download option): Efficiency-tuned variants  quick & dirty or slow but accurate Nucleic Acids Research, 2002, Vol. 30, No © 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 * Tools - Alignments

5 of 20 Choosing a MAFFT strategy quick & dirty slow but accurate Tools - Alignments

6 of 20 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 Tools - Alignments

7 of 20 PRANK Tools - Alignments

8 of 20 Classical alignment errors for HIV env Tools - Alignments CLUSTALW PRANK

9 of 20 PRANK Web server: Tools - Alignments

10 of 20 PRANK output If you need a different format – copy the results to the READSEQ sequence converter: Tools - Alignments

11 of 20 Downloadable PRANK  PRANK: A command-line program interface  PRANKSTER: A program with graphical user interface Tools - Alignments

12 1.Download the sequence files from the web-site Open "fahA.fas" in Notepad/Browser – 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 run the "auto" 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“ 5.If you have time, run PRANK web server (1)

13 of 20 Trees Reconstruction Methods

14 of 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 Tools - Trees

15 of 20 PhyML The most widely used maximum likelihood (ML) program Web server (& download) : Tools - Trees

16 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 and name) (2)

17 of 20 RAxML Web server: Similar maximum likelihood (ML) methodology as phyML, but much faster  Faster results  Better results in same run-time Tools - Trees

18 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 ) Save the resulting tree file as: "fahA.prank.phylip.raxml" (3)

19 of 20 FigTree: tree visualization and figure creation Manipulate a node Manipulate a clade Manipulate a taxon

20 1.In case tree are not ready yet… download tree from website 2.Open "fahA.prank.phylip_phyml_tree.txt" in FigTree 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" 4.Export a figure in PDF format (File  Export Graphic…) (4)

21 of 20 Final Questions… Thanks for your attention