1rst Daniel Jouvance-UPMC International School in Marine Biology (January 2011, Las Cruces, Chile) Genomes, phylogeny, and lateral gene transfer Computer Lab Yves Desdevises You can download programs, files, documents, and instructions from the following link: http://desdevises.free.fr/ISMB2011 Create a folder, and subfolders, on your computer to place input and output files, they tend to multiply very rapidly! Of course, you may use any other software you are familiar with. Take your time and do not hesitate to use help, example files and manuals from the various programs you will use. 1. Phylogeny from single and concatenated genes Software: SeaView, FigTree You can download from the website sequences from prasinovirus and microalgae. These sequences come from various genes: DNA Polymerase (DPO), Proliferating Cell Nuclear Antigen (PCNA), small and large subunits of the Ribonuclease Reductase (SSRR and LSRR), and Thymidine Synthase (TS). Another file contains the concatenation of these five genes. With SeaView (which contains a PhyML function for ML analysis, and uses a Phylip module for parsimony): -

Open SeaView, open the alignment

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You can select species (use the mouse to create a group: Species ➙ Create group) and/ or sites (Site ➙ Create set, then select with the mouse at the bottom of the window)

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You can try different tree reconstructions by varying methods and parameters via the Trees menu. Validate your tree using a bootstrap analysis. For model-based analyses (e.g. ML), you can set the model and parameters you obtained from ProtTest (http://darwin.uvigo.es/ software/prottest.html)

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Save your tree(s) via File in the tree window. You can save trees as text (Newick or Phylip format, that can be read with programs like FigTree) or graphic files (pdf). Do not forget to root your tree and show clade support values

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Compare topologies and support values for single and multiple gene trees

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2. Lateral Gene Transfer Software: SeaView, FigTree, Blast (via NCBI), Jane -

Find if the 4 different genes contained in the file CandidatesLGT.rtf have undergone lateral gene transfer (students can split in 4 groups to investigate the evolution of given genes). You need first to blast these genes against the nr dataset in GenBank: go to http://blast.ncbi.nlm.nih.gov/ and choose protein blast

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Copy the first best blast hits (BBH) in a text file in Fasta format with the candidate gene

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Choose some reference sequences (e.g. from the 3 domains of life) in the BBH list, or find some by looking in GenBank (http://www.ncbi.nlm.nih.gov/protein/), and add them to the Fasta file

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Open your Fasta file in SeaView, align the sequences (Align ➙ Align all, ClustalW2 or Muscle can be selected in the options). [Of course you can align the sequences and make the phylogenetic trees using other program]

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If needed, you can improve the alignment manually (Props ➙ Allow seq. Editing), and/or using GBlocks (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) by first exporting the alignment in Fasta format.

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Make a phylogenetic tree based on your alignment using ML or Distance methods in SeaView via Trees. Note that you can easily select sites and/or species via Site ➙ Create set and Species ➙ Create group

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By looking at the tree, is there any evidence of lateral gene transfer for the candidate sequence? From the host or another taxon?

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You can use this tree and a reference tree (such as the tree built for the DNA polymerase gene, Ref_dpo_tree.tre (that can be opened in SeaView) from the alignment Ref_dpo.tree.fasta) and the cophylogenetic analysis program Jane to infer a LGT scenario. For example, you can map a tree containing a candidate gene for a LGT, such as Acetolactate Synthase (AcS) or HSP70, on a reference tree built from DNA polymerase. You may have to use a tree with selected taxa, that you can built from the alignment in SeaView. To use Jane, open an association file such as file HSP70_DPO_LGT_Jane.nex (File ➙ Open Tree). You can modify event costs (such as duplication and transfer) via Settings ➙ Set Costs, and maximum transfer distance via the same menu. Population size and Number of Iterations are parameters of the search algorithm (the more the better, but the longer). Once solutions are computed, open them by clicking on the corresponding line

3. Evolution of a gene family (MCP) Software: SeaView, FigTree You will use the clp.nex sequence dataset, which contains capsid-like protein (clp) sequences from prasinoviruses and related phycodnavirus. The purpose is to study the evolution on the clp family across prasinoviruses. -

Build a phylogenetic tree (or several) as in 1.

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Interpret the resulting tree regarding the evolution of the clp gene family

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