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How to generate phylogenetic tree using the CART function

Programs needed. You need program(s) to generate multiple sequence alignment and phylogenetic trees.We recommend the MEGA program, which can be freely obtainable athttp://www.megasoftware.net/. Identifying a strain. We will use a Genbank entry (EF379568) as an example. Let's assume that this is yo

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How to generate phylogenetic tree using the CART function

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    1. How to generate phylogenetic tree using the CART function

    2. Programs needed You need program(s) to generate multiple sequence alignment and phylogenetic trees. We recommend the MEGA program, which can be freely obtainable at http://www.megasoftware.net/

    3. Identifying a strain We will use a Genbank entry (EF379568) as an example. Let’s assume that this is your own sequence.

    5. Identify Paste your own sequence in “Identify” page

    6. You should be re-directed to the “Results” page. To add your query sequence to the cart, click [Add query to cart] button. You will be re-directed to the “Cart” page.

    7. Now your cart has your query sequence. Go to the “Results” page to view the identification results again

    8. Now, click [Add to cart] button. This will add all phylogenetic neighbors to the cart. You will be re-directed to the “Cart” page.

    9. Now, your cart contains sequences of your query and phylogenetic neighbors.

    10. In addition, you can add sequences of a genus of your choice into the cart. Click the green button to add all species in the genus Marinobacterium (arrow below). You can add species in the other genera by clicking green buttons.

    11. Download sequence file (fasta format), and save it on your local hard disk. I will name our file “test.fasta”.

    12. Open sample.fasta with MEGA program. Align sequences by choosing “Alignment/Align by ClustalW”. Note that the EzTaxon entries are labeled (flanked) with “zZ”.

    13. After multiple alignment, YOU NEED TO TRIM ambiguously aligned positions. Select region (to be deleted) while holding left mouse button pressed. Then, choose “Edit/Delete” to delete selected region. Generally, trim both ends and internal regions, if necessary.

    14. Save trimmed alignment as “sample_align.meg”. Generate phylogenetic trees as you wish. MEGA offers the following treeing algorisms. Neighbor-joining Minimum evolution Maximum parsimony Bootstrap analysis (1,000 times recommended) A neighbor-joining tree with 1,000 bootstrap analysis is given at the left side. This tree is labeled with “zZ” flanked accession numbers by EzTaxon server. You can change the labels using various formats. To do this, first save the tree as a “newick” file. Choose “File/Export Current Tree(Newick)” in MEGA treeview mode. Save/export your tree as “sample.nwk“. Go back to EzTaxon server.

    15. To change labels in tree or any other text files, choose “Replace accession”. Then, select a format of your choice. We will select IJSEM format to convert “sample.nwk“ file. Save converted file as “sample.tre” and open it using MEGA or other tree viewing programs.

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