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Term Project part 3 - Tree Construction

GettingĀ started

The next step is to generate an informative phylogenetic tree containing your sequence(s) using the RDP "Tree" function. This involves selecting a series of sequences to include, generating a tree, then looking at the result so that you can go back & select additional sequences to include (or remove un-needed sequences). Once you've done this back and forth a few times, you'll end up with a nice tree that displays the relationship between your sequence(s) and other organisms.

Get a Java permission error?

Newer versions of Java (a programming language used by the RDP web site) have become increasingly strict about permissions, and so you may get a prompt about whether or not you wish to allow the RDP web site to run a Java program when the site is ready to show you your tree. You need to allow this. You may also need to add the RDP web site to a list of web sites that have this permission. To do this...

On a PC:
Open "Configure Java", which is located in the Java folder from the start menu, go to the Security tab, and select "Edit Site List". Enter "https://rdp.cme.msu.edu/treebuilder/viewer.spr" or (I think) just "https://rdp.cme.msu.edu". You'll need to restart your browser.

On a Mac:
From the Apple menu, select "System Preferences". At the bottom of the window there is an icon for Java - click this. It'll open a "Java Control Panel" window, then select the "Security" tab, and add "https://rdp.cme.msu.edu" to the Exception Site list. Restart your browser, and you should be good to go.

Is(Are) your sequence(s) selected?

  1. Click on the myRDP link in the menubar near the top of the page to bring up the "Overview" page.
  2. Your sequence(s) (they may no longer be at the top of the page) should still be "selected" (with a red "-" box in front of them). By now, the alignment process should be complete and there should be a "1" in the "A" column. If not, wait a while; if the server is busy, it can take a while. You can even come back tomorrow, but if you do you will have to re-select your sequences.

Are the best matches to your sequence(s) selected?

  1. If you just did the "Sequence match" analysis, did you select the best matches it found to your sequence? If not, or if you're not sure, so the Sequence Match again and make sure the sequences it found are selected.
  2. If you DID just do the "Sequence Match", and selected the top 20 matches, you're ready to proceed.

Selecting additional sequences

  1. Click on the "Browsers" link in the menubar at the top of the page. This takes you to the Taxonomic Browser, from which you can select the sequences you want to include in your tree. At the top level, each of the major phylogenetic branches of the Domains Bacteria and Archaea are listed. Notice that only a few Eukarya are listed. Notice the numbers after each name; these tell you the numbers of sequences in that group that you have selected so far, the total number of seqeunces in that group, and the number of search matches (we don't use this), respectively. Notice also that any group you have no selected sequences in is marked by a grey "+" icon. Any group you have some sequences selected from is marked by a half-and-half grey/red +/- icon. If you had ALL the sequences in a group selcted, it would have a red "-" icon.
  2. Scroll to the bottom of the page, and if necessary change the settings to:
    • Strain: Type
    • Source: Isolates
    • Size: >1200
    • Quality: Good
  3. Start with the Phylum you sequence and its matches are in - this will be the phylum marked by the red and grey icon. Click on the name of that phylum to look at the contents of this phylum - usually the classes and orders within the phylum. Groups you have selected sequences in are marked with the grey and red icon. Bore down through the classification to get to the species you've already selected as close matches to your unknown.
  4. Select all of the species (type strains only!) in the genera that were close matches to your sequence.
  5. At the "Lineage" at top of the page, click on the family (usually ending in -aceae), and select representatives from related genera as well.
  6. If you have less than 30 - 50 sequences selected so far, then continue to move up the taxonomy, selcting representatives of each group at each level of the taxonomy as you go, until you reach 40-50 sequences.
  7. Now select an outgroup - usually, this would be an organism just outside the taxonomy of all the other sequences in your tree so far. So, for example, if your selected sequences are all alpha-proteobacteria, you might select a gamma-proteobacterium as an outgroup - E.coli would be good choice.

Generating a draft tree

  1. When you're finished, click on the" Tree Builder" link in the menubar at the top of the page. Make sure the "Alignment model" is set to "RDPX-Bacteria", then change the outgroup to whatever sequence you chose from the outgroup, then click "CREATE TREE".
    If all goes well, the tree will be displayed after a few seconds. In some computer systems, it will ask you if you "trust" this Java app - you do, click OK or whatever to allow it tp run. If your sequence doen't show up in the tree, it may not be finished aligning. Use the commands shown above the tree to adjust the tree to your liking, and print out a copy.

    Here is an example of what the tree might look like:

Generating a final tree

  1. Now, have a close look at this tree; does it make sense? What could you do to make your tree more informative? For example, if the unrelated branches are very deep, and your close relatives all very shallow, might it not be nice to add some representatives into the gap between? Are there branches on your tree that just don't need to be included? Are there large gaps in the tree? Have you picked the right outgroup? Go back to the Browser to add or remove (by clicking the red "-" icon in front of the name) sequences in your selection.
  2. When you're done adjusting the sequences to include in your tree, click on the "Tree" link at the top of the page. Your old tree will still be here; click "Start over" to generate a new tree with your new list. Be sure to use the right outgroup! IIf all goes well, the tree will be displayed after a few seconds.
  3. Scrutinize this improved tree. How can if be improved further? Go back and add or remove sequences in the Browser, generate a new tree, and repeat until you're happy that your tree is as clear and informative as possible. When you've got the tree 'populated' the way you want, use the commands above the tree to adjust it to your liking, and print out or save a digital copy.

    Here is an example of what your tree might look like before you label it:

Annotating your trees

Annotations can be by hand on a printout of your trees, or in a drawing program on your computer. If you do it electronically, be sure to print a copy out when you're finished.

  1. Highlight your sequence(s) in the tree so it can be easily found.
  2. Use brackets or boxes to mark each phylgenetic group in your tree: Genera, Families, Orders, whatever. Be judicious in your labelings; all labels should make your tree easier to understand.
  3. Be sure to to indicate which sequences is your outgroup.

    Here is an example of what your tree might look like when you're finished: