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. 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.

1. Click on the myRDP link in the menubar at the top of the page to bring up the myRDP Overview page.
2. Your sequences (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.

1. Click on the "Browser" 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 Bacteria ("Phyla") are listed. Notice that only bacterial sequences are included in the RDP; no Eukarya, organelles, or Archaea. 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.
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 Aquificae. Click on the Aquificae name for a list of the sequences in this group in the database. Select a genus: Aquifex is a good choice, since we talked about it in class. In this case, there is only one sequence in this genus that matches your criteria: Aquifex pyrophilus. Check the box in front of the name to add it to your working list.
4. Click on the "Bacteria" link on the Lineage at the top of the page to go back to the top of the hierarchy.
5. Repeat this process with at least the major phyla we're talking about in class:
   
   

   Thermotogae	Deinococcus-Thermus	Chloroflexi	Cyanobacteria
   Chlorobi	alpha-proteobacteria	beta-proteobacteria	gamma-proteobacteria
   delta-proteobacteria	epsilon-proteobacteria	Firmicutes	Actinobacteria
   Planctomycetes	Chlamydiae	Spirochaetes	Bacteroidetes

   
   Feel free to choose the representatives you like, but it'll be easier for you to keep track of and interpret your trees if you try to choose familiar organisms, e.g. Escherichia coli for the gamma-proteobacteria.
6. Lastly, let's get an outgroup. You can use Aquifex if you wish, but perhaps more objective is to use a non-bacterium. On the list under "Domain Bacteria", click on "Archaea Outgroup", then select "Non-type Strain on the options at the bottom of the page, and "Refresh". This brings up Methanocaldococcus jannaschii, which you should select, then click on either the "Root" or "Bacteria" Lineage link at the top of the page.
7. When you're finished, click on the Tree 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 Methanocaldococcus jannaschii (or whatever sequence you chose from the outgroup), then click "CREATE TREE".
8. If all goes well, the tree will be displayed after a few seconds. 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:

Look over your tree - does it make sense based on what the Sequence Match and Classifier results were? Before you turn in your project, highlight your sequence(s) in the tree, and label each of the phyla.

1. Go back to your Sequence Match results summary page, and click the "view selectable matches" link after your sequence name. The best matches are listed taxonomically - select representatives from this list to include in your tree. Make sure to get the single best match, some good matches in that same group, and representatives from the other groups listed. Keep in mind that the Tree program has a limit of 50 sequences, and it gets slower & slower as the number of sequences increases.
2. Click "Save selection and return to summary" at the top of the page.
3. Repeat this with your other sequence, if you have two.
4. 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 working list. Be sure to use the right outgroup!
5. If all goes well, the tree will be displayed after a few seconds. 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 phylum-level branches are all very deep, and your close relatives all very shallow, might it not be nice to add some representatives into the gap between? What about adding a second representative to each phylum branch, as distant a relaative as possible, to "flesh out" each of these branches? Are there branches on your tree that just don't need to be inculded? Have you picked the right outgroup?
6. 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 a copy. Highlight your sequence(s) in the tree, and label each phylogenetic group.

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