Spring 2017
BIOL 312: Microbiology
A Town on Fire
Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire /
Instructor: Dr. Tammy Tobin
Susquehanna University
E-Mail:

Team Application Activity #4: Phylogenetic Analysis of Bacterial Communities.

Names of Team Members:

Introduction: During the last class period, you developed a phylogenetic tree from your metagenomics data, and then performed alpha diversity analyses. Today, you will complete your metagenomics analyses by using phylogenetic analysis to track community response to environmental parameters.

Instructions

We will now move to the analysis of our phylogenetic trees using FigTree. ( Your main goal in this analysis is to see if your taxon of interest is predominantly associated with a particular environmental parameter...and to get a feel for just how massively huge these metagenomic trees really are!

  1. Open the FigTree application (in Applications). You should get a window like the one shown to the left.
  2. In the top menu, click on “File”and then “Import Annotations”. Choose the rep_set1_tax_assignments.txt file that you will find in a folder entitled centralia_assigned_taxonomy in your Centralia_Case_Study folder on your desktop. This file contains the taxonomic information you will need, and tells FigTree which sequences came from each sample site.
  3. Import your tree file by clicking on “File”and then “Open.”Choose the rep_set1_aligned_pfiltered.tre file that you will see in the filtered_alignments folder in your Centralia_Case_Study folder.
  1. If all goes well your tree window will appear something like this:

  1. Next you will assign taxonomy. Click on Tip Labels,then under “Display”choose “unassigned”and under “Colour By”choose “1”. You have just told the program to label the tips of each branch with the taxonomic assignment, and to color the tip labels according to sample ID (borehole 1, 2 or 3). Your window should now look like the one below. If you click on Setup: Colour under the Tip Labels menu you will see a key that tells you which color represents which sample site. In this case, sample 1 is red, etc.

  1. As you have probably noticed, that is a huge tree! You are only going to be analyzing a small portion of it, but first you should change the view so you can see the taxa better. In the upper portion of the left menu you will see ‘Expansion’. Slide that bar back and forth. And the taxa will spread and contract vertically. You want to be able to see the taxa clearly, but not spread the tree out too much.
  1. Now search for your taxon by typing its name…or a portion of its name…in the upper right search window as shown below. If your taxon is there, you can scroll up and down and it will be highlighted as shown below for Sphingobacteria:

  1. You will probably need to “Zoom”(upper left menu) in a bit now to better see the entire portion of the tree with your taxon before completing the next step.
  2. Find a logical clade that includes your taxon and its nearest relatives. Select that clade by clicking on its root. The whole clade should be highlighted. If the wrong taxa are highlighted, click on a different root until you get what you want. Under “File”click on “Copy”.
  3. Open a new tree (File –Open), click on the empty window and paste your clade into the window (Edit –Paste). You should now see your subtree. Unfortunately, your tip labels and colors will all be gone, so reset those, and you should see something that looks like this:

  1. For some reason, the colors of the sample sites change as compared to the sample sites in your large tree. To see what the new colors represent, click on Setup: Colour under Tip Labels. You will see a key with the colors and their sample location designation. I recommend also changing from HSB spectrum to Fixed Colours (drop down menu under the key). The fixed colors are primary colors and a bit easier to see.
  2. Once you have your tree the way you want it, save it to your thumb drive by choosing ‘File -Save As’and naming it anything you want. The default file type is just fine. Once you have saved the file, you can export it as a .jpg that can be inserted into your final papers and presentations by going to ‘File –Export JPEG’and again saving to your thumb drive with a file name that makes sense to you.
  3. Congratulations!!! You are done!!