5. To open the text file in MEGA, simply go to the “File” tab at
the top right of the page, select “Open a File/Session,” and
navigate to the Notepad file that you previously saved. This
opens a window within MEGA 6 with the name of your
Notepad file as the title.
6. To transform the FASTA formatted text file data into .meg
format, select the “Utilities” drop-down menu on the upper
7. Select “Convert to MEGA format.” A pop-up box in the middle of the screen will prompt you to name and save the file.
When asked for the format of the data, make sure that you
choose FASTA from the drop-down menu in the pop-up.
Once you click “Save,” another box will appear letting you
name and save the file as a .meg file.
8. Now you must close the window with the converted data.
9. To open the data in MEGA 6 so that you can work with it, go
to the File menu, select “Open a File/Session,” and choose
the file that you just saved as a .meg file. You will be
prompted in a pop-up window to choose the type of data;
select “Nucleotide Sequence” (it is the first choice) and then
click on the “OK” button.
10. Another pop-up will appear asking if you are looking at
protein-encoding nucleotides; select “yes.”
11. Now your MEGA6 window should have a box in the upper
righthand corner that has a “T” and an “A” in it.
Choosing the Model
The next step in building a good phylogenetic tree is determining
the appropriate model of nucleotide substitution that should be
applied to the data. Follow the steps below to do this in MEGA 6.
1. Go to the “Analysis” tab of the top menu and click on it to
reveal a drop-down menu.
2. Scroll over the first option in the list “Models” and a submenu will appear.
3. In that submenu, select the first option: “Find the Best DNA/
Protein Models (ML).” A new pop-up will appear in the middle of the screen.
4. Leave the settings as they are and then select the “Compute” option.
5. A pop-up with a progress bar will appear as the different
nucleotide substitution models are tested for the data.
6. After completion, a pop-up box with a table containing the
results of the test will appear.
7. Typically, the best-fitting model will be the first in the table,
but always check to make sure that it is.
For a detailed description of how to build phylogenetic trees in
MEGA 5, please see Newman et al. (2016). Although the versions
are different, the main concepts of tree building in the program remain
There are several different ways that student learning can be assessed
after the process has been completed. Typically, I either have stu-
dents present their work in a scientific poster format or run a
mini-conference in lab or class where each student or group gives
a 10–15 minute presentation. The formats of both the poster and
the presentation are typical of scientific conferences, which include
Introduction, Methods, Results, and Discussion sections. Presenta-
tions like this are relatively easy to evaluate. There are a plethora of
rubrics available online that can be modified for your specific situa-
tion (many scientific societies have these on their websites).
Sample Assessment Questions
To ensure that students are understanding the basics of phylogenetic reconstruction and not just following the instructions blindly,
questions along the following lines may be asked:
1. What kind of information is available in GenBank?
2. Why must the sequences be aligned before you make a phylogenetic tree from them?
3. Why should you run a test to determine the best nucleotide
substitution model for your data set before you use it to build
4. What are the assumptions of the tree-building algorithm you
5. What is the purpose of the bootstrap values and what do they
6. What isolates/strains are most closely related in your tree?
Was this what you expected? Why or why not?
In our sample project, let’s examine the relationship between nine
Ranavirus species (Table 1). Following the steps outlined above,
I created a Notepad file and organized it into a readable FASTA format (Figure 1). I uploaded the file into MAFFT and the sequences
aligned (Figure 2). The aligned sequences were copied and pasted
into a new Notepad document, saved and uploaded into MEGA 6.
The aligned sequences were converted to MEGA format (Figure 3)
and saved as a .meg file. I then opened the .meg file in MEGA and
ran the test for the best-fitting model for nucleotide substitution.
The best fit was T92+I, which means that the Tamura 3- parameter
model with evolutionary invariability is the model that needs to be
chosen when building the phylogenetic tree ( i.e., no gamma distribution should be applied). For the neighbor-joining and minimum
evolution trees, it is not possible to select for evolutionary invariability at different sites, so the default setting (d: transitions and
transversions) was used in the substitutions to include section.
Next, three trees – a maximum-likelihood tree, a neighbor-joining
tree, and a minimum evolution tree – were constructed (Figures 4,
5, and 6). In all three trees, consensus trees were made by eliminating
all branches with < 50% support and each branch was bootstrapped
1000×. The branching pattern for all three trees is extremely similar.
However, there are some notable differences in sister taxa and in the
support of some of the branches. A summary of similarities and differences can be found in Table 2.
Challenges & Outputs
The biggest challenge with this project is getting students to ask
questions that are not overwhelming. Since a large amount of