MCB 272 : Phyml and Puzzle

You should answer the questions in red!

Preparation:

• both Phyml and tree-puzzle are already installed. If you install them on your own computer, you need to add their location to the PATH.
  To start the programs type puzzle or phyml at the commandline.
• You can either work on the files provided, or use your own dataset. For exercise one and two, you need to use the same dataset
  

1) ML on protein sequences using phyml

a) For a dataset of your choice or here use phyml and calculate the tree with the highest likelihood using a model for Among Site Rate Variation that has a proportion of invariant site estimated from the data, and that describes the remaining sites with 4 rate categories that are a discrete approximation of a continuous Gamma distribution whose shape parameter is estimated from the data.

• What dataset did you use?
• What was the estimated proportion of invariant sites?
• What was the estimated shape parameter?
• What does a shape parameter of this magnitude signify?

2) Using the same dataset and the same model in treepuzzle.
Use the tree from (1) as usertree (option k). Take you time in selecting the correct model ! (four rate categories plus invariant sites).

• Did all sequences pass the test for homogeneous composition?
• What was the proportion of invariant sites?
• What shape parameter was estimated?

2B) repeat the analyses from above, but estimate if a strict molecular clock is compatible with the data (option z). select the pinvar and alpha from the previous analysis.
For a large data set, this might take some time (about 20 minutes for archaea_euk.phy). Start it, once it is running, open a new ssh connection , and qrsh to a different node (preferred), alternatively you can send the process (running puzzle) into background. For the latter, stop the process in foreground by pressing down <ctrl> and <z> simultaneously. Then restart the process in background by typing
bg %1

While waiting, continue with 3 below.

• Where did treepuzzle place the root?
• Was the clock-model rejected? What was the difference in 2log(likelihood)?
• What else can you learn from the outfiel?

3) ML mapping

Use a dataset of your choice or use testseq5.phy.

The latter file contains vacuolar/archaeal ATPases from the following pro- and eukaryotes.

Daucus carota, Arabidopsis thaliana, Gossypium hirsutum are plants;

Acetabularia acetabulum is a green and Cyanidium caldarium is a red algae

Mus, Homo, Bos (mammals) Gallus (bird), Drosophila, Aedes (insects) are animals

Saccharomyces, Candida, Schizosaccharomyces, and Neurospora are fungi

Dictyostelium discoideum, Entamoeba, Plasmodium falciparum, Trypanosoma, Giardia are protists or protozoa

Sulfolobus acidocaldarius (70oC),Archaeoglobus fulgidus (83oC), Methanosarcina barkeri (30-37 oC), Methanosarcina mazeii (37oC), Methanococcus jannaschii (80oC), Haloferax volcanii (37oC), Halobacterium salinarium (ca37oC), Methanobacterium thermoautotrophicum (60-65oC), Desulfurococcus sp. (85-90 oC), Thermococcus sp (75+ oC) (archaea),

Enterococcus hirae (37oC), Borrelia burgdorferi (33-37oC), Thermus thermophilus (70-80oC), Deinococcus radiodurans (30oC) (Bacteria) are prokaryotes. (Usually Bacteria have an F- and not an A-ATPase. The bacteria probably obtained the archaeal/vacuolar type ATPase through horizontal gene transfer.)

The prokaryotes can be considered as outgroup for the eukaryotes.

Design a question that you can address using ml mapping.
For example:
Are the plants, green algae and red algae a monophyletic group?
Is Giardia the deepest branch among the eukaryotic sequences?
Do the animals group with the fungi, or do the fungi go with the plants?
After you have a question, figure out what your 4 groups should be (see the tree below). Only then start the program.

Before entering the final y to run the program, select that you want to run this on all possible quartets (option n, enter 0) with one sequence from each group.
Select a gamma distribution with 8 classes, enter the shape parameter as 0.6. 

This is an exercise in thinking and concentration. If you think too little, you will have to go back to the start several times.

The eukaryotic part of the tree calculated with phyml is as follows, the whole tree is here:

• What question did you ask? What is your answer?

4) Puzzleboot (only if you have time, and you want to try this).

Use a file of your choice (needs to be phylip formated).

Copy puzzleboot_mod.sh and puzzle.cmds into the directory where you want to run the script. .
CAREFUL: puzzleboot removes all outfiles and outtrees from the directory it runs in!

change permission of puzzleboot_mod.sh
chmod 755 puzzleboot_mod.sh

Change the responses in puzzle.cmds to that they correspond to the model you want to use. You probably want to fix pinvar and alpha to values you already estimated!

Hint: to help trouble shooting, run the puzzleboot first on the original data (one file), move to the bootstrap sample only after you are happy with the commands file.

Run seqboot on your data. To execute the script type:
./puzzleboot_mod.sh your_file_name_that_contains_the_bootstrappedsamples

The output consists of 100 distance matrices, run them through neighbor or fitch. You need to select the m option.

You get the support values with consense.