3. Steps of building a tree (Part II)¶

3.1. Make multiple sequence alignment for Globin gene family¶

Step 1:	Download globin.fasta from Blackboard and perform a MSA using MUSCLE (follow the steps we discussed last week).
Step 2:	Examine the alignment to make sure it is correct and no additional editing is needed.
Step 3:	Export the alignment as a fasta format file on your Desktop. Name it as globin_align

3.2. Find the best substitution model¶

Step 1:

Calculate the distance using different substitution models :a: Select Distance and then Compute Pairwise distance :b: Calculate distance using the following methods

i: No. of Differences

ii: p-distances

iii: Poisson model

Step 2:

Use the same alignment file and build three NJ trees using different substitution models:

a:	No. of Differences
b:	p-distances
c:	Poisson model

Step 3:	Best model based on ProtTest

3.3. Building Phylogenetic trees¶

Step 1:	Click on Phylogeny

Step 2:

Make Neighbor-Joining tree with Bootstrap 500 replicates

A:	What relationships can you see in the tree?
B:	What can you say about the statistical support for each relationship?
C:	What should be the out-group?

Step 3:	Save the tree as a pdf file by clicking on Image button

Step 4:

Build a tree using Parsimony method with 50 Bootstrap replicates (500 will be very slow).

A:	What relationships can you see in the tree?
B:	What can you say about the statistical support for each relationship?
C:	Do you see the same relationships that you saw with NJ tree?

Step 5:	Bayesian inference of phylogeny

Follow this link to MrBayes online server

A: Use the same alignment file

B: In MrBayes select Poisson amino acid model with equal rates of substitution.

C: Select prior parameters (e.g. equal, fixed frequencies for the states; equal probability for all topologies; unconstrained branch lengths).

D: Run 1,000,000 trials for Monte Carlo Markov Chain estimation of the posterior distribution.

E: Obtain phylogram

F: Export tree files

G: View in MEGA software