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


Step 2:  Use the same alignment file and build three NJ trees using different substitution models:


Step 3:  Best model based on ProtTest 

3.3. Building Phylogenetic trees¶
Step 1:  Click on Phylogeny 

Step 2:  Make NeighborJoining tree with Bootstrap 500 replicates


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).


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