A Beginner’s Guide to building a Phylogenetic Tree from Molecular Data with MEGA 11 (Molecular Evolutionary Genetics Analysis)

This guide outlines the steps required to build a phylogenetic tree from molecular data using the MEGA 11 software program, allowing a novice to start with a sequence of interest and build a tree illustrating the evolution of an appropriate set of homologs of that sequence. MEGA is available for use on PCs and Macs.

Source: https://apps4mac.com/mega-app-for-mac/

Steps

• Download and follow instructions to install MEGA — https://www.megasoftware.net/dload_mac_beta
• Download sequence file in FASTA format from NCBI — https://www.ncbi.nlm.nih.gov/
• Perform multiple alignment
• Export alignment into MEGA format
• Construct Phylogenetic tree

For my sequence of interest, I downloaded nucleotide sequences of the homo sapiens COI gene. The mitochondrial Cytochrome Oxidase Subunit 1(COI) gene is one of the most popular markers used for molecular systematics. Fragments of this gene are used to infer phylogenies, particularly the region near the 5’-end.

Sequence Alignment

In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences.

Aligned sequences of nucleotide or amino acid residues are typically represented as rows within a matrix. Gaps are inverted between the residues so that identical or similar characters are aligned in successive columns.

Alignment can be used when you want to perform phylogenetic studies, i.e when you want to study the evolutionary relationships between the sequences of interest.

1. Starting from the main MEGA window, select Data
2. Click on ‘Open a File/Session’ from the launch bar
3. Select downloaded sequence FASTA file to open
4. Click on ‘Align'
5. Click on the Muscle icon
6. Click ‘Align DNA'
7. Select All
8. Click ‘OK'
9. MUSCLE alignment might take a while…
10. Save

Building a Phylogenetic Tree

A phylogenetic tree is a branching diagram or a tree showing the evolutionary relationships among various biological species or other entities based upon similarities and differences in their physical or genetic characteristics. All life on Earth is part of a single phylogenetic tree, indicating common ancestry. https://en.m.wikipedia.org/wiki/Phylogenetic_tree

1. Go to Data
2. Click on ‘Open a File/Session’
3. Select previously saved aligned sequences in .mas format
4. Click on ‘Analyze'
5. A dialog will appear asking ‘Protein-coding nucleotide sequence data?’ Click Yes
6. The aligned .mas file appears in MEGA window
7. Click to open
8. Click on the ‘Save’ icon to export current data to a preferred format i.e .meta
9. Go to Phylogeny on the MEGA window
10. A dialog appears ‘Would you like to use the currently active data (sequence.mas)?’, Click Yes
11. Click ‘OK'
12. Phylogenetic tree is created. Save tree image to a file.