Step-by-Step Guide: Introduction to Phylogenetic Analysis with MEGA

Author: Dr. Itunuoluwa Isewon

Email: itunu.isewon@covenantuniversity.edu.ng

You have been provided with a fasta file called Aspergillus18S.fasta and Yeast18S.fasta

πŸ“₯ Dataset: Download the file for Aspergillus18S here

Download the file for Yeast18S.fasta here

Step 1: Import sequences into MEGA

  1. Launch MEGA X.
  2. File β†’ Open a File/Session… β†’ select Aspergillus18S.fasta.
  3. When prompted, choose Align.
  4. MEGA opens the Alignment Explorer showing your unaligned sequences.

Step 2 β€” Multiple Sequence Alignment (ClustalW)

In Alignment Explorer:

  1. Align β†’ Align by ClustalW .
  2. Click Options and set/confirm the parameters below, then Compute.

After alignment: β€’ Scroll through; 18S has conserved stems and variable loopsβ€”expect gaps mostly in variable regions.

β€’ If the first/last ~10–30 bases are gappy, select and Edit β†’ Delete Selected Sites (or Mask), so they don’t add noise.

Save the alignment: Data β†’ Export Alignment β†’ MEGA format (.meg) and also FASTA for records.

Step 3 β€” Build a Neighbor-Joining (NJ) tree

  1. Close Alignment Explorer (save when prompted).
  2. In the main MEGA window, choose Phylogeny β†’ Construct/Test Neighbor Joining Tree.
  3. In Analysis Preferences (the panel like your screenshot), set:

Analysis β€’ Scope β†’ All Selected Taxa (or choose a subset beforehand).

β€’ Statistical Method β†’ Neighbor-joining.

Phylogeny Test (support)

β€’ Test of Phylogeny β†’ Bootstrap method.

β€’ No. of Bootstrap Replications β†’ 1000.

Rule of thumb: β‰₯70% = moderate support, β‰₯90% = strong.

Substitution Model (Distances)

β€’ Substitutions Type β†’ Nucleotide.

β€’ Model/Method β†’ Maximum Composite Likelihood (MCL).

Alternatives: Tamura-Nei (TN93) or Kimura 2-parameter (K2P); try these if you want sensitivity analysis.

β€’ Substitutions to Include β†’ d: Transitions + Transversions (include both).

Rates and Patterns β€’ Rates among Sites β†’ Uniform Rates (appropriate for conserved 18S). If analyzing more variable loci (e.g., ITS), consider Gamma Distributed; MEGA will estimate the shape (Ξ±).

β€’ Pattern among Lineages β†’ Same (Homogeneous).

Data Subset to Use

β€’ Gaps/Missing Data β†’ Pairwise deletion (keeps more sites; good for rRNA with localized gaps). Complete deletion is stricter but can remove lots of signal.

β€’ Select Codon Positions β†’ Tick Noncoding Sites (coding positions have no effect for 18S).

  1. Click Compute to infer the NJ tree.

Step 4 β€” Inspect, root, and annotate

β€’ Rooting: If your file contains a clear outgroup (e.g., a non-Aspergillus fungus), Tree Explorer β†’ Tree β†’ Root on that taxon. If not, use Midpoint rooting.

β€’ Show bootstrap values: In Tree Explorer, View β†’ Display Option β†’ Show β†’ Bootstrap values.

β€’ Tidy labels: Edit β†’ Replace tip labels can shorten long headers; you kept accession+species, which is perfect.

β€’ Collapse weak nodes: Optionally collapse branches with support <50–70% to simplify.

Step 5 β€” Export & document

β€’ File β†’ Export Current Tree (Newick) β†’ Aspergillus18S_NJ.nwk.

β€’ Image export: File β†’ Export Image (PNG/PDF/SVG) at 300–600 dpi.

β€’ Save your .meg project so you can reopen without re-aligning.

Now try to repeat this process using the yeast18S.fasta