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MSA & Phylogeny

October 09, 2023 2022

Multiple Sequence Alignment (MSA) and phylogenetic tree reconstruction are core tasks in bioinformatics. This guide outlines the workflow using tools like MEGA.

Multiple Sequence Alignment (MSA)

1. Open MSA Tool: Launch your preferred MSA software (e.g., MEGA).
2. Reference Sequence: Set the reference sequence (e.g., “AFUA_5G01230”).
3. Initiate Alignment: Go to Align > Edit or Build Alignment.
4. Create New: Select Create a new alignment and choose Protein as the sequence type.
5. Add Sequences: Paste your sequences from a FASTA or text file into the alignment window.
6. Clean Up: Ensure all sequences are correctly labeled and delete any empty placeholder entries.
7. Perform Alignment: Navigate to Alignment > Align by ClustalW or Align by MUSCLE.
   • If prompted, select Select All and click OK.
   • Use the default alignment options for a standard analysis.
8. Export: Once complete, go to Data > Export Alignment and save it in the MEGA format (.meg).

MUSCLE Default Parameters

When using MUSCLE, the following parameters are typically used for protein alignments:

• Gap Penalties:
  • Gap Open: -2.90
  • Gap Extend: 0.00
  • Hydrophobicity Multiplier: 1.20
• Memory & Iterations:
  • Max Memory: 2048 MB
  • Max Iterations: 16
• Advanced Options:
  • Cluster Method (Iter 1, 2): UPGMA
  • Min Diagonal Length (Lambda): 24

Phylogenetic Tree Reconstruction

1. Start Analysis: Navigate to Phylogeny > Construct/Test Neighbor-Joining Tree.
2. Load Data: When prompted to use current data or preserve existing sessions, select No to ensure you are starting fresh with the newly aligned data.
3. Configure Parameters: In the reconstruction window, use the following settings for reliable results:

Reconstruction Parameters

• Analysis:
  • Scope: All selected taxa
  • Statistical Method: Neighbor-Joining
• Phylogeny Test:
  • Test Method: Bootstrap method (Recommended: 100 or 1000 replicates)
• Substitution Model:
  • Substitution Type: Amino Acid
  • Model/Method: Jones-Taylor-Thornton (JTT) model
• Rates and Patterns:
  • Rates Among Sites: Uniform Rates
  • Pattern Among Lineages: Homogeneous
• Data Subset Treatment:
  • Gaps/Missing Data: Pairwise Deletion
• System Resources:
  • Number of Threads: 4 (or adjust based on your CPU)

1. Compute: Click OK to begin the reconstruction. The resulting tree will provide insights into the evolutionary relationships between your sequences.

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