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MSA & Phylogeny

October 09, 2023 |

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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