How to Construct a Phylogenetic Tree from Nucleotide Sequences

🧬 How to Construct a Phylogenetic Tree from Nucleotide Sequences – Explained with Examples

🔍 Step 1: Collect and Download Nucleotide Sequences

🔧 Tool: NCBI GenBank

🐟 Example: Let’s say we want to study the evolutionary relationship of the Cytochrome c oxidase I (COI) gene among 5 fish species:

• Danio rerio (Zebrafish)

• Oreochromis niloticus (Tilapia)

• Carassius auratus (Goldfish)

• Oncorhynchus mykiss (Rainbow trout)

• Salmo salar (Atlantic salmon)

✅ Steps:

1. Go to NCBI Nucleotide

2. Search: COI gene [species name]

3. Click on a record, then choose Send to > File > FASTA

4. Repeat for all 5 species.

💾 You now have a FASTA file with sequences like:

>Danio_rerio_COI
ATGGCA...
>Oreochromis_niloticus_COI
ATGACA...
...

🧬 Step 2: Multiple Sequence Alignment (MSA)

🔧 Tool: Clustal Omega

✅ Steps:

1. Paste all 5 FASTA sequences into Clustal Omega

2. Click “Submit”

3. Download the alignment as Clustal (.aln) or FASTA (.fasta) format

🔍 Example Output (simplified):

Danio:         ATG-GCACTTGA---
Tilapia:       ATGGGCACTTGAGGA
Goldfish:      ATGGGCATTGGAGGA
...

This shows how similar or different the gene sequences are across species.

🧠 Step 3: Select a Model of Evolution

🔧 Tool: MEGA Software

1. Open MEGA > Models > Find Best DNA Model

2. Load the aligned file

3. Choose the best-fit model (e.g., Tamura-Nei, Jukes-Cantor, Kimura 2-parameter)

🧠 Why?

These models estimate mutation rates—some account for transitions/transversions, GC bias, etc.

🌳 Step 4: Construct the Phylogenetic Tree

🔧 Tool: MEGA

✅ Steps:

1. Open MEGA > Phylogeny > Construct/Test NJ Tree

2. Load aligned file

3. Choose method:

   • Neighbor-Joining (fast)

   • Maximum Likelihood (accurate)

4. Enable Bootstrap (1000 replicates recommended)

5. Run and view the tree

🌿 Example Tree (simplified output):

          ┌── Danio rerio
     ┌────┤
     │    └── Carassius auratus
─────┤
     │    ┌── Tilapia
     └────┤
          └── Oncorhynchus + Salmo (clustered)

🧠 Interpretation: Danio and Goldfish are closely related; Salmon and Trout cluster together, which makes sense based on evolutionary history.

🎨 Step 5: Edit and Visualize Tree

Tools:

• Use MEGA for basic visualization

• Use iTOL for interactive, colorful trees

✅ Add:

• Species names

• Bootstrap values

• Color groups

• Metadata (habitat, geography)

📂 Step 6: Export and Interpret Results

1. Save tree as .nwk, .png, or .svg

2. Interpret:

   • Clades = close evolutionary groups

   • Bootstrap > 70% = reliable branches

3. Use in:

   • Research

   • Presentation

🧾 References for Learning:

• Tamura, K., Stecher, G., Kumar, S. (2021). MEGA11: Molecular Evolutionary Genetics Analysis

• NCBI GenBank: https://www.ncbi.nlm.nih.gov/genbank/

• Clustal Omega MSA: https://www.ebi.ac.uk/Tools/msa/clustalo/

• iTOL Tree Viewer: https://itol.embl.de/

📌 Final Tips:

• Always align homologous genes

• Avoid partial or low-quality sequences

• Use more species for better phylogenetic resolution