Phylogenetic Analysis using PHYLIP – B.Sc. Bioinformatics Practical

 

Phylogenetic Analysis using PHYLIP – B.Sc. Bioinformatics Practical

Aim of the Experiment

To construct a phylogenetic tree and analyze evolutionary relationships among organisms using the PHYLIP software package.

Principle

Phylogenetic analysis determines evolutionary relationships based on similarities and differences in DNA or protein sequences.

The software PHYLIP (Phylogeny Inference Package) uses statistical methods such as:

• Distance methods (Neighbor-Joining)
• Maximum Parsimony
• Maximum Likelihood

Sequences are aligned, converted into distance matrices, and used to generate phylogenetic trees.

Requirements

• Computer system
• Sequence data (DNA or protein in FASTA format)
• Software:
  • PHYLIP
• Optional tools:
  • Sequence alignment tool (e.g., ClustalW)

Step-by-Step Procedure

Part A: Sequence Retrieval

Step 1: Retrieve Sequences

• Visit NCBI
• Download sequences (FASTA format) of related species

Part B: Sequence Alignment

Step 2: Perform Multiple Sequence Alignment

• Use alignment tool (e.g., ClustalW)
• Align sequences
• Save aligned file

Part C: Preparing PHYLIP Input

Step 3: Convert to PHYLIP Format

• PHYLIP requires specific format:

4 60
Seq1 ATGCGT...
Seq2 ATGAGT...
Seq3 ATGCCC...
Seq4 ATGTGT...

• First line: number of sequences and length

Part D: Phylogenetic Tree Construction

Step 4: Open PHYLIP Software

• Launch PHYLIP

Step 5: Generate Distance Matrix

• Select program: DNADIST (for DNA) or PROTDIST (for protein)
• Input aligned file
• Generate distance matrix

Step 6: Construct Tree

• Use:
  • NEIGHBOR (Neighbor-Joining method)
• Input distance matrix
• Generate tree file

Step 7: Bootstrap Analysis (Optional)

• Use SEQBOOT
• Generate replicates
• Assess reliability of tree

Part E: Tree Visualization

Step 8: Visualize Tree

• Use:
  • DRAWGRAM (tree diagram)
  • DRAWTREE (unrooted tree)

Typical Phylogenetic Tree

5

Step 9: Interpret Tree

• Identify:
  • Common ancestors
  • Branch lengths
  • Evolutionary relationships
  • Closely related species

Step 10: Record Observations

• Number of sequences
• Method used
• Tree type (rooted/unrooted)
• Key relationships observed

Result

A phylogenetic tree was successfully constructed using PHYLIP showing evolutionary relationships among selected organisms.

 Precautions

• Use properly aligned sequences
• Ensure correct PHYLIP format
• Avoid gaps/errors in alignment
• Use appropriate evolutionary model

Applications

• Evolutionary biology studies
• Species classification
• Comparative genomics
• Disease evolution tracking
• Conservation biology

 Viva Voce Questions (with Answers)

1. What is phylogenetic analysis?
   Study of evolutionary relationships.
2. What is PHYLIP?
   A phylogeny analysis software package.
3. What is Neighbor-Joining method?
   Distance-based tree construction method.
4. What is a phylogenetic tree?
   Diagram showing evolutionary relationships.
5. What is bootstrapping?
   Method to test reliability of tree.
6. What is alignment?
   Arrangement of sequences to identify similarity.
7. Which database provides sequences?
   NCBI
8. What is a rooted tree?
   Tree with a common ancestor.