FASTA Sequence Analysis and Pairwise Sequence Alignment. Kale PB (Molecular Biology and Biotechnology)

Practical 2: FASTA Sequence Analysis and Pairwise Sequence Alignment

Kale PB (Molecular Biology ology and Biotechnology)

🎯 Aim: To analyze nucleotide sequences in FASTA format and perform Pairwise Sequence Alignment using the Needleman-Wunsch (Global Alignment) and Smith-Waterman (Local Alignment) algorithms via EMBL-EBI tools.

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📥 Sequence Dataset & Quick Links

Use the sequences retrieved in Practical 1 corresponding to the following NCBI accession numbers. You can access the direct NCBI entries below:

NCBI: PP948896.1 NCBI: PP948895.1 NCBI: PP948894.1
NCBI: PP948893.1 NCBI: PP948892.1 NCBI: PP948891.1

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💻 Requirements

• Internet-enabled computer
• Web browser
• Text editor (e.g., Notepad, VS Code, or TextEdit)
• Saved FASTA files from Practical 1 (PP948896.1.fasta through PP948891.1.fasta)

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🛠️ Procedure

Step 1: FASTA Sequence Inspection and Parsing

1. Open your downloaded FASTA file PP948896.1.fasta in a plain text editor.
2. Identify the two essential structural components of a FASTA file:
   • Header line: Begins with a > character followed by the accession number and description.
   • Sequence data: The lines following the header containing single-letter nucleotide codes (A, T, C, G, N).
3. Count the sequence length (total base pairs) manually or using your text editor's character counter (excluding header line and line breaks).
4. Calculate the basic nucleotide composition for PP948896.1 by counting occurrences of A, T, C, and G.
5. Calculate GC content using the formula:

GC % = [ (Count of G + Count of C) / Total Base Pairs ] × 100

6. Repeat this composition calculation for sequence PP948895.1.

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Step 2: Global Pairwise Alignment using Needleman-Wunsch (EMBL-EBI EMBOSS Needle)

1. Open your web browser and navigate to the EMBOSS Needle tool: https://www.ebi.ac.uk/Tools/psa/emboss_needle/
2. Select DNA as the sequence type.
3. Under STEP 1 - Enter your input sequences:
   • Sequence 1: Paste the contents of PP948896.1.fasta or upload the file.
   • Sequence 2: Paste the contents of PP948895.1.fasta or upload the file.
4. Keep the default parameters:
   • Matrix: DNAfull / EDNAFULL
   • Gap Open Penalty: 10.0
   • Gap Extend Penalty: 0.5
5. Click Submit.

📷 Representation of the EMBOSS Needle submission form showing Sequence 1 and Sequence 2 fields entered with input FASTA records from NCBI

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Step 3: Local Pairwise Alignment using Smith-Waterman (EMBL-EBI EMBOSS Water)

1. Open a new tab and navigate to the EMBOSS Water tool: https://www.ebi.ac.uk/Tools/psa/emboss_water/
2. Select DNA as the sequence type.
3. Enter PP948896.1.fasta as Sequence 1 and PP948895.1.fasta as Sequence 2.
4. Keep default parameters (Matrix: EDNAFULL, Gap Open: 10.0, Gap Extend: 0.5).
5. Click Submit.

📷 Representation of the EMBOSS Water interface showing results.

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Step 4: Compare Alignments Across Multiple Accession Pairs

Perform pairwise alignments (both Needle and Water) for the following additional pairs using your local files:

• Pair A: PP948896.1 vs PP948895.1
• Pair B: PP948894.1 vs PP948893.1 
• Pair C: PP948892.1 vs PP948891.1

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📊 Step 5: Record the Observations

Record your FASTA analysis and pairwise alignment outputs in the observation tables below:

Table 1: Sequence Composition Summary

Accession Number	Sequence Length (bp)	Count (A)	Count (T)	Count (G)	Count (C)	Calculated GC Content (%)
PP948896.1
PP948895.1
PP948894.1
PP948893.1
PP948892.1
PP948891.1

Table 2: Pairwise Alignment Results Comparison

Sequence Pair	Algorithm	Score	Alignment Length	Identity (%)	Similarity (%)	Gaps (%)
PP948896.1 vs PP948895.1	EMBOSS Needle (Global)
PP948896.1 vs PP948895.1	EMBOSS Water (Local)
PP948894.1 vs PP948893.1	EMBOSS Needle (Global)
PP948894.1 vs PP948893.1	EMBOSS Water (Local)
PP948892.1 vs PP948891.1	EMBOSS Needle (Global)
PP948892.1 vs PP948891.1	EMBOSS Water (Local)

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🎓 Expected Learning Outcomes

After completing this practical, students should be able to:

• Identify and manually validate the header structure and sequence body of a FASTA file.
• Calculate nucleotide counts and GC content from raw FASTA strings.
• Differentiate between Global Alignment (Needleman-Wunsch) and Local Alignment (Smith-Waterman) approaches.
• Interpret pairwise alignment metrics including alignment score, percentage identity, similarity, and gap penalties.
• Select the appropriate pairwise alignment strategy depending on sequence length equality and conserved local domains.

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✅ Result

FASTA format characteristics were analyzed, and pairwise sequence alignments were successfully executed for accession pairs (PP948896.1, PP948895.1, PP948894.1, PP948893.1, PP948892.1, PP948891.1). Alignment metrics including identity, gaps, and scores were documented and evaluated.