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.

Exploration of NCBI Nucleotide Database and Sequence Retrieval. PB Kale (Molecular Biology and Biotechnology), Mahatma Phule Krushi Vidyapeeth, Rahuri

 Students' Self-Open-Learning Bioinformatics Lab

Exploration of NCBI Nucleotide Database and Sequence Retrieval

PB Kale (Molecular Biology and Biotechnology), Mahatma Phule Krushi Vidyapeeth, Rahuri

 

Aim: To explore the NCBI Nucleotide database, retrieve nucleotide sequences using accession numbers, examine their annotation, and download sequences in FASTA and GenBank formats.

Sequence Dataset: Use the following accession numbers; reference PP948896.1, PP948895.1, PP948894.1, PP948893.1, PP948892.1, PP948891.1

Requirements:

1. Internet-enabled computer
2. Web browser
3. NCBI account (optional)

Procedure:

1. Open NCBI Nucleotide

Go to NCBI Nucleotide.

Image 1: Screenshot of the NCBI Nucleotide homepage with the search box highlighted.

2. Search the accession number-

Enter PP948896.1 in the search box and click Search.

Note: Repeat the procedure for all six accession numbers.

See image 1, showing PP948896.1 entered in the NCBI search box.

3. Examine the sequence record.

Open the record and note:

• Accession and version
• Definition/title
• Organism
• Sequence length
• Molecule type
• References
• FEATURES: viz., gene, CDS, mRNA, etc. (learn for recording the data)

Image 2: Annotated GenBank format record with ACCESSION, VERSION, SOURCE/ORGANISM, FEATURES, and ORIGIN labelled.

4. View the GenBank format

Select GenBank from the display-format options.

Understand the difference:

• GenBank: sequence + biological annotation
• FASTA: sequence + identifier/header

5. Retrieve the FASTA sequence

Select FASTA and observe the sequence.

Use Send to → File → FASTA to save the sequence.

Save the six records as:

PP948896.1.fasta

PP948895.1.fasta

PP948894.1.fasta

PP948893.1.fasta

PP948892.1.fasta

PP948891.1.fasta

Image 3: Screenshot of the FASTA record and send to → File → FASTA option.

6. Record the observations: Complete the table and elaborate in the results section

Accession	Organism	Length (bp)	Gene/Feature	CDS	Sequence downloaded	Any other Feature/ Remark
PP948896.1
PP948895.1
PP948894.1
PP948893.1
PP948892.1
PP948891.1

 

7. Explore related information

For one record, examine available links to Gene, Protein, Taxonomy, BioProject, BioSample, or PubMed, as applicable.

Expected Learning

Students should be able to:

• Search NCBI using an accession number.
• Interpret a nucleotide/GenBank record.
• Identify sequence versus annotation.
• Retrieve and save FASTA sequences.
• Understand accession number and version.
• Record sequence information systematically for subsequent BLAST, MSA, phylogenetic, and primer-design exercises (further experiments).

Result/ interpretation:

The nucleotide sequences corresponding to PP948896.1, PP948895.1, PP948894.1, PP948893.1, PP948892.1 and PP948891.1 were successfully retrieved and documented from the NCBI Nucleotide database.

Do you know?

You can retrieve all the targeted sequences from NCBI

Refer to the link and select all accessions to view/retrieve the data in different file formats:

Link: https://www.ncbi.nlm.nih.gov/nuccore/?term=Kale+PB+AND+Bactrocera

You can try the same way we used in the experiment to download the data and use it for further analysis and documentation.

Reference:

1.       Collection of the nucleotide sequences: https://www.ncbi.nlm.nih.gov/nuccore/?term=Kale+PB+AND+Bactrocera

2.       NCBI Handbook & Tutorials (https://www.ncbi.nlm.nih.gov/guide/training-tutorials/).

Understanding Drought in Sorghum: What Science Reveals (PB Kale and Prachi Mahajan, Mahatma Phule Krishi Vidyapeeth, Rahuri)

Understanding Drought in Sorghum: What Science Reveals

(PB Kale and Prachi Mahajan, Mahatma Phule Krishi Vidyapeeth, Rahuri)

🌾 Why Sorghum Matters

Sorghum (Sorghum bicolor) is a lifeline crop for millions of farmers living in semi-arid and arid regions across Asia and Africa. In countries like India, where agriculture often depends on unpredictable rainfall, sorghum provides:

• Food security (as a staple grain)
• Fodder for livestock
• Raw material for industries

Its natural ability to survive under harsh conditions makes it a “climate-resilient crop.” However, even sorghum is not immune to drought stress, which remains one of the biggest challenges to its productivity.

 

🌦️ The Problem: Drought Stress

Drought affects plants at multiple levels:

Development of Trait-Specific SSR Primers: A Step-by-Step Guide [PB Kale, Molecular Biology and Biotechnology, Mahatma Phule Krishi Vidyapeeth Rahuri]

 Development of Trait-Specific SSR Primers: A Step-by-Step Guide 

(A practical guide for research students)

PB Kale, Molecular Biology and Biotechnology, Mahatma Phule Krishi Vidyapeeth Rahuri

Simple Sequence Repeats (SSRs), also known as microsatellites, are short tandem repeats of 1–6 nucleotides widely distributed across genomes. Because of their high polymorphism, co-dominant inheritance, and reproducibility, SSR markers remain valuable tools in genetic diversity studies, QTL mapping, and marker-assisted selection. In many postgraduate research projects, particularly in plant biotechnology and molecular breeding, students develop trait-specific SSR primers based on genes associated with target traits such as drought tolerance, heat stress, disease resistance, or nutritional quality.

The following step-by-step guide outlines the general workflow used in research laboratories to develop gene-based SSR markers using sequence databases and bioinformatics tools.

1. Identify Candidate Genes Associated with the Trait

Guide to Download, Analyse Gene Expression at NCBI GEO [PB Kale, Molecular Biology and Biotechnology, Mahatma Phule Krishi Vidyapeeth Rahuri]

 Steps and Guide to Download and Analyze Gene Expression Data from NCBI GEO 

PB Kale, Molecular Biology and Biotechnology, Mahatma Phule Krishi Vidyapeeth Rahuri

Protocol 1: To Download Datasets

1. Open Web Browser:

   - Launch your preferred web browser (e.g., Chrome, Firefox, Safari).

   - In the address bar, type in the URL: [https://www.ncbi.nlm.nih.gov/geo/](https://www.ncbi.nlm.nih.gov/geo/) and press Enter. This will direct you to the Gene Expression Omnibus (GEO) homepage, a public repository for gene expression data.

   

2. Search for Datasets:

   - On the GEO homepage, locate the search bar.

   - Enter the keyword(s) relevant to your research or the specific GEO accession number (a unique identifier for a dataset) into the search box.

   - Click the 'Search' button to initiate the search. The website will display a list of search results matching your query.

3. View Search Results:

   - Browse through the search results. Each result will typically include a brief description and a hyperlink.