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Lecture 9... Basic Local Alignment Tool (BLAST) - 1

BLAST (Basic Local Alignment Search Tool) is a bioinformatics tool for comparing nucleotide or protein sequences to identify similarities that may indicate functional or evolutionary relationships. It includes various types such as BLASTn, BLASTp, and tBLASTx, and its workflow involves inputting sequences, selecting databases, and executing searches to generate results. Key features include alignment scores, e-values, and applications range from gene identification to drug discovery, though it has limitations in sensitivity and computational demands.

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15 views11 pages

Lecture 9... Basic Local Alignment Tool (BLAST) - 1

BLAST (Basic Local Alignment Search Tool) is a bioinformatics tool for comparing nucleotide or protein sequences to identify similarities that may indicate functional or evolutionary relationships. It includes various types such as BLASTn, BLASTp, and tBLASTx, and its workflow involves inputting sequences, selecting databases, and executing searches to generate results. Key features include alignment scores, e-values, and applications range from gene identification to drug discovery, though it has limitations in sensitivity and computational demands.

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Adnan Jaffar
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Basic Local

Alignment Tool
(BLAST)

Mr. M. Mushahid

ABG-619
Introduction to BLAST
• What is BLAST?
• BLAST (Basic Local Alignment Search Tool) is a bioinformatics tool
used to compare nucleotide or protein sequences to a database of
sequences.

• It identifies regions of similarity, which may indicate functional,


structural, or evolutionary relationships.
Types of BLAST

• BLASTn: Compares nucleotide sequences.

• BLASTp: Compares protein sequences.

• BLASTx: Translates nucleotide sequences into proteins and compares them.

• tBLASTn: Searches a nucleotide database with a protein query.

• tBLASTx: Compares the six-frame translations of two nucleotide sequences.


Workflow of BLAST

• Input Sequence:
• Query can be DNA, RNA, or protein.
• Format: FASTA or plain text.
• Database Selection:
• Choose a relevant database (e.g., NCBI nt, nr, RefSeq, Swiss-Prot).
• Algorithm Settings:
• Adjust parameters like word size, scoring matrix (e.g., PAM, BLOSUM), and e-value
threshold.
• Search Execution:
• The tool aligns the query with database sequences.
• Output Generation:
• Results include alignments, scores, and statistical significance.
Key Features of BLAST

• Alignment Score:
• Indicates the quality of the alignment between query and database
sequences.
• E-value (Expect Value):
• Represents the number of matches expected by chance.
• Lower e-value = higher significance.
• Percent Identity:
• Proportion of matching residues in the alignment.
• Query Coverage:
• Fraction of the query sequence aligned with a database sequence.
Applications of BLAST

• Gene Identification:
• Identify homologous genes across species.
• Function Prediction:
• Predict protein function by identifying conserved domains.
• Evolutionary Studies:
• Trace phylogenetic relationships and evolutionary history.
• Primer Design:
• Verify primer specificity in PCR experiments.
• Metagenomics:
• Classify microbial communities by comparing sequence reads.
• Drug Discovery:
• Identify potential drug targets through sequence similarity.
Interpreting BLAST Results

• Reading the Alignment:


• Look for high alignment scores and low e-values.
• Evaluating Hits:
• Focus on hits with high percent identity and query coverage.
• Multiple Alignments:
• BLAST provides a list of potential matches ranked by their scores.
• Beware of False Positives:
• Validate findings with other tools or methods.
Practical Demonstration

• Step 1: Access BLAST (e.g., NCBI BLAST).


• Step 2: Input a query sequence in FASTA format.
• Step 3: Choose the appropriate BLAST program and database.
• Step 4: Customize parameters if needed.
• Step 5: Submit the query and interpret the output.
Limitations of BLAST

• Sensitivity decreases with highly divergent sequences.

• Computationally intensive for large databases.

• Does not provide structural or functional context.


Tips for Effective BLAST Use

• Use specific databases to improve relevance.

• Adjust algorithm parameters for optimal performance.

• Combine BLAST with other tools (e.g., phylogenetic analysis).


Summary

• BLAST is an essential tool for sequence analysis with diverse


applications.

• Proper interpretation of BLAST results requires understanding key


metrics like alignment scores, e-values, and query coverage.

• Regular updates in databases and algorithms enhance BLAST's power


and accuracy.

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