Beginner’s Guide to BLAST

1. What is BLAST and Why Is It Needed?

BLAST (Basic Local Alignment Search Tool) is a powerful and widely used bioinformatics program that allows users to compare a nucleotide (DNA/RNA) or protein sequence—the query—against databases of known sequences[1][2][3]. The primary purpose is to identify regions of local similarity, which can provide insight into gene or protein function, structure, and evolutionary relationships[1][3][4].

For example, if you sequence a novel gene or protein and want to learn more about its possible function or origin, BLAST helps by:

Identifying closely related known sequences
Analyzing evolutionary relationships and taxonomy
Locating protein domains and mapping genes
Aiding in species identification and DNA mapping

🧬 “BLAST is an algorithm that uses heuristic methods to find regions of local similarity between biological sequences, thereby enabling comparisons with sequence databases.”[5]

2. Different Types of BLAST

There are several BLAST programs, each suited to different types of queries and databases. Here’s a summary of the main BLAST types[3][6][7]:

Tool	Query	Database	Purpose
blastn	Nucleotide	Nucleotide	Finds similar DNA/RNA sequences
blastp	Protein	Protein	Compares protein sequences
blastx	Nucleotide (translated)	Protein	Translates DNA sequence (6 frames) and searches protein matches
tblastn	Protein	Nucleotide (translated)	Compares protein against a translated DNA database
tblastx	Nucleotide (translated)	Nucleotide (translated)	Compares protein-coding potential of novel DNA

Use Cases:

blastn is used for identifying sequence similarity between DNA/RNA sequences and is vital for evolutionary analysis.
blastp compares amino acid sequences of proteins.
blastx helps when you suspect a coding region in your DNA but are unsure of the reading frame.
tblastn allows you to find possible coding regions for a protein in nucleotide data.
tblastx is useful when comparing potentially protein-coding regions in novel DNA against other species[6][3].

3. Popular Platforms Offering BLAST

🔹 NCBI BLAST (Web Version)

Website: NCBI BLAST web tool[8]

Use by selecting the appropriate program (e.g., blastn, blastp) and submitting a FASTA sequence. You can compare against databases like nr, refseq, or swissprot.

Important output metrics:

Max/Bit Score: Strength of match
% Identity: Similarity of query to a hit i.e. a database entry
E-value: Statistical significance (lower is better)

Applications: Identifying gene/protein functions, finding homologs, and exploring evolutionary relationships[1][8][3][1][8][3].

🔹 BLAST+ (Command Line Tool)

Suitable for large-scale, high-throughput or automated sequence analyses. BLAST+ provides a suite of command-line tools that can be run locally. This is essential for:

Analyses involving proprietary or large datasets.
Integrating BLAST searches into bioinformatics workflows.
No dependency on internet access

Installers and databases can be downloaded from NCBI, and tools are supported across different platforms. BLAST+ offers flexibility, database management, and can run without the restrictions of the web version[9][10][11][9][10][11].

🔹 EBI BLAST (European Bioinformatics Institute)

Website: EBI BLAST

The EMBL-EBI also provides web interfaces to BLAST and related tools, supporting nucleotide and protein alignments with direct integration to many European sequence databases[12][13][12][13].

4. Interpreting BLAST Results

When you run BLAST, the output is typically broken down into:

Graphical overview of alignment regions
Ranked list of hits with statistics
Detailed sequence alignments

Key Metrics:

Score/Bit score: Reflects alignment strength—the higher, the better[1][3][1][3].
E-value: Represents the number of expected hits by chance—a lower E-value means greater significance.
% Identity: Indicates the degree of similarity between query and database hit.

      📊 BLAST results help identify homologous sequences, trace evolutionary history, and locate conserved domains across species.
    