🔍 Sequence Similarity Search

BLAST Sequence Similarity Search

Basic Local Alignment Search Tool (BLAST) is the standard tool for database similarity searches of nucleotide and amino acid sequences. Select the appropriate program and access NCBI BLAST directly.

blastn
Nucleotide BLAST

Search nucleotide sequences against nucleotide databases (e.g., nt). Most commonly used for gene identification, primer specificity checking, and clone verification. Megablast (fast, high-identity) and Discontiguous Megablast (cross-species comparison) are also available.

Nucleotide Query → Nucleotide DB
Open blastn →
blastp
Protein BLAST

Search amino acid sequences against protein databases (nr, RefSeq, Swiss-Prot, PDB, etc.). Used for protein function prediction, domain analysis, and identification of homologous proteins across species. PSI-BLAST (iterative search, distant homology detection) and PHI-BLAST (pattern-specified) are also available.

Amino Acid Query → Protein DB
Open blastp →
blastx
Translated BLAST (nt → aa DB)

Translates nucleotide sequences in all 6 reading frames and searches protein databases. Useful for functional prediction of novel genes, EST sequence analysis, and checking whether coding sequences exist in unannotated regions.

Nucleotide Query → Translate(6frame) → Protein DB
Open blastx →
tblastn
Translated BLAST (aa → nt DB)

Searches amino acid sequences against nucleotide databases translated in all 6 reading frames. Effective for finding genes homologous to known proteins in unannotated genomes or ESTs.

Amino Acid Query → vs Translate(6frame) → Nucleotide DB
Open tblastn →
tblastx
Translated BLAST (nt → nt, both translated)

Translates both query and database sequences in all 6 frames for comparison. The most sensitive method for detecting distantly related genes conserved at the amino acid level even when nucleotide similarity is low. Computationally expensive; not suitable for long sequences.

Nucleotide Query (translated) → Nucleotide DB (translated)
Open tblastx →
📋 Program Selection Guide
Program Query Database Sensitivity Typical Use Case
blastn Nucleotide Nucleotide ★★★ (close relatives) Gene identification, primer verification, clone confirmation
blastp Amino Acid Protein ★★★★ Protein function prediction, homologous protein identification
blastx Nucleotide Protein ★★★★ Novel gene function prediction, EST analysis
tblastn Amino Acid Nucleotide ★★★★ Homologous gene search in unannotated genomes
tblastx Nucleotide Nucleotide ★★★★★ (highest sensitivity) Conserved gene search across distant species (slow)
💡 Search Tips
🎯 Understanding E-value
E-value is the expected frequency of equivalent scores in random sequences. Below 10⁻⁵ indicates significant homology. Below 10⁻¹⁰ is highly reliable.
⚡ Megablast vs blastn
Use Megablast (fast) for same-species or closely related searches. Choose Discontiguous Megablast for cross-species or low-similarity searches.
🧬 Scoring Matrix
Use BLOSUM80 for closely related sequences, BLOSUM45 for distant ones. The default BLOSUM62 is general-purpose and suitable for most cases.
🌍 Organism Filter
Enter Homo sapiens, Mus musculus, etc. in the "Organism" field to restrict your search to a specific organism's database.
🔗 Saving Results with RID
Note the RID (Request ID) shown after submitting a BLAST search. You can use it to retrieve the same results page later (usually retained for 36 hours).
📊 Low Complexity Filter
Enabling the filter for repetitive sequences and low-complexity regions (e.g., polyA) is recommended. Disabling it increases false-positive hits.
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