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Run a local BLAST search against a custom database

Build a BLAST database from a FASTA file and query it locally with blastn — no web submission required.

SSSudipta SardarUpdated July 8, 2026

Prerequisites

  • BLAST+ installed (`makeblastdb`, `blastn`)
  • A subject FASTA file
  • A query FASTA file

1Build the database

Index your subject sequences into a nucleotide BLAST database.

bash
makeblastdb -in subject.fasta -dbtype nucl -out subjectdb

Expected output

Building a new DB, current time: ...
Adding sequences from FASTA; added 42 sequences in 0.03 seconds.

2Run the search

Query the database and request tabular output for easy parsing.

bash
blastn -query query.fasta -db subjectdb -outfmt 6 -out hits.tsv

Expected output

query1	subject3	98.5	200	3	0	1	200	1	200	1e-95	355

Troubleshooting

BLAST Database error: No alias or index file found

The -db value must match the -out prefix from makeblastdb, without a file extension.

No hits returned

Confirm query and subject are the same molecule type (nucl vs prot) and lower -evalue if sequences are short.

References