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How to Blast Protein Sequence in Linux?

Step 1: Create a BLAST Database from Your Protein File

If you haven't already created a BLAST database from the NbT2T.pep.fasta file, you need to do so using the makeblastdb command. This command generates the necessary index files for BLAST to search against.

Example Command to Create a Protein Database:

  1. Navigate to the directory where your NbT2T.pep.fasta file is located, or provide the full path to the file.
  2. Run the makeblastdb command to create a BLAST database.
makeblastdb -in NbT2T.pep.fasta -dbtype prot -out NbT2T_db

Explanation of parameters:

  • -in NbT2T.pep.fasta: This is your input protein sequence file in FASTA format.
  • -dbtype prot: Since you are working with a protein sequence database, specify prot.
  • -out NbT2T_db: This is the name of the output database, which will generate several files like NbT2T_db.nhr, NbT2T_db.nin, and NbT2T_db.nsq.

Step 2: Verify Database Files Exist

After running makeblastdb, you should see the following files in the directory:

  • NbT2T_db.nhr
  • NbT2T_db.nin
  • NbT2T_db.nsq

These files are the database indexes and are required by BLAST to perform the search. Ensure that they exist in the directory where you are running the BLAST search.

Step 3: Check the BLAST Search Path

If you're still encountering the error after creating the database, it's possible that BLAST cannot locate the database files. Make sure that the search path you're providing to the blastp or blastx command points to the correct location of the database files.

For example, if you created the database in the current directory, use the following command to run the BLAST search:

blastp -query protein.fasta -db NbT2T_db -out results.txt -outfmt 6

If your database is located in a different directory, provide the full or relative path to the database:

blastp -query protein.fasta -db /path/to/NbT2T_db -out results.txt -outfmt 6

Make sure you replace /path/to/NbT2T_db with the correct directory where your BLAST database files (NbT2T_db.nhr, NbT2T_db.nin, NbT2T_db.nsq) are located.

Step 4: Ensure Correct File Extensions

Sometimes, the error can occur if the file extensions do not match or if there is a typo in the file name. Verify that the database file (NbT2T.pep.fasta) is indeed in the right format and that the extension is .fasta or .fa. Also, ensure that the files created by makeblastdb are named consistently with the -out prefix you specified.

Step 5: Run BLAST Search Again

Once you've verified that the database is properly created and the search path is correct, run the BLAST search again:

blastp -query protein.fasta -db NbT2T_db -out results.txt -outfmt 6

Additional Troubleshooting Tips:

  1. Check for file permissions: Ensure that the database files (NbT2T_db.*) are readable and accessible by the user running the BLAST command.
  2. Ensure the database is created properly: If you suspect the database was not created correctly, delete the old database files and re-run makeblastdb.

Summary of Key Points:

  1. Create the BLAST database using makeblastdb: 
    makeblastdb -in NbT2T.pep.fasta -dbtype prot -out NbT2T_db
  2. Verify the existence of the .nhr, .nin, and .nsq index files.
  3. Ensure you provide the correct path to the database when running the BLAST command.
  4. Run the BLAST search: 
    blastp -query protein.fasta -db NbT2T_db -out results.txt -outfmt 6

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