🧬 BLAST - Using CMD! 🧬

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🚀 Running BLAST from the Command Line

BLAST can also be executed directly through the command line (CMD or Terminal), which is faster and more flexible for batch processing. Below are the steps to install and run BLAST locally on Windows.

Step 1: Download BLAST+

• Go to the official NCBI BLAST+ download page: Download BLAST+
• Download the ZIP file for your operating system (e.g., win64 for 64-bit Windows).
• Extract the folder (e.g., ncbi-blast-2.x.x+) to a location such as C:\Program Files\BLAST+.

Step 2: Add BLAST+ to PATH

• Open System Properties → Environment Variables.
• Find Path, click Edit, and add the bin folder inside your BLAST installation (e.g., C:\Program Files\BLAST+\bin).
• Open a new CMD window and check installation by typing:

  blastn -version

Step 3: Prepare Query and Database

• Place your query file in FASTA format, e.g., query.fasta, inside your working directory.
• If using a local database, format it first using makeblastdb:

  makeblastdb -in my_sequences.fasta -dbtype nucl -out mydb

• If using NCBI’s remote database, you can use -db nt -remote directly.

• If you want to read about BLAST Read the full blog about BLAST →
  

• And if you want to know everything about BLAST and not interested in reading then Watch BLAST Tutorial on YouTube

Watch Code Explanation on YouTube

Following is the explanation of a command to run BLAST using command prompt terminal> It also explains some alternatives options to get required results of BLAST.

blastn -query query.fasta -db nr -out results.txt -outfmt 6 -evalue 1e-5 -num_threads 4 -max_target_seqs 10 -word_size 11
    

📝 Explanation

• 1. blastn
  Chooses the BLAST program type.
  Variants:
  • blastn → Nucleotide vs Nucleotide
  • blastp → Protein vs Protein
  • blastx → Translate nucleotide query → search protein db
  • tblastn → Protein query → search translated nucleotide db
  • tblastx → Translated nucleotide vs translated nucleotide


• 2. -query query.fasta
  Input file containing your query sequence(s) in FASTA format.
  Alternative: Provide a full path or use -query - to paste sequences directly.


• 3. -db mydb
  Database against which you’re searching. Must be formatted with makeblastdb if local.
  Alternatives:
  • -db nt or -db nr with -remote → Use NCBI’s databases.
  • -subject subject.fasta → Compare directly to a FASTA file.


• 4. -out results.txt
  File to save results.
  Alternatives:
  • Omit → results print directly in CMD.
  • Redirect manually: > results.txt.


• 5. -outfmt 6
  Output format.
  Common choices:
  • 0 → Pairwise (default, human-readable)
  • 5 → XML
  • 6 → Tabular
  • 7 → Tabular + comments
  • 10 → CSV
  • 11 → BLAST archive


• 6. -evalue 1e-5
  Expect value threshold (smaller = stricter).
  Alternatives:
  • -evalue 1e-10 → very strict.
  • -evalue 10 → default, less strict.


• 7. -num_threads 4
  Number of CPU threads to use.
  Default = 1. Use more if your system supports it.


• 8. -max_target_seqs 10
  Limit the number of hits shown per query.
  Alternatives:
  • 1 → Only best hit.
  • 500 → Extensive results.
  Default = 500.


• 9. -word_size 11
  Seed word length for finding matches.
  Smaller = more sensitive but slower.
  Common values:
  • Nucleotide BLAST → default = 11, can reduce to 7 for short queries.
  • Protein BLAST → default = 3.

⚡ Optional but Useful Parameters

• -perc_identity 90 → Only report matches with ≥90% identity.
• -qcov_hsp_perc 80 → Require ≥80% query coverage per HSP.
• -task blastn → Choose variant:
  • blastn (default)
  • blastn-short (short reads)
  • megablast (fast, highly similar)
  • dc-megablast (cross-species)
• -remote → Run search on NCBI’s servers instead of locally.

The output below shows the BLAST results for the provided DNA sequence.

Note: Click on the pictue zoom in to get better view or you can download the image by using the button at the bottom of image.

Download Output

For full details straight from NCBI, read the official BLAST+ command-line manual.

Source: NCBI Bookshelf — BLAST® Command Line Applications User Manual.