Content developed by Kristine Lacek

BLAST (Basic Local Alignment Search Tool)

  • Finds regions of similarity between sequences
  • Compares a query sequence to a database
  • Identifies closest matches
  • Uses local alignment
  • Finds the best matching regions, not full-length alignment
  • Widely used in bioinformatics
    • Gene identification
    • Species confirmation
    • Contamination checks

Different BLAST Programs

  • blastn
    • Nucleotide vs nucleotide database
  • blastp
    • Protein vs protein database
  • blastx
    • Translated nucleotide vs protein database
  • tblastn
    • Protein vs translated nucleotide database
  • megablast
    • Optimized for highly similar sequences

Databases

  • NCBI BLAST
  • GISAID BLAST
  • Custom Blast Database (CLI)

NCBI BLAST

GISAID BLAST

BLAST Databases Search Considerations

  • Databases: NCBI BLAST, GISAID BLAST, Custom Blast Database (CLI)
  • NCBI GenBank: open source, INSDC database
  • GISAID: access control, but ingests INSDC samples
  • For a more complete set of flu samples, which BLAST is better?
  • When might an open-source BLAST search be better?

Sample database 1

Sample database 2

Interpreting BLAST output

  • Percent Identity → How similar the aligned region is
  • E-value → Probability the match occurred by chance
    • Lower = more significant
  • Bit Score → Alignment strength (higher = better)
  • Alignment Length → How much of the query matched

BLAST Table

Running BLAST from the CLI

  • BLAST+ is the command-line version
    • Installed locally for large datasets or automation
    • More reproducible than web BLAST
    • Exact parameters and databases can be recorded
  • Common workflow
    • Prepare database → run BLAST → interpret output

Example command:

blastn -query query.fasta \
       -db nt \
       -out results.txt

Creating and Using Local Databases

  • Download or prepare reference FASTA
    • Example: novel influenza genomes for diagnostics efficacy
  • Create a BLAST database 
    makeblastdb -in reference.fasta \
          -dbtype nucl
  • Run BLAST against local database 
    blastn -query sample.fasta \
     -db reference.fasta \
     -out results.txt
  • Faster and ideal for targeted analysis
  • Especially useful in viral genomics

Useful CLI Options

  • Control output format
    • -outfmt 6 → tabular format (easy to parse)
  • Limit results
    • -max_target_seqs 5
  • Adjust sensitivity
    • -evalue 1e-5
  • Set number of threads
    • -num_threads 8