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Basics of BLAST

Basics of BLAST. Basic BLAST Search - What is BLAST? - The framework of BLAST - Different protocols of BLAST - The database you can search - Where can I BLAST?. What is BLAST?. BLAST stands for B asic L ocal A lignment S earch T ool NCBI- BLAST vs. WU- BLAST BLAST ! = BLAT

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Basics of BLAST

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  1. Basics of BLAST • Basic BLAST Search - What is BLAST? - The framework of BLAST - Different protocols of BLAST - The database you can search - Where can I BLAST?

  2. What is BLAST? • BLAST stands for Basic Local Alignment Search Tool • NCBI- BLAST vs. WU- BLAST • BLAST ! = BLAT • Why is BLAST popular? - Good balance of sensitivity and speed - Reliability - Flexibility

  3. The Framework of BLAST (1) • Scoring matrix - high BLOSUM (low PAM)  closely related sequences - low BLOSUM (high PAM)  distantly related sequences - Default is BLOSUM 62

  4. BLOSUM Matrix • BLOSUM = BLOcks SUbstitution Matrix http://helix.biology.mcmaster.ca/721/distance/node10.html

  5. The Framework of BLAST (2) • Sequence Alignment - W (word size); blastn 11; others 2 or 3 - G (gap open penalty); blastn 5; others 11 - E (gap extension penalty); blastn 2; others 1 • Statistic Interpretation - e (threshold for expectation value) default 10

  6. BLAST Protocols • The most common BLAST search includes fiveprotocols:

  7. BLASTN • BLASTN - The query is a nucleotide sequence. - The database is a nucleotide database - No conversion is done on the query or database • DNA :: DNA homology - Mapping oligos to a genome - Cross-species sequence exploration - Annotating genomic DNA with ESTs

  8. BLASTP • BLASTP - The query is an amino acid sequence - The database is an amino acid database - No conversion is done on the query or database • Protein :: Protein homology - Protein function exploration - Novel gene  makes parameters more sensitive

  9. BLASTX • BLASTX - The query is a nucleotide sequence - The database is an amino acid database - All six reading frames are translated on the query and used to search the database • Coding nucleotide seq :: Protein homology - Gene finding in genomic DNA - Annotating ESTs (and Shotgun Sequence)

  10. TBLASTN • TBLASTN - The query is an amino acid sequence - The database is a nucleotide database - All six frames are translated in the database and searched with the protein sequence • Protein :: Coding Nucleotide DB homology - Mapping a protein to a genome - Mining ESTs (Shotgun DNA) for protein similarities

  11. TBLASTX • TBLASTX - The query is a nucleotide sequence - The database is a nucleotide database - All six frames are translated on the query and on the database • Coding :: Coding homology - For searching distantly related species - Sensitive but expensive

  12. BLAST output • List of Sequences with scores – Raw score, higher is better (length dependant) – Expect Value, smaller is better (length and database size independent) • List of alignments

  13. The Databases (1) • Genbank NR (protein and nucleotide versions) Non-redundant large databases (compile and remove dups) Anyone can submit, you can call your sequence anything Quality low; names can be meaningless • EST databases Short single reads of cDNA clones Other short single reads High error rates

  14. The Databases (2) • Swissprot Curated from literature REAL proteins; REAL functions; small; • Genomic Databases Human, Mouse, Drosophila, Arabidopsis etc NCBI, species specific web pages

  15. Where Can I run BLAST? • Three choices: – NCBI (www.ncbi.nih.gov) databases updated constantly (daily); very slow at times – Goose web (goose.wustl.edu/blast/blast.html) –command line (blastall)

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