BLAST Tutorial: Exploring Sequence Databases and Analyzing Similarity Searches

1. Tutorial 3 BLAST • What is BLAST? • Basic Local Alignment Search Tool • Is a set of similarity search programs designed to explore sequence databases.  • What are similarity searches good for? • One sequence by itself is not informative; it must be analyzed by comparative methods against existing sequence databases to develop hypothesis concerning relatives and function Database Query BLAST program

2. BLAST Databases

3. http://www.ncbi.nlm.nih.gov/BLAST/

4. Place Query Choose Database ?

5. BLASTN Databases http://www.ncbi.nlm.nih.gov/BLAST/blastcgihelp.shtml#nucleotide_databases

6. Place Query Choose Database Optimize similarity level of the search ? Limit output size Threshold for results significance Primary word match (16-64 nt) Reward and penalty for matching and mismatching bases Cost to create and extend a gap Remove low information content Limit search to specific organism

7. Search for homologous to chick “olfactory receptor 6” gene

8. Global Alignments Local Alignments Query sequence Matched Areas of database sequences

9. Sequence description E value Score(bits) Sequence Identifier Identity Coverage

10. Score andE value Identities and gaps Strand

11. Multiple hits on a same subject

12. Design of the BLAST survey • Consider your research question: • Are you looking for an particular gene in a particular species?: BLAST against the genome of that species. • Are you looking for additional members of a gene family across all species? : BLAST against the gene collection database. • Are you looking for exact motif matches? : increase gap penalty or use megablast.

13. Score and E-value Score (S): (identities + mismatches)-gaps Bit Score (S’): Score Depends on search space Depends on scoring system Database length(bp) Query length(bp)

14. Score and E-value • The score is a measure of the similarity of the query • to the sequence shown. • The E-value is a measure of the reliability of the score. • The definition of the E-value is: The probability due to chance, that there is another alignment with a similarity greater than the given S score.

15. Score and E-value • The Size of the E-value • The typical threshold for a good E-value from a BLAST • search is E=10-6≈e-6 or lower. • The reason for such low values is that an E=0.001 in a • million entry database would still leave 1000 entries due • to chance. An E=e-6 would only leave one entry due to • chance.

17. Exercise Calculate the S, S’ and E for the following BLAST hit: ACGTCGATCGAGCT |||||||| ||||| AGGTCGTC-GAGGT Given the following parameters: Query length: 150 • =1.37 K=0.711 Average Sequence length in database: 270 Number of sequences in database: 4,554,026 S: (Id+MM)-GP S = 13-1 = 12 S’= (1.37*12 – ln(0.711))/ln(2) S’= 16.44 + 0.341 /0.693 S’= 24.2

18. Exercise Calculate the S, S’ and E for the following BLAST hit: ACGTCGATCGAGCT |||||||| ||||| AGGTCGTC-GAGGT Given the following parameters: Query length: 150 • =1.37 K=0.711 Average Sequence length in database: 270 Number of sequences in database: 4,554,026 E= 0.711x150x270x4,554,026xe-1.37*12 E= 131135455683x7.24e-8 E= 9504.27

19. Exercise What will be the minimal score in order to achieve a significant E value (e-6~10-6)? 131135455683e-1.37S=10-6 ln (131135455683e-1.37S)=ln(10-6) ln (131135455683)+ln(e-1.37S)=-13.81 25.6-1.37S=-13.81 S= =-13.81-25.6/-1.37 S≈ 28.76

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