1. David Wishart
February 18th, 2004
Lecture 3
BLAST
(c) 2004 CGDN

2. BLAST Basic Local Alignment Search Tool
A heuristic method for performing local alignments through searches of high scoring segment pairs (HSP’s)
1st to use statistics to predict significance of initial matches -
Offers both sensitivity and speed

3. BLAST
Looks for clusters of nearby or locally dense “similar or homologous” k-tuples
Uses “look-up” tables to shorten search time
Uses larger “word size” than FASTA to accelerate the search process
Performs both Global and Local alignment
Fastest and most frequently used sequence alignment tool -- THE STANDARD
Lecture 3.1

4. Uses of BLAST
Identifying species With the use of BLAST, you can possibly correctly identify a species and/or find homologous species.
Locating domains When working with a protein sequence you can input it into BLAST, to locate known domains within the sequence of interest.
Establishing phylogeny Using the results received through BLAST you can create a phylogenetic tree using the BLAST web-page. It should be noted that phylogenies based on BLAST alone are less reliable than other purpose-built computational phylogenetic methods, so should only be relied upon for "first pass" phylogenetic analyses.
DNA mapping When working with a known species, and looking to sequence a gene at an unknown location, BLAST can compare the chromosomal position of the sequence of interest, to relevant sequences in the database(s).
Comparison When working with genes, BLAST can locate common genes in two related species, and can be used to map annotations from one organism to another.

5. BLAST Access NCBI BLAST Canadian Bioinformatics Resource BLAST
Canadian Bioinformatics Resource BLAST
European Bioinformatics Institute BLAST

6. Lecture 3.1

7. Lecture 3.1

8. Lecture 3.1

9. Different Flavours of BLAST
BLASTP - protein query against protein DB
BLASTN - DNA/RNA query against GenBank (DNA)
BLASTX - 6 frame trans. DNA query against proteinDB
TBLASTN - protein query against 6 frame GB transl.
TBLASTX - 6 frame DNA query to 6 frame GB transl.
PSI-BLAST - protein ‘profile’ query against protein DB
PHI-BLAST - protein pattern against protein DB

10. Other BLAST Services
MEGABLAST - for comparison of large sets of long DNA sequences
RPS-BLAST - Conserved Domain Detection
BLAST 2 Sequences - for performing pairwise alignments for 2 chosen sequences
Genomic BLAST - for alignments against select human, microbial or malarial genomes

11. Running NCBI BLAST
Lecture 3.1

12. MT0895
MMKIQIYGTGCANCQMLEKNAREAVKELGIDAEFEKIKEMDQILEAGLTALPGLAVDGELKIMGRVASKEEIKKILS

13. Running NCBI BLAST OR OR
Paste in sequence (FASTA format, raw sequence or type in GI or accession number)
>Mysequence MT0895 KIQIYGTGCANCQMLEKNAREAVKELGIDAEFEKIKEMDQILEAGLTALPGLAVDGELKIDS OR
> KIQIYGTGCANCQMLEKNAREAVKELGIDAEFEKIKEMDQILEAGLTALPGLAVDGELKIDS OR
KIQIYGTGCANCQMLEKNAREAVKELGIDAEFEKIKEMDQILEAGLTALPGLAVDGELKIDS
Lecture 3.1

14. Running NCBI BLAST
Choose a range of interest in the sequence “set subsequences” (not usually used)
Select the database from pull-down menu (usually choose nr = non-redundant)
Leave “Options” unchanged (use defaults)

15. Running NCBI BLAST
Select Database
Lecture 3.1

16. Running NCBI BLAST
Click BLAST!
Lecture 3.1

17. Formatting Results
Lecture 3.1

18. BLAST Format Options
Lecture 3.1

19. BLAST Output
Lecture 3.1

20. BLAST Output
Lecture 3.1

21. BLAST Output
Lecture 3.1

22. BLAST Output
Lecture 3.1

23. BLAST Output
Lecture 3.1

24. BLAST Output
Lecture 3.1

25. BLAST Parameters Identities - No. & % exact residue matches
Positives - No. and % similar & ID matches
Gaps - No. & % gaps introduced
Score - Summed HSP score (S)
Bit Score - a normalized score (S’)
Expect (E) - Expected # of chance HSP aligns

26. Conclusions
BLAST is the most important program in bioinformatics (maybe all of biology)
BLAST is based on sound statistical principles (key to its speed and sensitivity)
A basic understanding of its principles is key for using/interpreting BLAST output
Use NBLAST or MEGABLAST for DNA
Use PSI-BLAST for protein searches