1. BLAST Workshop
Maya Schushan
June 2009

2. Workshop OUTLINE Part 1: Introduction and motivation
How does BLAST work?
Part 2:
BLAST programs
Sequence databases
Work Steps
Extract and analyze results

3. will be considered as homologous
Why BLAST?
Finding homologous
Homology- similarity between sequences that result from a common ancestor.
Sequences look alike  probably have the same function and structure.
Use a sequence as a search query in order to find homologous sequences in a data base.
Save time! – exploit the knowledge you have about your homologues, and conclude about your query.
More then:
25% for proteins
70% for nucleotides
will be considered as homologous

4. Identify sequence motifs
Why BLAST?
Finding homologous
Identify sequence motifs

5. Why BLAST? Finding homologous
Find out which region are evolutionary conserved  important for function and\or structure

6. Why BLAST? Finding homologous
Construct phylogenetic trees  understand the evolution of the sequence’s family

7. Why BLAST? Finding homologous
Inferring function for a novel sequence 
learning from previous data available for homologous sequences

8. Why BLAST? Finding homologous
Finding out if your protein sequence has a structure (or a close homologue has one….)

9. How does BLAST work?
What Is An Alignment? Before we can understand how BLAST works, we first have to understand the principles of sequence alignment….

10. How does BLAST work? What Is An Alignment? VLSPADKTNVKAAWAKVGAHAAGHG
Comparing 2 (pairwise) or more (multiple) sequences.
Searching for a series of identical or similar characters in the sequences.
VLSPADKTNVKAAWAKVGAHAAGHG
||| | | |||| | ||||
VLSEAEWQLVLHVWAKVEADVAGHG

11. How does BLAST work? ? or What Is An Alignment? T C A T G C A T T G
A process of lining-up 2 or more sequences to achieve maximum level of identity, in order to find homologies.
T C A T G
C A T T G ?
T C A T G
C A T T G
T C A T G
C A T T G or

12. How does BLAST work? What Is An Alignment?
S = ACTG S’ = AC_TG S’ = ACTG S’ = ACTG
T = AGT T’ = A_GT_ T’ = AGT_ T’ = _AGT
Good: Identical characters- match.
Bad: Different characters- mismatch; gap (InDel).
Each pair of characters gets a value, depending on its identity.
The similarity score of the alignment is the sum of pair values.

13. Example: Aligning Two Globins
How does BLAST work?
General Alignment Methodology
What Is An Alignment?
Example: Aligning Two Globins
Human Hemoglobin (HH):
VLSPADKTNVKAAWGKVGAHAGYEG
Sperm Whale Myoglobin (SWM):
VLSEGEWQLVLHVWAKVEADVAGHG

14. Example: Aligning Two Globins
How does BLAST work?
What Is An Alignment?
Example: Aligning Two Globins
No Gaps:
Percent identity: 36
Percent similarity: 40
(HH) VLSPADKTNVKAAWGKVGAHAGYEG
(SWM) VLSEGEWQLVLHVWAKVEADVAGHG

15. Example: Aligning Two Globins
How does BLAST work?
What Is An Alignment?
Example: Aligning Two Globins
With Gaps:
Gaps: 2
Percent identity: (instead of 36 without gaps)
Percent similarity: (instead of 40 without gaps)
(HH) VLSPADKTNVKAAWGKVGAH-AGYEG
(SWM) VLSEGEWQLVLHVWAKVEADVAGH-G

16. How does BLAST work? What Is An Alignment? Alignment Scoring
1. Assume independent mutation model
2. Score at each position
Positive if the same/similar
Negative if different or gap
3. Score of an alignment is sum of position score
Each position considered separately

17. How does BLAST work? What Is An Alignment? Scoring Matrix
A matrix n  n : n=4 for DNA, n=20 for proteins
Each entry matrix defines the score for observing the two letters in the alignment
Positive if likely to change
Negative otherwise T C G A 1 -5

18. How does BLAST work? What Is An Alignment? DNA scoring matrices
Transitions – purine to purine or pyrmidine to pyrmidine
(4 possibilities)
Transversions – purine to pyrmidine or pyrmidine to purine (8 possibilities)
By chance alone transversions should occur twice as often as transitions.
De-facto transitions are more frequent than transversions.

19. How does BLAST work? What Is An Alignment? DNA scoring matrices T C G
From To 2 -4 -6
Transversion
Transition
Match

20. Proteins scoring matrices
How does BLAST work?
What Is An Alignment?
Proteins scoring matrices
Observation: some substitutions are more frequent than others, e.g., chemically similar amino acids
As for DNA, protein matrices define the probabilities of change between the different amino acids
Popular matrices are based on empirical data: PAM & BLOSUM

21. How does BLAST work? What Is An Alignment? PAM Matrices
PAM matrices are based on sequences with 85% identity.
The changes are “accepted” by natural selection
1 PAM unit:
the probability of 1 point mutation per 100 residues.
Multiplying PAM1 by itself gives higher PAMs matrices that are suitable for larger evolutionary distance.

22. How does BLAST work? What Is An Alignment? BLOSUM Matrices
Based on BLOCKS database:
Low BLUSOM numbers for distant sequences,
High BLUSOM numbers for similar sequence
BLOSUMn is based on sequences that shared at least n percent identity, generally:
BLOSUM62 for general use
BLOSUM80 for close relations
BLOSUM45 for distant relations

23. Proteins scoring matrices
How does BLAST work?
What Is An Alignment?
Proteins scoring matrices
Closer sequences
PAM100 = BLOSUM90
PAM120 = BLOSUM80
PAM160 = BLOSUM60
PAM200 = BLOSUM52
PAM250 = BLOSUM45
Distant sequences

24. How do we calculate gap scores
How does BLAST work?
How do we calculate gap scores
Same substitution scores are applied on gapped and ungapped local alignments.
Appropriate gap scores have been selected over the years by trial and error  default gap scores
If you wish to apply a different scoring matrix-
No grantee that the gap scores will remain appropriate!!!!
large penalty for opening and much smaller one for extending it are most effective

25. How does BLAST work? What Is An Alignment? Scoring
The final score of the alignment is the sum of the positive scores and penalty scores:
+ Number of Identities
+ Number of Similarities
- Number of Gap insertions
- Number of Gap extensions
Alignment score
Scoring Matrix
Gap penalties

26. BLAST (Basic Local Alignment Search Tool)
How does BLAST work?
BLAST (Basic Local Alignment Search Tool)
Goal: A fast search for homologues in a huge database
The underlying hypothesis: when two sequences are similar there are short ungapped regions of high similarity between them
The heuristic:
Discard irrelevant sequences
Perform exact local alignment only with the remaining sequences
Altschul, S.F.,Gish, W., Miller, W., Myers, E.W., and Lipman,D.J(1990) “basic local alignment search
tool” J. Mol. Biol. 215:

27. Searching a sequence database
How does BLAST work?
Searching a sequence database
Idea:
In order to find homologous sequences to a sequence of interest, one should compute its pairwise alignment against all known sequences in a database, and detect the best scoring significant homologs
Query sequence - the sequence with which we are searching
Hit – a sequence found in the database, suspected as homologous

28. How does BLAST work? The parameters-
W : Word size – find W-mers in target/query
2-3 for aa, 6-11 for nucleotides.
T : Threshold – focus on pairs scoring >T
usually 11-13
X : Drop-off – stop extending when loss >X
S : Score – the final score of segment pair

29. How does BLAST work? The algorithm: s t
Align a query sequence with the database.
Find “hits”: short word pairs of length W with an ungapped alignment score of at least T.
Extend alignments until score drops more than X below hitherto best score
Consumes most of the processing
time (>90%) s t

30. How do we discard irrelevant sequences quickly?
How does BLAST work?
How do we discard irrelevant sequences quickly?
Divide the database into words of length w (default: w = 3 for protein and w = 7 for DNA)
Save the words in a look-up table that can be searched quickly
WTD
TDF
DFG
FGY
GYP …
WTDFGYPAILKGGTAC

31. BLAST: discarding sequences
How does BLAST work?
BLAST: discarding sequences
When the user enters a query sequence, it is also divided into words
Search the database for consecutive neighboring words
neighbor words are defined according to a scoring matrix (e.g., BLOSUM62 for proteins) with a certain cutoff level
GFC (20)
GFB
GPC (11)
WAC (5)

32. Look for a seed: hits on the same diagonal which can be connected
How does BLAST work?
Neighbor word
Look for a seed: hits on the same diagonal which can be connected A
At least 2 hits on the same diagonal with distance which is smaller than a predetermined cutoff
Database record
This is the filtering stage – many unrelated hits are filtered, saving lots of time!
Query

33. Try to extend the alignment
How does BLAST work?
Try to extend the alignment
Stop extending when the score of the alignment drops X beneath the maximal score obtained so far
Discard segments with score < S
ASKIOPLLWLAASFLHNEQAPALSDAN
JWQEOPLWPLAASOIHLFACNSIFYAS
Score=15
Score=17
Score=14

34. How does BLAST work? Two-Hit Gapped BLAST
The new gapped BLAST algorithm:
Start with the two hit method-
(a) find two hits of score higher then T,
within a distance A.
(b) invoke an ungapped extension on the second hit.
If the HSP generated has an expected score:
(a) Trigger a gapped extension
(b) If the final score has a significant E-value – report the gapped alignment.

35. The result – local alignment
How does BLAST work?
The result – local alignment
The result of BLAST will be a series of local alignments between the query and the different hits found

36. How does BLAST work? The scoring system
BLAST uses BLOSSOM62 as the scoring matrix to perform the alignment (default).

37. How does BLAST work? E-value Small E-value  better score
To asses the bits score we calculate E-value:
E-value = The expected number of HSP’s with a score of at least S:
For each score S there is a specific E-value.
Small E-value  better score

38. In practice – BLAST uses estimations.
How does BLAST work?
E-value
Theoretically, we could trust any result with an E-value ≤ 1
In practice – BLAST uses estimations.
E-values of 10-4 and lower indicate a significant homology.
E-values between 10-4 and 10-2 should be checked (similar domains, maybe non-homologous).
E-values between 10-2 and 1 do not indicate a good homology

39. How does BLAST work? PSI-BLAST Step 1:
Set a standard protein-protein BLAST search (BLOSUM62)
Build a position specific scoring matrix (PSSM) according to MSA of the alignment results with low E-value.
Step 2:
Set a BLAST search using the PSSM to evaluate the alignment. PSSM vs. DB instead of seq vs. DB
Update the PSSM according to the new result
Go back to the beginning of step two or stop.

40. How does BLAST work? PSI-BLAST The difference-
The score for aligning a letter with a pattern position is
given by the matrix itself!
The matrix is of the length of the original seq. (L* 20)
No theory for deriving gap costs  Gap scores the same
as the one in the 1st iteration 1 2 3 4 5 6 7 8 9 A .1 .3 .2 .8 D .6 .4 L .7 .9

41. The power of PSI-BLAST:
How does BLAST work?
The power of PSI-BLAST:
A much sensitive scoring system .
each position has its own pattern probabilities .
Different weight to conserved positions.
Important motifs are bounded
Lowers the level of random noise.
Finding distant relatives.

42. How does BLAST work? Lets sum up…
Blast is a fast way to find homologues
No analytic theory that estimates the statistical significance of gapped alignments
Gap scores have been selected by trial and error.
applying different scoring matrix  No grantee for gap scores
PSI-BLAST finds weak homologues fast