1. Construction of Substitution matrices
BLOSUM
BLOCKS SUBSTITUTION MATRIX
PAM
POINT ACCEPTED MUTATIONS

2. Substitution matrices
Substitution matrix contains values proportional to the probability that amino acid A mutates into amino acid B for all pairs of amino acids through a period of evolution
Substitution matrices are constructed from a large and diverse sample of sequence alignments

3. How to construct substitution matrices
Multiple alignment of well studies gene sequences from different species
use orthologs: functionally similar
observed substitutions tend to preserve functions
minimal gaps

4. How to construct substitution matrices ?
Tabulate substitutions
A to A: 9867 times
A to R: 2 times
A to N: 9 times
etc….

5. How to construct substitution matrices ?

6. Construction of Substitution matrices
BLOSUM

7. Construction of Substitution matrices
BLOSUM

8. How to construct substitution matrices ?
Substitution matrix score =
Log Observed mutation rate in alignment
Expected random mutation rate

9. How do we find the random mutation rate?

10. The random mutation rate
compute the overall occurrence of an amino acid in a protein database

11. The random mutation rate
compute the overall occurrence of an amino acid in a protein database

12. The random mutation rate
Example:
Expected random mutation rate is 1 in and observed mutation rate of W to R is 1 in 10
Score = log (0.1/0.0001) = log (1000) = +3

17. Calculating BLOSUM62 scores

18. Calculating BLOSUM62 scores

19. Calculating BLOSUM62 scores

20. Calculating BLOSUM62 scores

21. Calculating BLOSUM62 scores

22. Calculating BLOSUM62 scores

23. Calculating BLOSUM62 scores

24. Calculating BLOSUM62 scores

25. Calculating BLOSUM62 scores

26. Calculating BLOSUM62 scores

27. PAM matrices [1 point mutation per 100 amino acids]
Point Accepted Mutations
[1 point mutation per 100 amino acids]
does not take into account different evolutionary rates between conserved and non-conserved regions

28. PAM1 is 1% average change in amino acids

30. Why use substitution matrices?????

31. Why use substitution matrices?
Database searches

32. Database searching

33. Database searching

34. Database searching
Query Sequence; Database sequences

35. Database searching: Filtering
Dynamic programming is computationally expensive
Apply DP to sequence pairs that are likely to be similar
find short words: query-database
DNA 7-28bases (BLAST?)
PROTEIN 3 amino acids (BLAST?)

36. BLAST
Basic Local Alignment Search Tool
Heuristic method?

47. Blast output parameter
E value

48. E value
number of alignments one can expect see by chance.
Number of alignments having the same or greater score.
Dependent on size of database and length of query seq.