Pairwise sequence alignment

1. Pairwise sequence alignment Urmila Kulkarni-Kale Bioinformatics Centre, University of Pune, Pune 411 007. urmila@bioinfo.ernet.in

2. Bioinformatics Databases • Collection of records • DNA sequences: GenBank, EMBL • Protein sequences: NBRF-PIR, SWISSPROT • organized to permit search and retrieval • Text-based searching: Entrez, SRS • Authors, Keywords • Sequence-based searching: BLAST, FASTA • allow processing and reorganization • Alignments, finding patterns • help to discover patterns

3. Heuristic approaches:local sequence alignment • Two main Heuristic Local Alignment Algorithms: • BLAST and FASTA. • They are significantly faster but do not guarantee • to find the optimal alignment.

4. How to analyse sequences? • Analysis of single sequence • Composition • Location of pattern • Profile of properties such as hydrophilicity, hydrophobicity • Comparison with self • Repeats • Comparison with one or more sequences • Sequence and/or structural similarity • Evolutionary relationship (homology)

5. Basis for Sequence comparison • Theory of evolution: • gene sequences have evolved/derived from a common ancestor • Proteins that are similar in sequence are likely to have similar structure and function

6. WHAT IS ALIGNMENT? Alignments are useful organizing tools because they provide pictorial representation of similarity / homology in the protein or nucleic acid sequences.

7. Sample Alignment • SEQ_A: GDVEKGKKIFIMKCSQ • SEQ_B: GCVEKGKIFINWCSQ There are two possible linear alignments • GDVEKGKKIFIMKCSQ | ||||| GCVEKGKIFINWCSQ 2. GDVEKGKKIFIMKCSQ |||| ||| GCVEKGKIFINWCSQ

8. The optimal alignment GDVEKGKKIFIMKCSQ | ||||| ||| ||| GCVEKGK-IFINWCSQ Insertion of one break maximizes the identities.

9. Theoretical background • Alignment is the method based on the theoretical view that the two sequences are derived from each other by a number of elementary transformations – • Mutations (residue substitution) • Insertion/deletion • Slide function

10. TransformationsSubstitution, Addition/deletion, Slide function • The most homologous sequences are those which can be derived from one another by the smallest number of such transformations. • How to decide “the smallest number of transformation?” • Therefore alignments are an optimization problem.

11. Terminology • Identity • Similarity • Homology

12. Identity • Objective and well defined • Can be quantified • Percent • The number of identical matches divided by the length of the aligned region

13. What is Similarity? • Objective and well defined • Can be quantified by using the ‘scoring schemes’ • Percent • The number of “similar matches” divided by the length of the aligned region Protein similarity could be due to – • Evolutionary relationship • Similar two or three dimensional structure • Common Function

14. What is Homology? Homologous proteins may be encoded by- • Same genes in different species • Genes that have transferred between the species • Genes that have originated from duplication of ancestral genes.

15. Difference between Homology and Similarity • Similarity does not necessarily imply Homology. • Homology has a precise definition: having a common evolutionary origin. • Since homology is a qualitative description of the relationship, the term “% homology” has no meaning. • Supporting data for a homologous relationship may include sequence or structural similarities, which can be described in quantitative terms. • % identities, rmsd

16. An optimal alignment AALIM AAL-M A sub-optimal alignment AALIM AA-LM

17. Global Alignment

18. Local Alignment

19. Needleman & Wunsch algorithm • JMB (1970). 48:443-453. • Maximizes the number of amino acids of one protein that can be matched with the amino acids of other protein while allowing for optimum deletions/insertions. • Based on theory of random walk in two dimensions

20. Random walk in two dimensions • 3 possible paths • Diagonal • Horizontal • Vertical • Optimum path • Diagonal

21. N & W Algorithm • The optimal alignment is obtained by maximizing the similarities and minimizing the gaps. GLOSSARY 1. PROTEINS The words composed of 20 letters 2. LETTER is an element other than NULL 3. NULL is an symbol “-” i.e. the GAP 4. GAPS Run of nulls which indicates the deletion(s) in one sequence and insertion(s) in other sequence

22. Contd../ 5. SCORING Assigns a value to each possible MATRIX pair of Amino acids. Examples of matrices are UN, MD, GCM, CSW, UP. 6. PENALTY There are two types of penalties. • Matrix Bias: is added to every cell of the scoring matrix and decides the size of the break. Also called Gap continuation penalty. • Break Penalty: Applied every time a gap is inserted in either sequence.

23. Unitary Matrix • Simplest scoring scheme • Amino acids pairs are classified into 2 types: • Identical • Non-identical • Identical pairs are scored 1 • Non-identical pairs are scored 0 • Less effective for detection of weak similarities … . . .

24. N & W definitions/variables • A,B Two sequences under comparison • M,L lengths of two sequences • A(i) ith amino acid in sequence A • B(j) jth amino acid in sequence B • MAT is a two dimensional array used to compare all possible pair combinations of sequence A and B. • SM(i,j) The cell that represents a pair combination that contains A(i) and B(j). • In a simplest way • SM (i,j) = 1; if A(i) = B(j) • SM(i,j) = 0; if A(I)  B(j)

25. MAT(i,j)=SM(Ai,Bj)+max(x,y,z) where X= row max along the diagonal– penalty Y = column max along the diagonal – penalty Z= next diagonal: MAT (i+1,j+1) GDVEKGKKIFIMKCSQ | ||||| ||| ||| GCVEKGK-IFINWCSQ

26. Trace back GDVEKGKKIFIMKCSQ | ||||| ||| ||| GCVEKGK-IFINWCSQ

27. Generation of Random sequences: How & Why • Obtain randomized sequences such that – • Length & composition is same • Why randomisation? • To filter chance similarity from biologically significant ones • To obtain statistical scores

28. Contd../ • Real Score ( R ) • Similarity Score of real sequences • Mean Score ( M ) • Average similarity score of randomly permuted sequences • Standard deviation ( Sd ) • Standard deviation of the similarity scores of randomly permuted sequences. • Alignment Score ( A ) • A = (R-M)/sd • Alignment score is expressed as number of standard deviation units by which the similarity score for real sequences (R) exceeds the average similarity score (M) of randomly permuted sequences

29. Significant Alignment Score • A< 3Sd • No homology • A> 3-6 Sd • May /may not be similar OR homologous • Need additional evidence to prove similarity/homology. • A> 6 Sd • Sequence are similar and may be homologous • Additional experimental evidence required to prove homology. • A> 9 Sd • Homology could be deduced from sequence alignment studies alone.

30. Calculation ofNormalized Alignment Score ( # Ident * 10) + (# C *25) – (# B * 20) NAS = ----------------------------------------------------* 100 Length of Alignment

31. Sample output

32. An example of high scoring alignment (7.55 sd) that actually shares no structural similarity between citrate synthase (2cts) and transthyritin (2paba). Note completely different secondary structures.

33. The distribution of S.D. scores for 100,000 optimal alignments of length >20 between proteins of unrelated three-dimensional structure

34. Evolutionary processOrthologues Gene X • A single Gene X is retained as the species diverges into two separate species • Genes in two species are Orthologues Gene X Gene X

35. Evolutionary processParalogues: genes that arise due to duplication Gene X • Single gene X in one species is duplicated • As each gene gathers mutations, it may begin to perform new function or may specialize in carrying out functions of ancestral genes • These genes in a single species are paralogues • If the species diverges, the daughter species may maintain the duplicated genes, therefore each species contain an Orthologue and a Paralogue to each gene in other species Gene X Gene X Gene A Gene B

36. Orthologous sequences are homologous sequences in different species that have a common origin Distinction of Orthologoes is a result of gradual evolutionary modifications from the common ancestor Perform same function in different species Paralogous sequences are homologous sequences that exists within a species They have a common origin but involve gene duplication events to arise Purpose of gene duplication is to use sequence to implement a new function Perform different functions Homologous/Orthologous/Paralogous sequences

37. Local Sequence Alignment Using Smith-Waterman Dynamic Programming Algorithm

38. Significance of local sequence alignment In locating common domains in proteins Example: transmembrane proteins, which might have different ends sticking out of the cell membrane, but have common 'middleparts' For comparing long DNA sequences with a short one Comparing a gene with a complete genome For detecting similarities between highly diverged sequences which still share common subsequences (that have little or no mutations).

39. Performs an exhaustive search for optimal local alignment • Modification of Needleman-Wunsch algorithm: • Negative weighting of mismatches • Matrix entries non-negative • Optimal path may start anywhere (not just first / last row/column) • After the whole path matrix is filled, the optimal local alignment is simply given by a path starting at the highest score overall in the path matrix, containing all the contributing cells until the path score has dropped to zero. Local sequence alignment

40. Smith-Waterman Algorithm

41. Example of local alignment

42. Scoring the alignment using BLOSUM50 matrix Gap penalty: -8

43. Summary: S & W • Fill the matrix using a similarity scoring matrix • Implement the dynamic programming algorithm • Find the maximal value in the matrix • Trace back from that value until a 0 value is reached • As we can start a new alignment anywhere the scores • cannot be negative. • Trace-back is started at the highest values rather than at • the lower right hand corner. • Trace-back is stopped as soon as a zero is encountered.