Pairwise sequence alignments

Pairwise sequence alignments Volker Flegel VI, October 2003 Page 1

Outline Introduction Definitions Biological context of pairwise alignments Computing of pairwise alignments Some programs VI, October 2003 Page 2

Importance of pairwise alignments Sequence analysis tools depending on pairwise comparison Multiple alignments Profile and HMM making (used to search for protein families and domains) 3D protein structure prediction Phylogenetic analysis Construction of certain substitution matrices Similarity searches in a database VI, October 2003 Page 3

Goal Sequence comparison through pairwise alignments Goal of pairwise comparison is to find conserved regions (if any) between two sequences Extrapolate information about our sequence using the known characteristics of the other sequence THIO_EMENI GFVVVDCFATWCGPCKAIAPTVEKFAQTY G ++VD +A WCGPCK IAP +++ A Y??? GAILVDFWAEWCGPCKMIAPILDEIADEY Extrapolate??? THIO_EMENI SwissProt VI, October 2003 Page 4

Do alignments make sense? Evolution of sequences Sequences evolve through mutation and selection Selective pressure is different for each residue position in a protein (i.e. conservation of active site, structure, charge, etc.) Modular nature of proteins Nature keeps re-using domains Alignments try to tell the evolutionary story of the proteins Relationships Same Sequence Same Origin Same Function Same 3D Fold VI, October 2003 Page 5

Example of an alignment - Textual view Two similar regions of the Drosophila melanogaster Slit and Notch proteins 970 970 980 980 990 990 1000 1000 1010 1010 1020 1020 SLIT_DROME SLIT_DROME FSCQCAPGYTGARCETNIDDCLGEIKCQNNATCIDGVESYKCECQPGFSGEFCDTKIQFC FSCQCAPGYTGARCETNIDDCLGEIKCQNNATCIDGVESYKCECQPGFSGEFCDTKIQFC..:.:..:.: :. :. :.: :.:...:.:...:.:.... : : :.. :.. : : ::.. ::.... :.: :.: ::..:. ::..:. :. :. :. :. : : NOTC_DROME NOTC_DROME YKCECPRGFYDAHCLSDVDECASN-PCVNEGRCEDGINEFICHCPPGYTGKRCELDIDEC YKCECPRGFYDAHCLSDVDECASN-PCVNEGRCEDGINEFICHCPPGYTGKRCELDIDEC 740 740 750 750 760 760 770 770 780 780 790 790 VI, October 2003 Page 6

Example of an alignment - graphical view Comparing the tissue-type and urokinase type plasminogen activators Tissue-Type plasminogen Activator Urokinase-Type plasminogen Activator URL: www.isrec.isb-sib.ch/java/dotlet/dotlet.html VI, October 2003 Page 7

Some definitions Identity Proportion of pairs of identical residues between two aligned sequences. Generally expressed as a percentage, this value strongly depends on how the two sequences are aligned. Similarity Proportion of pairs of similar residues between two aligned sequences. The residue similarity is determined by a substitution matrix. This value strongly depends on how the two sequences are aligned, as well as on the substitution matrix used. Homology Two sequences are homologous if and only if they have a common ancestor. It's either yes or no. There is no percentage of homology!!! Homologous sequences do not necessarily serve the same function...... Nor are they always highly similar: structure may be conserved while sequence is not. VI, October 2003 Page 8

More... Consider: a set S (say, globins: G) a test T that tries to detect members of S (for example, through a pairwise comparison with another globin). Globins True positives True negatives False positives False negatives G G X G G X G G G X G G X Matches VI, October 2003 Page 9

... definitions True positive A protein is a true positive if it belongs to S and is detected by t. True negative A protein is a true negative if it does not belong to S and is not detected by t. False positive A protein is a false positive if it does not belong to S and is (incorrectly) detected by t. False negative A protein is a false negative if it belongs to S and is not detected by t (but should be). VI, October 2003 Page 10

Even more definitions! Sensitivity Ability of a method to detect positives, irrespective of how many false positives are reported. Selectivity Ability of a method to reject negatives, irrespective of how many false negatives are rejected. True positives Greater sensitivity Less selectivity True negatives False positives False negatives Less sensitivity Greater selectivity VI, October 2003 Page 11

Pairwise sequence alignment Concept of sequence alignment Pairwise Alignment: Explicit mapping between the residues of 2 sequences deletion Seq A GARFIELDTHELASTFA-TCAT Seq B GARFIELDTHEVERYFASTCAT errors / mismatches insertion - Tolerant to errors (mismatches, insertion / deletions or indels) - Evaluation of the alignment in a biological concept (significance) VI, October 2003 Page 12

Pairwise sequence alignment Number of alignments There are many ways to align two sequences Consider the sequence fragments below: a simple alignment shows some conserved portions CGATGCAGACGTCA CGATGCAAGACGTCA but also: CGATGCAGACGTCA CGATGCAAGACGTCA Number of possible alignments for 2 sequences of length 1000 residues: more than 10 600 gapped alignments (Avogadro 10 24, estimated number of atoms in the universe 10 80 ) VI, October 2003 Page 13

Alignment evaluation What is a good alignment? We need a way to evaluate the biological meaning of a given alignment Intuitively we "know" that the following alignment: CGAGGCACAACGTCA CGATGCAAGACGTCA is better than: ATTGGACAGCAATCAGG ACGATGCAAGACGTCAG We can express this notion more rigorously, by using a scoring system VI, October 2003 Page 14

Scoring system Simple alignment scores A simple way (but not the best) to score an alignment is to count 1 for each match and 0 for each mismatch. CGAGGCACAACGTCA CGATGCAAGACGTCA Score: 12 ATTGGACAGCAATCAGG ACGATGCAAGACGTCAG Score: 5 VI, October 2003 Page 15

Introducing biological information Importance of the scoring system discrimination of significant biological alignments Based on physico-chemical properties of amino-acids Hydrophobicity, acid / base, sterical properties,... Scoring system scales are arbitrary Based on biological sequence information Substitutions observed in structural or evolutionary alignments of well studied protein families Scoring systems have a probabilistic foundation Substitution matrices In proteins some mismatches are more acceptable than others Substitution matrices give a score for each substitution of one amino-acid by another VI, October 2003 Page 16

Substitution matrices (log-odds matrices) Example matrix (Leu, Ile): 2 (Leu, Cys): -6... For a set of well known proteins: Align the sequences Count the mutations at each position For each substitution set the score to the log-odd ratio Ê log Á Ë observed expected by chance ˆ Positive score: the amino acids are similar, mutations from one into the other occur more often than expected by chance during evolution Negative score: the amino acids are dissimilar, the mutation from one into the other occurs less often than expected by chance during evolution PAM250 From: A. D. Baxevanis, "Bioinformatics" VI, October 2003 Page 17

Matrix choice Different kind of matrices: PAM series (M. Dayhoff, 1968, 1972, 1978) Percent Accepted Mutation. A unit introduced by Dayhoff et al. to quantify the amount of evolutionary change in a protein sequence. 1.0 PAM unit, is the amount of evolution which will change, on average, 1% of amino acids in a protein sequence. A PAM(x) substitution matrix is a look-up table in which scores for each amino acid substitution have been calculated based on the frequency of that substitution in closely related proteins that have experienced a certain amount (x) of evolutionary divergence. Based on 1572 protein sequences from 71 families Old standard matrix:pam250 VI, October 2003 Page 18

Matrix choice Different kind of matrices: BLOSUM series (Henikoff S. & Henikoff JG., PNAS, 1992) Blocks Substitution Matrix. A substitution matrix in which scores for each position are derived from observations of the frequencies of substitutions in blocks of local alignments in related proteins. Each matrix is tailored to a particular evolutionary distance. In the BLOSUM62 matrix, for example, the alignment from which scores were derived was created using sequences sharing no more than 62% identity. Sequences more identical than 62% are represented by a single sequence in the alignment so as to avoid over-weighting closely related family members. Based on alignments in the BLOCKS database Standard matrix: BLOSUM62 VI, October 2003 Page 19

Matrix choice Different kind of matrices: Limitations Substitution matrices do not take into account long range interactions between residues. They assume that identical residues are equal (a residue at the active site has other evolutionary constraints than the same residue outside of the active site) They assume evolution rate to be constant. VI, October 2003 Page 20

Alignment score Amino acid substitution matrices Example: PAM250 Most used: Blosum62 Raw score of an alignment TPEA _ _ APGA Score = 1 + 6 + 0 + 2 = 9 VI, October 2003 Page 21

Gaps Insertions or deletions Proteins often contain regions where residues have been inserted or deleted during evolution There are constraints on where these insertions and deletions can happen (between structural or functional elements like: alpha helices, active site, etc.) Gaps in alignments GCATGCATGCAACTGCAT GCATGCATGGGCAACTGCAT can be improved by inserting a gap GCATGCATG--CAACTGCAT GCATGCATGGGCAACTGCAT VI, October 2003 Page 22

Gap opening and extension penalties Costs of gaps in alignments We want to simulate as closely as possible the evolutionary mechanisms involved in gap occurrence. Example Two alignments with identical number of gaps but very different gap distribution. We may prefer one large gap to several small ones (e.g. poorly conserved loops between well-conserved helices) CGATGCAGCAGCAGCATCG CGATGC------AGCATCG CGATGCAGCAGCAGCATCG CG-TG-AGCA-CA--AT-G gap opening gap extension Gap opening penalty Counted each time a gap is opened in an alignment (some programs include the first extension into this penalty) Gap extension penalty Counted for each extension of a gap in an alignment VI, October 2003 Page 23

Gap opening and extension penalties Example With a match score of 1 and a mismatch score of 0 With an opening penalty of 10 and extension penalty of 1, we have the following score: CGATGCAGCAGCAGCATCG CGATGC------AGCATCG CGATGCAGCAGCAGCATCG CG-TG-AGCA-CA--AT-G gap opening gap extension 13 x 1-10 - 6 x 1 = -3 13 x 1-5 x 10-6 x 1 = -43 VI, October 2003 Page 24

Statistical evaluation of results Alignments are evaluated according to their score Raw score It's the sum of the amino acid substitution scores and gap penalties (gap opening and gap extension) Depends on the scoring system (substitution matrix, etc.) Different alignments should not be compared based only on the raw score It is possible that a bad long alignment gets a better raw score than a very good short alignment => we need a normalized score to compare alignments! (p-value, e-value) Normalized score Is independent of the scoring system Allows the comparison of different alignments Units: expressed in bits VI, October 2003 Page 25

Statistical evaluation of results Statistics derived from the scores p-value Probability that an alignment with this score or better occurs by chance in a database of this size The closer the p-value is towards 0, the better the alignment e-value (expectation value) Number of matches with this score one can expect to find by chance in a database of this size The closer the e-value is towards 0, the better the alignment VI, October 2003 Page 26

Diagonal plots or Dotplot Concept of a Dotplot Produces a graphical representation of similarity regions. The horizontal and vertical dimensions correspond to the compared sequences. A region of of similarity stands out as a diagonal. Urokinase-Type plasminogen Activator Tissue-Type plasminogen Activator VI, October 2003 Page 27

Dotplot construction Simple example A dot is placed at each position where two residues match. The color of the dot can be chosen according to the substitution value in the substitution matrix Note T H E F A S T C A T T H E F A T C A T THEFA-TCAT THEFASTCAT This method produces dotplots with too much noise to be useful The noise can be reduced by calculating a score using a window of residues The score is compared to a threshold or stringency VI, October 2003 Page 28

Dotplot construction Window example Each window of the first sequence is aligned (without gaps) to each window of the 2nd sequence T H E F A S T C A T T H E F A T C A T HEF THE CAT HEF THE Score: 13-5 -3 A color is set into a rectangular array according to the score of the aligned windows VI, October 2003 Page 29

Dotplot limitations It's a visual aid. The human eye can rapidly identify similar regions in sequences. It's a good way to explore sequence organization. It does not provide an alignment. Urokinase-Type plasminogen Activator Tissue-Type plasminogen Activator VI, October 2003 Page 30

Creating an alignment Relationship between alignment and dotplot An alignment can be seen as a path through the dotplot diagramm. Seq Seq B B Seq Seq A A-CA-CA ACA--CA A-CA-CA ACA--CA ACCAAC- A-CCAAC ACCAAC- A-CCAAC VI, October 2003 Page 31

Finding an alignment Alignment algorithms An alignment program tries to find the best alignment between two sequences given the scoring system. This can be seen as trying to find a path through the dotplot diagram including all (or the most visible) diagonals. Alignment types Global Alignment between the complete sequence A and the complete sequence B Local Alignment between a sub-sequence of A an a subsequence of B Computer implementation (Algorithms) Dynamic programming Global Needleman-Wunsch Local Smith-Waterman VI, October 2003 Page 32

Global alignment (Needleman-Wunsch) Example Global alignments are very sensitive to gap penalties Global alignments do not take into account the modular nature of proteins Tissue-Type plasminogen Activator Urokinase-Type plasminogen Activator Global alignment: VI, October 2003 Page 33

Local alignment (Smith-Waterman) Example Local alignments are more sensitive to the modular nature of proteins They can be used to search databases Urokinase-Type plasminogen Activator Local alignments: Tissue-Type plasminogen Activator VI, October 2003 Page 34

Optimal alignment extension How to optimally extend an optimal alignment An optimal alignment up to positions i and j can be extended in 3 ways. Keeping the best of the 3 guarantees an extended optimal alignment. Seq A Seq B a 1 a 2 a 3... a i-1 a i b 1 b 2 b 3... b j-1 b j s Seq A a 1 a 2 a 3... a i-1 a i a i+1 Seq B b 1 b 2 b 3... b j-1 b j b j+1 Score = Score ij + Subst i+1 j+1 b j+1 d Seq A Seq B a 1 a 2 a 3... a i-1 a i b 1 b 2 b 3... b j-1 b j a i+1 - Score = Score ij - gap d Seq A Seq B a 1 a 2 a 3... a i-1 a i b 1 b 2 b 3... b j-1 b j - b j+1 Score = Score ij - gap We have the optimal alignment extended from i and j by one residue. VI, October 2003 Page 35

Exact algorithms Simple example (Needleman-Wunsch) Scoring system: Match score: 2 Mismatch score: -1 Gap penalty: -2 2 + 2 G A T T A 0-2 -4-6 -8-10 0-2 G -2 2 0-2 -4-6 A -4 0 4 2 0-2 F (i-1,j-1) s (xi,yj) F (i-1,j) -d F (i,j-1) -d F(i,j) F(i,j): score at position i, j s(x i,y j ): match or mismatch score (or substitution matrix value) for residues x i and y j d: gap penalty (positive value) Note A -6-2 2 3 1 2 0-2 T -8-4 0 4 5 3 T -10-6 -2 2 6 4 C -12-8 -4 0 4 5 GA-TTA GAATTC We have to keep track of the origin of the score for each element in the matrix. This allows to build the alignment by traceback when the matrix has been completely filled out. Computation time is proportional to the size of sequences (n x m). VI, October 2003 Page 36

Concluding remarks Substitution matrices and gap penalties introduce biological information into the alignment algorithms. It is not because two sequences can be aligned that they share a common biological history. The relevance of the alignment must be assessed with a statistical score. There are many ways to align two sequences. Do not blindly trust your alignment to be the only truth. Especially gapped regions may be quite variable. Sequences sharing less than 20% similarity are difficult to align: You enter the Twilight Zone (Doolittle, 1986) Alignments may appear plausible to the eye but are no longer statistically significant. Other methods are needed to explore these sequences (i.e: profiles) Know your data! At the very end, YOU will decide, not the computer Web resources LALIGN - pairwise sequence alignment: www.ch.embnet.org/software/lalign_form.html PRSS - alignment score evaluation: www.ch.embnet.org/software/prss_form.html VI, October 2003 Page 37