2. Multiple Sequence Alignment benchmarking, pattern recognition and Phylogeny Introduction to bioinformatics 2008 Lecture 11 C E N T R F O R I N T E G R A T I V E B I O I N F O R M A T I C S V U E

3. Evaluating multiple alignments There are reference databases based on structural information: e.g. BAliBASE and HOMSTRAD Conflicting standards of truth –evolution –structure –function With orphan sequences no additional information Benchmarks depending on reference alignments Quality issue of available reference alignment databases Different ways to quantify agreement with reference alignment (sum-of-pairs, column score) “Charlie Chaplin” problem

4. Evaluating multiple alignments As a standard of truth, often a reference alignment based on structural superpositioning is taken These superpositionings can be scored using the root-mean- square-deviation (RMSD) of atoms that are equivalenced (taken as corresponding) in a pair of protein structures. Typically, C atoms only are used for superpositioning (main-chain trace).

5. BAliBASE benchmark alignments Thompson et al. (1999) NAR 27, 2682. 8 categories: cat. 1 - equidistant cat. 1 - equidistant cat. 2 - orphan sequence cat. 2 - orphan sequence cat. 3 - 2 distant groups cat. 3 - 2 distant groups cat. 4 – long overhangs cat. 4 – long overhangs cat. 5 - long insertions/deletions cat. 5 - long insertions/deletions cat. 6 – repeats cat. 6 – repeats cat. 7 – transmembrane proteins cat. 7 – transmembrane proteins cat. 8 – circular permutations cat. 8 – circular permutations

6. BAliBASE BB11001 1aab_ref1Ref1 V1 SHORT high mobility group protein BB11002 1aboA_ref1 Ref1 V1 SHORT SH3 BB11003 1ad3_ref1 Ref1 V1 LONG aldehyde dehydrogenase BB11004 1adj_ref1 Ref1 V1 LONG histidyl-trna synthetase BB11005 1ajsA_ref1 Ref1 V1 LONG aminotransferase BB11006 1bbt3_ref1 Ref1 V1 MEDIUM foot-and-mouth disease virus BB11007 1cpt_ref1 Ref1 V1 LONG cytochrome p450 BB11008 1csy_ref1 Ref1 V1 SHORT SH2 BB11009 1dox_ref1 Ref1 V1 SHORT ferredoxin [2fe-2s]......

7. T-Coffee: correctly aligned Kinase nucleotide binding sites

8. Scoring a single MSA with the Sum-of-pairs (SP) score Sum-of-Pairs score Calculate the sum of all pairwise alignment scores This is equivalent to taking the sum of all matched a.a. pairs The latter can be done using gap penalties or not Good alignments should have a high SP score, but it is not always the case that the true biological alignment has the highest score.

9. Evaluation measures QueryReference Column score Sum-of-Pairs score What fraction of the MSA columns in the reference alignment is reproduced by the computed alignment What fraction of the matched amino acid pairs in the reference alignment is reproduced by the computed alignment

10. Evaluating multiple alignments

11.  SP BAliBASE alignment nseq * len Evaluating multiple alignments Charlie Chaplin problem

13. T-coffee global, local or both

14. Comparing T-coffee with other methods

15. BAliBASE benchmark alignments

16. Summary Individual alignments can be scored with the SP score. –Better alignments should have better SP scores –However, there is the Charlie Chaplin problem A test and a reference multiple alignment can be scored using the SP score or the column score (now for pairs of alignments) Evaluations show that there is no MSA method that always wins over others in terms of alignment quality

17. Pattern Recognition Introduction to bioinformatics 2008 C E N T R F O R I N T E G R A T I V E B I O I N F O R M A T I C S V U E

18. Patterns Some are easy some are not Knitting patterns Cooking recipes Pictures (dot plots) Colour patterns Maps In 2D and 3D humans are hard to be beat by a computational pattern recognition technique, but humans are not so consistent

19. Example of algorithm reuse: Data clustering Many biological data analysis problems can be formulated as clustering problems –microarray gene expression data analysis –identification of regulatory binding sites (similarly, splice junction sites, translation start sites,......) –(yeast) two-hybrid data analysis (experimental technique for inference of protein complexes) –phylogenetic tree clustering (for inference of horizontally transferred genes) –protein domain identification –identification of structural motifs –prediction reliability assessment of protein structures –NMR peak assignments –......

20. Data Clustering Problems Clustering: partition a data set into clusters so that data points of the same cluster are “similar” and points of different clusters are “dissimilar” Cluster identification -- identifying clusters with significantly different features than the background

21. Application Examples Regulatory binding site identification: CRP (CAP) binding site Two hybrid data analysis l Gene expression data analysis These problems are all solvable by a clustering algorithm

22. Multivariate statistics – Cluster analysis 1234512345 C1 C2 C3 C4 C5 C6.. Raw table Any set of numbers per column Multi-dimensional problems Objects can be viewed as a cloud of points in a multidimensional space Need ways to group the data

23. Multivariate statistics – Cluster analysis Dendrogram Scores Similarity matrix 5×5 1234512345 C1 C2 C3 C4 C5 C6.. Raw table Similarity criterion Cluster criterion Any set of numbers per column

24. Comparing sequences - Similarity Score - Many properties can be used: Nucleotide or amino acid composition Isoelectric point Molecular weight Morphological characters But: molecular evolution through sequence alignment

25. Multivariate statistics – Cluster analysis Now for sequences Phylogenetic tree Scores Similarity matrix 5×5 Multiple sequence alignment 1234512345 Similarity criterion Cluster criterion

26. Human -KITVVGVGAVGMACAISILMKDLADELALVDVIEDKLKGEMMDLQHGSLFLRTPKIVSGKDYNVTANSKLVIITAGARQ Chicken -KISVVGVGAVGMACAISILMKDLADELTLVDVVEDKLKGEMMDLQHGSLFLKTPKITSGKDYSVTAHSKLVIVTAGARQ Dogfish –KITVVGVGAVGMACAISILMKDLADEVALVDVMEDKLKGEMMDLQHGSLFLHTAKIVSGKDYSVSAGSKLVVITAGARQ Lamprey SKVTIVGVGQVGMAAAISVLLRDLADELALVDVVEDRLKGEMMDLLHGSLFLKTAKIVADKDYSVTAGSRLVVVTAGARQ Barley TKISVIGAGNVGMAIAQTILTQNLADEIALVDALPDKLRGEALDLQHAAAFLPRVRI-SGTDAAVTKNSDLVIVTAGARQ Maizey casei -KVILVGDGAVGSSYAYAMVLQGIAQEIGIVDIFKDKTKGDAIDLSNALPFTSPKKIYSA-EYSDAKDADLVVITAGAPQ Bacillus TKVSVIGAGNVGMAIAQTILTRDLADEIALVDAVPDKLRGEMLDLQHAAAFLPRTRLVSGTDMSVTRGSDLVIVTAGARQ Lacto__ste -RVVVIGAGFVGASYVFALMNQGIADEIVLIDANESKAIGDAMDFNHGKVFAPKPVDIWHGDYDDCRDADLVVICAGANQ Lacto_plant QKVVLVGDGAVGSSYAFAMAQQGIAEEFVIVDVVKDRTKGDALDLEDAQAFTAPKKIYSG-EYSDCKDADLVVITAGAPQ Therma_mari MKIGIVGLGRVGSSTAFALLMKGFAREMVLIDVDKKRAEGDALDLIHGTPFTRRANIYAG-DYADLKGSDVVIVAAGVPQ Bifido -KLAVIGAGAVGSTLAFAAAQRGIAREIVLEDIAKERVEAEVLDMQHGSSFYPTVSIDGSDDPEICRDADMVVITAGPRQ Thermus_aqua MKVGIVGSGFVGSATAYALVLQGVAREVVLVDLDRKLAQAHAEDILHATPFAHPVWVRSGW-YEDLEGARVVIVAAGVAQ Mycoplasma -KIALIGAGNVGNSFLYAAMNQGLASEYGIIDINPDFADGNAFDFEDASASLPFPISVSRYEYKDLKDADFIVITAGRPQ Lactate dehydrogenase multiple alignment Distance Matrix 1 2 3 4 5 6 7 8 9 10 11 12 13 1 Human 0.000 0.112 0.128 0.202 0.378 0.346 0.530 0.551 0.512 0.524 0.528 0.635 0.637 2 Chicken 0.112 0.000 0.155 0.214 0.382 0.348 0.538 0.569 0.516 0.524 0.524 0.631 0.651 3 Dogfish 0.128 0.155 0.000 0.196 0.389 0.337 0.522 0.567 0.516 0.512 0.524 0.600 0.655 4 Lamprey 0.202 0.214 0.196 0.000 0.426 0.356 0.553 0.589 0.544 0.503 0.544 0.616 0.669 5 Barley 0.378 0.382 0.389 0.426 0.000 0.171 0.536 0.565 0.526 0.547 0.516 0.629 0.575 6 Maizey 0.346 0.348 0.337 0.356 0.171 0.000 0.557 0.563 0.538 0.555 0.518 0.643 0.587 7 Lacto_casei 0.530 0.538 0.522 0.553 0.536 0.557 0.000 0.518 0.208 0.445 0.561 0.526 0.501 8 Bacillus_stea 0.551 0.569 0.567 0.589 0.565 0.563 0.518 0.000 0.477 0.536 0.536 0.598 0.495 9 Lacto_plant 0.512 0.516 0.516 0.544 0.526 0.538 0.208 0.477 0.000 0.433 0.489 0.563 0.485 10 Therma_mari 0.524 0.524 0.512 0.503 0.547 0.555 0.445 0.536 0.433 0.000 0.532 0.405 0.598 11 Bifido 0.528 0.524 0.524 0.544 0.516 0.518 0.561 0.536 0.489 0.532 0.000 0.604 0.614 12 Thermus_aqua 0.635 0.631 0.600 0.616 0.629 0.643 0.526 0.598 0.563 0.405 0.604 0.000 0.641 13 Mycoplasma 0.637 0.651 0.655 0.669 0.575 0.587 0.501 0.495 0.485 0.598 0.614 0.641 0.000 How can you see that this is a distance matrix?

28. Multivariate statistics – Cluster analysis Dendrogram/tree Scores Similarity matrix 5×5 1234512345 C1 C2 C3 C4 C5 C6.. Data table Similarity criterion Cluster criterion

29. Multivariate statistics – Cluster analysis Why do it? Finding a true typology Model fitting Prediction based on groups Hypothesis testing Data exploration Data reduction Hypothesis generation But you can never prove a classification/typology!

30. Cluster analysis – data normalisation/weighting 1234512345 C1 C2 C3 C4 C5 C6.. Raw table Normalisation criterion 1234512345 C1 C2 C3 C4 C5 C6.. Normalised table Column normalisationx/max Column range normalise(x-min)/(max-min)

31. Cluster analysis – (dis)similarity matrix Scores Similarity matrix 5×5 1234512345 C1 C2 C3 C4 C5 C6.. Raw table Similarity criterion D i,j = (  k | x ik – x jk | r ) 1/r Minkowski metrics r = 2 Euclidean distance r = 1 City block distance

32. (dis)similarity matrix D i,j = (  k | x ik – x jk | r ) 1/r Minkowski metrics r = 2 Euclidean distance r = 1 City block distance EXAMPLE: lengthheightwidth Cow11173 Cow 2745 Euclidean dist. = sqrt(4 2 + 3 2 + -2 2 ) = sqrt(29) = 5.39 City Block dist. = |4|+|3|+|-2| = 9 4 3 -2

33. Cluster analysis – Clustering criteria Dendrogram (tree) Scores Similarity matrix 5×5 Cluster criterion Single linkage - Nearest neighbour Complete linkage – Furthest neighbour Group averaging – UPGMA Neighbour joining – global measure, used to make a Phylogenetic Tree

34. Cluster analysis – Clustering criteria Dendrogram (tree) Scores Similarity matrix 5×5 Cluster criterion Four different clustering criteria: Single linkage - Nearest neighbour Complete linkage – Furthest neighbour Group averaging – UPGMA Neighbour joining (global measure) Note: these are all agglomerative cluster techniques; i.e. they proceed by merging clusters as opposed to techniques that are divisive and proceed by cutting clusters

35. Cluster analysis – Clustering criteria 1.Start with N clusters of 1 object each 2.Apply clustering distance criterion iteratively until you have 1 cluster of N objects 3.Most interesting clustering somewhere in between Dendrogram (tree) distance N clusters1 cluster Note: a dendrogram can be rotated along branch points (like mobile in baby room) -- distances between objects are defined along branches

36. Single linkage clustering (nearest neighbour) Char 1 Char 2

37. Single linkage clustering (nearest neighbour) Char 1 Char 2

38. Single linkage clustering (nearest neighbour) Char 1 Char 2

39. Single linkage clustering (nearest neighbour) Char 1 Char 2

40. Single linkage clustering (nearest neighbour) Char 1 Char 2

41. Single linkage clustering (nearest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the smallest distance between that point and any point in the cluster

42. Single linkage clustering (nearest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the smallest distance between that point and any point in the cluster

43. Single linkage clustering (nearest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the smallest distance between that point and any point in the cluster

44. Single linkage clustering (nearest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the smallest distance between that point and any point in the cluster

45. Single linkage clustering (nearest neighbour) Single linkage dendrograms typically show chaining behaviour (i.e., all the time a single object is added to existing cluster) Let C i and C j be two disjoint clusters: d i,j = Min(d p,q ), where p  C i and q  C j

46. Complete linkage clustering (furthest neighbour) Char 1 Char 2

47. Complete linkage clustering (furthest neighbour) Char 1 Char 2

48. Complete linkage clustering (furthest neighbour) Char 1 Char 2

49. Complete linkage clustering (furthest neighbour) Char 1 Char 2

50. Complete linkage clustering (furthest neighbour) Char 1 Char 2

51. Complete linkage clustering (furthest neighbour) Char 1 Char 2

52. Complete linkage clustering (furthest neighbour) Char 1 Char 2

53. Complete linkage clustering (furthest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the largest distance between that point and any point in the cluster

54. Complete linkage clustering (furthest neighbour) Char 1 Char 2 Distance from point to cluster is defined as the largest distance between that point and any point in the cluster

55. Complete linkage clustering (furthest neighbour) More ‘structured’ clusters than with single linkage clustering Let C i and C j be two disjoint clusters: d i,j = Max(d p,q ), where p  C i and q  C j

56. Clustering algorithm 1.Initialise (dis)similarity matrix 2.Take two points with smallest distance as first cluster (later, points can be clusters) 3.Merge corresponding rows/columns in (dis)similarity matrix 4.Repeat steps 2. and 3. using appropriate cluster measure when you need to calculate new point-to-cluster or cluster-to-cluster distances until last two clusters are merged

57. Average linkage clustering ( Unweighted Pair Group Mean Averaging -UPGMA) Char 1 Char 2 Distance from cluster to cluster is defined as the average distance over all within-cluster distances

58. UPGMA Let C i and C j be two disjoint clusters: 1 d i,j = ————————  p  q d p,q, where p  C i and q  C j |Ci| × |Cj| In words: calculate the average over all pairwise inter-cluster distances CiCi CjCj

59. Multivariate statistics – Cluster analysis Phylogenetic tree Scores Similarity matrix 5×5 1234512345 C1 C2 C3 C4 C5 C6.. Data table Similarity criterion Cluster criterion

60. Multivariate statistics – Cluster analysis Scores 5×5 1234512345 C1 C2 C3 C4 C5 C6 Similarity criterion Cluster criterion Scores 6×6 Cluster criterion Make two-way ordered table using dendrograms

61. Multivariate statistics – Two-way cluster analysis 1425314253 C4 C3 C6 C1 C2 C5 Make two-way (rows, columns) ordered table using dendrograms; This shows ‘blocks’ of numbers that are similar

62. Multivariate statistics – Two-way cluster analysis

63. Multivariate statistics – Principal Component Analysis (PCA) 1234512345 C1 C2 C3 C4 C5 C6 Similarity Criterion: Correlations 6×6 Calculate eigenvectors with greatest eigenvalues: Linear combinations Orthogonal Correlations Project data points onto new axes (eigenvectors) 1 2

64. Multivariate statistics – Principal Component Analysis (PCA)

65. Evolution/Phylogeny Introduction to bioinformatics 2008 C E N T R F O R I N T E G R A T I V E B I O I N F O R M A T I C S V U E

66. “Nothing in Biology makes sense except in the light of evolution” (Theodosius Dobzhansky (1900-1975)) “Nothing in bioinformatics makes sense except in the light of Biology” Bioinformatics

67. Evolution Most of bioinformatics is comparative biology Comparative biology is based upon evolutionary relationships between compared entities Evolutionary relationships are normally depicted in a phylogenetic tree

68. Where can phylogeny be used For example, finding out about orthology versus paralogy Predicting secondary structure of RNA Predicting protein-protein interaction Studying host-parasite relationships Mapping cell-bound receptors onto their binding ligands Multiple sequence alignment (e.g. Clustal)

69. DNA evolution Gene nucleotide substitutions can be synonymous (i.e. not changing the encoded amino acid) or nonsynonymous (i.e. changing the a.a.). Rates of evolution vary tremendously among protein- coding genes. Molecular evolutionary studies have revealed an ∼ 1000-fold range of nonsynonymous substitution rates (Li and Graur 1991). The strength of negative (purifying) selection is thought to be the most important factor in determining the rate of evolution for the protein-coding regions of a gene (Kimura 1983; Ohta 1992; Li 1997).

70. DNA evolution “Essential” and “nonessential” are classic molecular genetic designations relating to organismal fitness. –A gene is considered to be essential if a knock-out results in (conditional) lethality or infertility. –Nonessential genes are those for which knock-outs yield viable and fertile individuals. Given the role of purifying selection in determining evolutionary rates, the greater levels of purifying selection on essential genes leads to a lower rate of evolution relative to that of nonessential genes This leads to the observation: “What is important is conserved”.

71. Reminder -- Orthology/paralogy Orthologous genes are homologous (corresponding) genes in different species Paralogous genes are homologous genes within the same species (genome)

72. Old Dogma – Recapitulation Theory (1866) Ernst Haeckel: “Ontogeny recapitulates phylogeny” Ontogeny is the development of the embryo of a given species; phylogeny is the evolutionary history of a species http://en.wikipedia.org/wiki/Recapitulation_theory Haeckels drawing in support of his theory: For example, the human embryo with gill slits in the neck was believed by Haeckel to not only signify a fishlike ancestor, but it represented a total fishlike stage in development. However,gill slits are not the same as gills and are not functional.

73. Phylogenetic tree (unrooted) human mousefugu Drosophila edge internal node leaf OTU – Observed taxonomic unit

74. Phylogenetic tree (unrooted) human mousefugu Drosophila root edge internal node leaf OTU – Observed taxonomic unit

75. Phylogenetic tree (rooted) human mouse fugu Drosophila root edge internal node (ancestor) leaf OTU – Observed taxonomic unit time

76. How to root a tree Outgroup – place root between distant sequence and rest group Midpoint – place root at midpoint of longest path (sum of branches between any two OTUs) Gene duplication – place root between paralogous gene copies f D m h Dfmh f D m h Dfmh f-  h-  f-  h-  f-  h-  f-  h-  5 3 2 1 1 4 1 2 1 3 1

77. Combinatoric explosion Number of unrooted trees = Number of rooted trees =

78. Combinatoric explosion # sequences# unrooted# rooted trees trees 211 313 4315 515105 6105945 794510,395 810,395135,135 9135,1352,027,025 102,027,02534,459,425

79. Tree distances humanx mouse6 x fugu7 3 x Drosophila14 10 9 x human mouse fugu Drosophila 5 1 1 2 6 human mousefuguDrosophila Evolutionary (sequence distance) = sequence dissimilarity 1 Note that with evolutionary methods for generating trees you get distances between objects by walking from one to the other.

80. Phylogeny take home messages Orthology/paralogy Rooted/unrooted trees, how to root trees Combinatorial explosion in number of possible tree topologies (not taking branch lengths into account)