1. BASIC METHODOLOGIES OF ANALYSIS: SUPERVISED ANALYSIS: HYPOTHESIS TESTING USING CLINICAL INFORMATION (MLL VS NO TRANS.) IDENTIFY DIFFERENTIATING GENES Basic methodologies1 SUPERVISED METHODS CAN ONLY VALIDATE OR REJECT HYPOTHESES. CAN NOT LEAD TO DISCOVERY OF UNEXPECTED PARTITIONS. UNSUPERVISED: EXPLORATORY ANALYSIS NO PRIOR KNOWLEDGE IS USED EXPLORE STRUCTURE OF DATA ON THE BASIS OF CORRELATIONS AND SIMILARITIES

2. AIMS: ASSIGN PATIENTS TO GROUPS ON THE BASIS OF THEIR EXPRESSION PROFILES IDENTIFY DIFFERENCES BETWEEN TUMORS AT DIFFERENT STAGES IDENTIFY GENES THAT PLAY CENTRAL ROLES IN DISEASE PROGRESSION EACH PATIENT IS DESCRIBED BY 30,000 NUMBERS: ITS EXPRESSION PROFILE

3. UNSUPERVISED ANALYSIS GOAL A: FIND GROUPS OF GENES THAT HAVE CORRELATED EXPRESSION PROFILES. THESE GENES ARE BELIEVED TO BELONG TO THE SAME BIOLOGICAL PROCESS. GOAL B: DIVIDE TISSUES TO GROUPS WITH SIMILAR GENE EXPRESSION PROFILES. THESE TISSUES ARE EXPECTED TO BE IN THE SAME BIOLOGICAL (CLINICAL) STATE. CLUSTERING, SORTING Unsupervised analysis

4. Giraffe DEFINITION OF THE CLUSTERING PROBLEM

5. CLUSTER ANALYSIS YIELDS DENDROGRAM Dendrogram1 T (RESOLUTION) LINEAR ORDERING OF DATA

6. Giraffe + Okapi BUT WHAT ABOUT THE OKAPI?

7. STATEMENT OF THE PROBLEM GIVEN DATA POINTS X i, i=1,2,...N, EMBEDDED IN D - DIMENSIONAL SPACE, IDENTIFY THE UNDERLYING STRUCTURE OF THE DATA. AIMS:PARTITION THE DATA INTO M CLUSTERS, POINTS OF SAME CLUSTER - "MORE SIMILAR“ M ALSO TO BE DETERMINED! GENERATE DENDROGRAM, IDENTIFY SIGNIFICANT, “STABLE” CLUSTERS "ILL POSED": WHAT IS "MORE SIMILAR"? RESOLUTION

8. CLUSTER ANALYSIS YIELDS DENDROGRAM Dendrogram2 T STABILITY T LINEAR ORDERING OF DATA YOUNG OLD

9. CENTROID (REPRESENTATIVE) –SELF ORGANIZED MAPS (KOHONEN 1997; (GENES: GOLUB ET. AL., SCIENCE 1999) –K-MEANS (GENES; TAMAYO ET. AL., PNAS 1999) AGGLOMERATIVE HIERARCHICAL -AVERAGE LINKAGE (GENES: EISEN ET. AL., PNAS 1998) PHYSICALLY MOTIVATED –DETERMINISTIC ANNEALING (ROSE ET. AL.,PRL 1990; GENES: ALON ET. AL., PNAS 1999) –SUPER-PARAMAGNETIC CLUSTERING (SPC)(BLATT ET.AL. GENES: GETZ ET. AL., PHYSICA 2000,PNAS 2000) --COUPLED MAPS (ANGELINI ET. AL., PRL 2000) CLUSTERING METHODS Clustering methods

10. INFORMATION THEORY –AGGLOMERATIVE INFORMATION BOTTLENECK (TISHBY ET. AL.) LINEAR ALGEBRA –SPECTRAL METHODS (MALIK ET. AL.) MULTIGRID BASED METHODS (BRANDT ET. AL., ) CLUSTERING METHODS (Cont) Clustering methods

11. Centroid methods – K-means i = 1,...,N DATA POINTS, AT X i = 1,...,K CENTROIDS, AT Y ASSIGN DATA POINT i TO CENTROID ; S i = COST E: E(S 1, S 2,...,S N ; Y 1,...Y K ) = MINIMIZE E OVER S i, Y

12. K-means “GUESS” K=3

13. K-means Iteration = 0 Start with random positions of centroids.

14. K-means Iteration = 1 Start with random positions of centroids. Assign each data point to closest centroid

15. K-means Iteration = 1 Start with random positions of centroids. Assign each data point to closest centroid Move centroids to center of assigned points

16. K-means; algorithm to find minima Iteration = 3 Start with random positions of centroids. Assign each data point to closest centroid Move centroids to center of assigned points Iterate till minimal cost

17. E=Total Sum of Squares vs K

18. Result depends on initial centroids’ position Fast algorithm: compute distances from data points to centroids O(N) operations (vs O(N 2 )) Must preset K Fails for non-spherical distributions K-means - Summary

19. 5 24 13 Agglomerative Hierarchical Clustering 3 1 4 2 5 Distance between joined clusters Dendrogram The dendrogram induces a linear ordering of the data points at each step merge pair of nearest clusters initially – each point = cluster Need to define the distance between the new cluster and the other clusters. Single Linkage: distance between closest pair. Complete Linkage: distance between farthest pair. Average Linkage: average distance between all pairs or distance between cluster centers Need to define the distance between the new cluster and the other clusters. Single Linkage: distance between closest pair. Complete Linkage: distance between farthest pair. Average Linkage: average distance between all pairs or distance between cluster centers

21. COMPACT WELL SEPARATED CLOUDS – EVERYTHING WORKS

22. 2 flat clouds 2 FLAT CLOUDS - SINGLE LINKAGE WORKS

23. average linkage

25. filament SINGLE LINKAGE SENSITIVE TO NOISE

26. filament SINGLE LINKAGE SENSITIVE TO NOISE

27. filament with one point removed SINGLE LINKAGE SENSITIVE TO NOISE

28. Hierarchical Clustering - Summary Results depend on distance update method Greedy iterative process NOT robust against noise No inherent measure to identify stable clusters Average Linkage – the most widely used clustering method in gene expression analysis

29. nature 2002 breast cancer

30. how many clusters? 3 LARGE MANY small SuperParamagnetic Clustering (SPC) toy problem SPC

31. other methods

32. Graph based clustering Undirected graph: Vertices (nodes). Edges. A cut. J i,j i j

33. Graph based clustering (cont.) i=1,2,...N data points = vertices (nodes) of graph J i,j – weight associated with edge i,j 5 1 8 J 5,8 J i,j depends on distance D i,j J i,j D i,j A cut in the graph represents a clustering solution (partition).

34. = cut edge high cost (high resolution) low cost (low resolution) COST OF A CUT, I.E, PARTITION = WEIGHTS OF ALL CUT EDGES

35. highest cost = sum of all edges. each point is a cluster lowest cost = 0 One cluster. Conclusion –minimization/maximization of the cost are meaningless

36. Clustering: The SPC spirit M.Blatt, S.Weisman and E.Domany (1996) SPC’s idea – consider ALL cuts, i.e. partitions {S}. Each partition appears with probability p({S}). Measure the correlation between points i,j connected by an edge, over all partitions: Cij = probability that the edge i-j was NOT cut. {S} 1 : p({S} 1 ) i j i j i j i j {S} 2 : p({S} 2 ){S} 3 : p({S} 3 ){S} 4 : p({S} 4 ) Cij = p({S} 2 )+ p({S} 3 )+ p({S} 4 )

37. Clustering: The SPC spirit (cont) We have a graph, whose edge values are the correlations 0.45 0.75 1 0.8 0.9 0.2 0.45 0.850.7 0.9 1 Create the clustering solution by deleting edges for which Cij < 0.5

38. What is p({S}) ? COST OF {S} = H({S}) CORRESPONDS TO THE RESOLUTION SOUNDS REASONABLE TO FIND A SOLUTION FOR EACH VALUE OF THE COST/RESOLUTION, E. FIX H=E, AND GENERATE PARTITIONS FOR WHICH H({S})=E. P({X}) =1/(# PARTITIONS WITH H({S})=E), IF H({X})=E 0 OTHERWISE

39. What is p({S}) ? (Cont) Due to computational issues it is easier to generate partitions for with an AVERAGE cost E: INSTEAD OF FINDING PARTITIONS WITH H({S})=E FIND PARTITIONS WITH =E P({X})=exp [-H({X})/T ] /Z Boltzmann distribution T is the temperature = the resolution parameter

40. Outline of SPC Go over resolutions T (minT to maxT is steps of deltaT): Generate thousands (Cycles) of partitions with average cost that corresponds to the current resolution. Calculate pair correlations : C i,j (T). Clusters(T): connected components of C i,j > 0.5 Map data to a graph G.

41. Example: N=4800 points in D=2 Super-Paramagnetic Clustering (SPC)

42. Output of SPC Size of largest clusters as function of T Dendrogram Stable clusters “live” for large  T A function  (T) that peaks when stable clusters break

43. Identify the stable clusters

44. Same data - Average Linkage Examining this cluster No analog to  (T)

45. Advantages of SPC Scans all resolutions (T) Robust against noise and initialization - calculates collective correlations. Identifies “natural” (  ) and stable clusters (  T) No need to pre-specify number of clusters Clusters can be any shape Can use distance matrix as input (vs coordinates)