1. Normalization Review and Cluster Analysis Class web site: http://statwww.epfl.ch/davison/teaching/Microarrays/ Statistics for Microarrays

2. Normalization Methods Global adjustment (e.g. median or mean for a set of genes) Intensity-dependent -- shift M by c=c(A); e.g. c(A) made with lowess Within print-tip group normalization to correct for spatial bias Both print-tip and intensity-dependent adjustment: perform LOWESS fits to the data within print-tip groups

3. Print-tip-group normalization

5. Normalization: Which Spots to use? The LOWESS lines can be run through many different sets of points, and each strategy has its own implicit set of assumptions justifying its applicability. For example, the use of a global LOWESS approach can be justified by supposing that, when stratified by mRNA abundance, a) only a minority of genes are expected to be differentially expressed, or b) any differential expression is as likely to be up- regulation as down-regulation. Pin-group LOWESS requires stronger assumptions: that one of the above applies within each pin-group. The use of other sets of genes, e.g. control or housekeeping genes, involve similar assumptions.

6. Pre-processed cDNA Gene Expression Data On p genes for n slides: p is O(10,000), n is O(10-100), but growing, Genes Slides Gene expression level of gene 5 in slide 4 = Log 2 ( Red intensity / Green intensity) slide 1slide 2slide 3slide 4slide 5 … 1 0.46 0.30 0.80 1.51 0.90... 2-0.10 0.49 0.24 0.06 0.46... 3 0.15 0.74 0.04 0.10 0.20... 4-0.45-1.03-0.79-0.56-0.32... 5-0.06 1.06 1.35 1.09-1.09... These values are conventionally displayed on a red (>0) yellow (0) green (<0) scale.

7. Cluster analysis Used to find groups of objects when not already known “Unsupervised learning” Associated with each object is a set of measurements (the feature vector) Aim is to identify groups of similar objects on the basis of the observed measurements

8. Clustering Gene Expression Data Can cluster genes (rows), e.g. to (attempt to) identify groups of co- regulated genes Can cluster samples (columns), e.g. to identify tumors based on profiles Can cluster both rows and columns at the same time

9. Clustering Gene Expression Data Leads to readily interpretable figures Can be helpful for identifying patterns in time or space Useful (essential?) when seeking new subclasses of samples Can be used for exploratory purposes

10. Similarity Similarity s ij indicates the strength of relationship between two objects i and j Usually 0 ≤ s ij ≤1 Correlation-based similarity ranges from –1 to 1

11. Problems using correlation

12. Dissimilarity and Distance Associated with similarity measures s ij bounded by 0 and 1 is a dissimilarity d ij = 1 - s ij Distance measures have the metric property (d ij +d ik ≥ d jk ) Many examples: Euclidean, Manhattan, etc. Distance measure has a large effect on performance Behavior of distance measure related to scale of measurement

13. Partitioning Methods Partition the objects into a prespecified number of groups K Iteratively reallocate objects to clusters until some criterion is met (e.g. minimize within cluster sums of squares) Examples: k-means, partitioning around medoids (PAM), self-organizing maps (SOM), model-based clustering

14. Hierarchical Clustering Produce a dendrogram Avoid prespecification of the number of clusters K The tree can be built in two distinct ways: –Bottom-up: agglomerative clustering –Top-down: divisive clustering

15. Agglomerative Methods Start with n mRNA sample (or G gene) clusters At each step, merge the two closest clusters using a measure of between-cluster dissimilarity which reflects the shape of the clusters Examples of between-cluster dissimilarities: –Unweighted Pair Group Method with Arithmetic Mean (UPGMA): average of pairwise dissimilarities –Single-link (NN): minimum of pairwise dissimilarities –Complete-link (FN): maximum of pairwise dissimilarities

16. Divisive Methods Start with only one cluster At each step, split clusters into two parts Advantage: Obtain the main structure of the data (i.e. focus on upper levels of dendrogram) Disadvantage: Computational difficulties when considering all possible divisions into two groups

17. Partitioning vs. Hierarchical Partitioning –Advantage: Provides clusters that satisfy some optimality criterion (approximately) –Disadvantages: Need initial K, long computation time Hierarchical –Advantage: Fast computation (agglomerative) –Disadvantages: Rigid, cannot correct later for erroneous decisions made earlier

18. Generic Clustering Tasks Estimating number of clusters Assigning each object to a cluster Assessing strength/confidence of cluster assignments for individual objects Assessing cluster homogeneity

20. Estimating Number of Clusters

21. (BREAK)

22. Bittner et al. It has been proposed (by many) that a cancer taxonomy can be identified from gene expression experiments.

23. Dataset description 31 melanomas (from a variety of tissues/cell lines) 7 controls 8150 cDNAs 6971 unique genes 3613 genes ‘strongly detected’

24. This is why you need to take logs!

25. After logging…

26. How many clusters are present?

27. ‘cluster’ unclustered Average linkage hierarchical clustering, melanoma only 1-  =.54

28. Issues in Clustering Pre-processing (Image analysis and Normalization) Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

29. Issues in Clustering Pre-processing (Image analysis and Normalization) Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

30. Filtering Genes All genes (i.e. don’t filter any) At least k (or a proportion p) of the samples must have expression values larger than some specified amount, A Genes showing “sufficient” variation –a gap of size A in the central portion of the data –a interquartile range of at least B Filter based on statistical comparison –t-test –ANOVA –Cox model, etc.

31. Issues in Clustering Pre-processing (Image analysis and Normalization) Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

32. ‘cluster’ unclustered Average linkage hierarchical clustering, melanoma only

33. ‘cluster’ control unclustered Average linkage hierarchical clustering, melanoma & controls

34. Issues in clustering Pre-processing Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

35. Complete linkage (FN) hierarchical clustering

36. Single linkage (NN) hierarchical clustering

37. Issues in clustering Pre-processing Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

38. Divisive clustering, melanoma only

39. Divisive clustering, melanoma & controls

40. Partitioning methods K-means and PAM, 2 groups BittnerK-meansPAM# samples 11110 111111 122122 212212 018018 222222 112112 121121 106106 2225

41. BittnerK-meansPAM# samples 11111 112112 121121 221221 261261 2224 2223322233 2332323323 3133131331 1241312413 3333

42. Issues in clustering Pre-processing Which genes (variables) are used Which samples are used Which distance measure is used Which algorithm is applied How to decide the number of clusters K

43. How many clusters K? Many suggestions for how to decide this! Milligan and Cooper (Psychometrika 50:159- 179, 1985) studied 30 methods Some new methods include GAP (Tibshirani ) and clest (Fridlyand and Dudoit) Applying several methods yielded estimates of K = 2 (largest cluster has 27 members) to K = 8 (largest cluster has 19 members)

44. cluster unclustered Average linkage hierarchical clustering, melanoma only

45. Summary Buyer beware – results of cluster analysis should be treated with GREAT CAUTION and ATTENTION TO SPECIFICS, because… Many things can vary in a cluster analysis If covariates/group labels are known, then clustering is usually inefficient

46. Acknowledgements IPAM Group, UCLA: Debashis Ghosh Erin Conlon Dirk Holste Steve Horvath Lei Li Henry Lu Eduardo Neves Marcia Salzano Xianghong Zhao Others: Sandrine Dudoit Jane Fridlyand Jose Correa Terry Speed William Lemon Fred Wright