1. Frédéric Schütz Frederic.Schutz@isb-sib.ch Statistics and bioinformatics applied to –omics technologies Part II: Integrating biological knowledge Center for Integrative Genomics University of Lausanne, Switzerland Bioinformatics Core Facility Swiss Institute of Bioinformatics

2. Class prediction 1-19 Gene Ontology analysis 20-29 Geneset analysis (GSEA, etc) 30-39 Contents Slides

3. Class discovery and class prediction Example: patients from which we obtained measurements (e.g. gene expression) Class discovery Gene 1 Gene 2 Find natural groups in the data (e.g. sets of patients with similar gene expression) Class prediction Given previous measurements for which the grouping is known (red and blue), can we predict the group to which a new observation belongs ? Gene 1 Gene 2 ?

4. Many questions in biology and medicine are “class prediction” questions: –Does a patient have a predisposition for a given disease ? –What is the prognosis for this patient ? –What will be the response of this patient to a given drug ? –Is this tumour benign or malign ? –What type is this tumour ? –Which treatment should be used ? Why do we want to do class prediction ?

5. Class prediction: easy case Gene 1 Gene 2 Classify everything on this side as “red” Classify everything on this side as “blue” Threshold

6. Example Pierre Farmer et al. Identification of molecular apocrine breast tumours by microarray analysis. Oncogene (2005) 24, 4660–4671 Blue points represent “oestrogen receptor (ER) status positive” determined by immunohistochemistry.

7. Class prediction: in practice Gene 1 Gene 2 The two groups are not perfectly separated (and this is still a pretty good case…) One variable (gene) is not sufficient to assign patients to groups Remember that with microarrays, we are not talking about just 2 measurements, but several 10,000s.

8. Goal: assign objects (e.g. patients) to classes based on some measurements (e.g. gene expression) Typically, in a microarray setting: –10s or (at best) 100s of patients –10,000s genes Unsupervised learning: nothing is known about the grouping of the data, and we try to find natural groups in the data Supervised learning: the classes are predefined; we use previously labelled objects to create a procedure for classification of future observations. Discrimination in general

9. K-nearest neighbours Linear Discriminant Analysis Classification trees Support Vector Machines (SVM) etc. Some supervised analysis methods

10. Example: 3-nearest neighbours Gene 1 Gene 2 Red or blue ?

11. Example: 3-nearest neighbours Gene 1 Gene 2 2 red vs 1 blue: the point is assigned to “red”

12. Choose a value for k (typical values: 3 or 5); in practice it can be chosen using the learning data (value that produces the best result) Find the k observations in the learning set that are closest to the new, unknown, observation Predict the class by a majority vote, that is, choose the class that is most common among the neighbours. Very simple method, with surprisingly good performance K-nearest neighbours

13. Suggested by R.A. Fisher in 1935 Procedure to find a linear combination of the observed variables that best separates (discriminates) two classes of objects. Using the “new variable”, objects from the same class are close together, and objects from different class are further away. Straightforward to calculate Can easily be extended to more than two classes Similar idea to Principal Component Analysis (PCA) Often forgotten in favour of PCA Linear Discriminant Analysis

14. Back to the easy case Gene 1 Gene 2 Classify everything on this side as “red” High value of the discriminant Classify everything on this side as “blue” Low value of the discriminant Threshold Discriminant = Gene 1

15. Linear Discriminant Analysis: Example Gene 1 Gene 2 The two groups are well separated Neither Gene1 nor Gene2 is able to discriminate between the two categories

16. Linear Discriminant Analysis: Example Gene 1 Gene 2 However, the linear combination L = Gene1 + Gene2 discriminates well between the two groups Blue points tend to have smaller L values Red points tend to have bigger L values Low values High values

17. Linear Discriminant Analysis: Example Gene 1 Gene 2 A threshold is set in between the mean of the two groups Points with a value L above the threshold are classified as red Points with a value L below the threshold are classified as blue Low values High values Threshold

18. Caveats: Overfitting It is easy to create classifiers which fit the training data perfectly It is harder to find classifiers which still work as well when validated on new data A classifier must ALWAYS be tested on data independent from the one used to actually train the classifier. This is particularly important in microarray analysis: –Few samples –Many different measurements If not careful, it is always possible to find a classifier that works well for your training data !

19. Caveats: Overfitting Gene 1 Gene 2 Classify everything in this region as red Perfect classifier for this data Probably not so good with any new data

20. Many microarray experiments produce lists of genes that are significantly differently expressed between two conditions (gene comparison). In some (rare) cases, only a few genes are of interest, and they can easily be examined and validated. In most cases, however, a long list of differentially expressed genes is returned, and these genes can not be considered individually. It is harder to obtain biological understanding from this data. One strategy: consider the functional annotation of the differentially expressed genes. Question: what do these genes have in common that could be of interest ? Gene Ontology analysis

21. Collaborative effort to address the need for consistent descriptions of gene products in different databases. Three structured, controlled vocabularies (ontologies) that describe gene products in terms of their associated –biological processes –cellular components –molecular functions in a species-independent manner. Reminder: Gene Ontology (GO) project (From http://www.geneontology.org/)

22. Example (From http://www.geneontology.org/) PPARA, NR1C1, PPAR: Peroxisome proliferator-activated receptor alpha (TAS: Traceable Author Statement, IPI: Inferred from Physical Interaction)

23. Example of GO analysis 10,000 genes in total 10% 1000 genes differentially expressed Simple microarray experience: WT vs KO The microarray has 10,000 genes, 100 of which have GO annotation “fatty acid transport” I obtain 1000 differentially expressed genes (10% of all genes) 90% If my experiment has nothing to do with “fatty acid transport”, I expect in average about 10% of genes (or 10) to be differentially expressed. If this proportion is higher, it means the list of differentially-expressed genes is enriched in “fatty acid transport” genes If the difference is significant, it suggests a link between differential expression and this GO annotation: genes with this annotation are more likely to be differentially expressed than others This indicates that this biological process may be related to my KO experiment.

24. 10,000 genes in total 10% 1000 genes differentially expressed 90% 10 (10%) 90 (90%) Number of genes “fatty acid transport” 100 (100%) 0 (0%) Looks like a random distribution No apparent association Strong association ?...

25. Statistical analysis Assume that I found 20 differential expression with the GO annotation of interest. Count the numbers of genes with the GO annotation or not, and compare with differential expression: A statistical test such as Fisher’s exact test can tell us what is the probability of observing this result (or more extreme) if there is no association between the rows and columns In this case, this probability (p-value) is 0.002 This indicates that this biological process may be important in the difference between WT and KO. Differentially expressed Not D.E.Total “Fatty acid transport”2080100 Others98089809900 Total1000900010000

26. In practice One can either suggest a GO annotation and see if it is enriched in the list of differentially expressed genes Or we may want to go “fishing” and try all potentially interesting GO annotations to see if any of them is enriched. Easy to do Multiple services available on the web –User indicates the list of genes differentially expressed –Returns the most significant GO annotations

27. Microarray with about 22,000 genes We look at the 1% of the genes that are most different between different subtypes of cancer. Which processes are likely to be different between these subtypes ? –Those for which more than 1% of the genes are differentially expressed are good candidates Gene Ontology analysis: example. I Pierre Farmer et al. Identification of molecular apocrine breast tumours by microarray analysis. Oncogene (2005) 24, 4660–4671 Prop. 5% 19% 10% 3% 5% 4%

28. To apply this GO analysis, we need first to define a list of differentially expressed genes. This usually means calculating a “score” (e.g. p- value), and selecting a cut-off point. While there are some traditional cut-off points (0.001, 0.01 or the “magical” 0.05), they remain fairly arbitrary –Is there really a difference between a gene associated with a p-value of 0.049 and another one with a p-value of 0.051 ? Gene Ontology analysis: example. II

29. Some genes may be differentially expressed, but the change may be so small (lost in the noise) that it will not appear in the list. However, the difference in expression may appear at the level of a set of genes rather than individual genes Set of genes may correspond e.g. to co-regulated genes, or genes belonging to the same pathway If the change of expression is consistent across genes in the set, it may indicate that the set is of interest, even if no individual gene shows a significant difference. Gene Ontology analysis: example. III

30. Gene set enrichment analysis (GSEA)

31. Series of papers describing a method for analyzing the expression of sets of genes Software available, along with a database of biologically relevant gene sets Relatively hot topic in bioinformatics/statistics: many differerent papers and methods published on the topic, with small or large differences GSEA usually refers to this particular program, but sometimes indicates any such method which examines sets of genes.

32. Principle of GSEA We have a list of genes sorted according to a given measure (score for differential expression, correlation to a phenotype, etc) Among this list, we have a smaller set of genes of interest (e.g. all belonging to a given pathway) Is the smaller set distributed randomly in the sorted list of genes ? –If yes, the set is less likely to be of interest –If no, it may indicate that the function represented by the set is linked with the measure.

33. Principle of GSEA (most methods) All genes, sorted High values (e.g. upregulated) Low values (e.g. down-regulated) Position in the list of genes of our set of interest The location of the genes of our set of interest within the list seem random (uniform); the set does not appear to be linked with differential expression.

34. Principle of GSEA (most methods) All genes, sorted High values (e.g. upregulated) Low values (e.g. down-regulated) Position in the list of genes of our set of interest Link with up-regulation Position in the list of genes of our set of interest Link with down-regulation

35. Statistical analysis “Random walk”: –The list of genes is walked down from left to right –Everytime a gene belong to our list S, the score goes up –Everytime a gene does not belong to the list, it goes down If the genes of the set are uniformly distributed, the score will never go very high (“up” soon followed by a “down”) If the genes are distributed together, the score will go higher before getting back to 0. Using a permutation test, a p- value can be associated to the geneset. From fig. 1 of Subramanian et al. PNAS 2005; 102; 15545-15550

36. Statistical analysis How can we summarise and assess an apparent link between a set and differential expression ? Each method uses different statistics Original GSEA method based on the Kolmogorov- Smirnov test (compare the distribution of genes with a uniform distribution) Later replaced by an “Enrichment Score” (similar but weighted)

37. Example mRNA expression profiles from lymphoblastoid cell lines derived from 15 males and 17 females Identify gene sets correlated with the difference between males and females (False Discovery Rate) From table 2 of Subramanian et al. PNAS 2005; 102; 15545-15550

38. Example Gene expression patterns from a collection of 50 cancer cell lines p53 regulates gene expression in response to various signals of cellular stress 33 cell lines carry a mutation on the p53 gene, and 17 are normal. From table 2 of Subramanian et al. PNAS 2005; 102; 15545-15550

39. Conclusions GeneSet Enrichment Analysis methods have quickly become widespread in the microarray community. Intuitive method Can be used to confirm an association known or suspected… (use a given geneset) … or to go “fishing” for unknown association (use a database of genesets) More generally, microarray analysis uses more and more this external biological knowledge.