From genes to functional blocks in the study of biological systems Fátima Al-Shahrour, Joaquín Dopazo National Institute of Bioinformatics, Functional Genomics node Department of Bioinformatics, Centro de Investigación Príncipe Felipe, and Functional genomics node, INB, Valencia, Spain

Two-steps functional interpretation statistic - + A Metabolism Transport... Reproduction test A B B Metabolism Transport... Reproduction test t-test Genes are selected based on their experimental values and... 2 Enrichment in functional terms is tested (FatiGO, GoMiner, etc.) 2 2

Two-steps approach reproduces pre-genomics paradigms experiments interpretation test no pass Context and cooperation between genes is ignored experiments test interpretation test.... ::::

Cooperative activity of genes can be detected and related to a macroscopic observation statistic - + A B GO 1 GO 2 GO 3 Ranking: A list of genes is ranked by their differential expression between two experimental conditions A and B (using fold change, a t-test, etc.) Distribution of GO: Rows GO 1, GO 2 and GO 3 represent the position of the genes belonging to three different GO terms across the ranking. The first GO term is completely uncorrelated with the arrangement, while GOs 2 and 3 are clearly associated to high expression in the experimental conditions B and A, respectively. Note that genes can be multi-functional

A previous step of gene selection causes loss of information and makes the test insensitive statistic - + A B GO 1 GO 2 If a threshold based on the experimental values is applied, and the resulting selection of genes compared for over-abundance of a functional term, this migh not be found. t-test with two tails. p<0.05 Significantly over-expressed in B Significantly over-expressed in A Classes expressed as blocks in A and B Very few genes selected to arrive to a significant conclussion on GOs 1 and 2

A previous step of gene selection causes loss of information and makes the test insensitive statistic - + A B GO 1 GO 2 The main problem is that the two-steps approach cannot distinguish between these two different cases. We put both sides of the partition into two bags and destroy the structure of the data. t-test with two tails. p<0.05 Significantly over-expressed in B Significantly over-expressed in A up down GO no GO Same contingency table for GO 1 and GO 2 !!

FatiScan, a segmentation test, provides an easy approach to directly test functional terms statistic - + A B p1p1 p2p2 p3p3 GOs can be directly tested by a segmentation test. A series of partitions of the list are performed (p 1, p 2, p 3 …) and the GO terms for each functional class in the upper part are compared to the corresponding ones in the lower part by a Fisher test. Asymmetrical distributions of terms towards the extremes of the list will produce significant values of the test. Finally, p-values are adjusted by FDR Al-Shahrour et al., 2005 Bioinformatics E.g., term 2, partition p 1 up down GO no GO GO 1 GO 2 GO 3

Obtaining significant results statistic - + A B p1p1 p2p2 p3p3 For each GO term (T), different partitions (P) are tested. TxP p-values of tests to be adjusted for multiple testing. Empirical results suggest that 20 to 50 partitions optimally find significant asymmetrical distributions of terms Al-Shahrour et al., 2005 Bioinformatics term background

Nested inclusive analysis Levels from 9 up to 3 are tested. The deepest significant level is reported

% Genes with the specific GO annotation for each partition U U L - + L

Case study: functional differences in a class comparison experiment B 17 with normal tolerance to glucose (NTG) A 8 with impaired tolerance (IGT) + 18 with type 2 diabetes mellitus (DM2) A B No one single gene shows significant differential expression upon the application of a t-test Nevertheless, many pathways, and functional blocks are significantly activated/deactivated (Mootha et al., 2003)

Beyond discrete variables: Survival data Microarrays 34 samples from tumours of hypopharyngeal cancer (GEO GDS1070) Cox Proportional- Hazards model to study how the expression of each gene across patients is related to their survival Gen risk Gen1 5.8 Gen2 5’6 Gen3 5.4 Gen4 5.2 Gen5 5.2 Gen6 5.0 …… …. Gen Gen Survival + Survival GEPAS t-rex tool Since FatiScan depends only on a list of ordered genes, and not on the original experimental values, it can be applied to different experimental designs

Functional analysis of a time series in P. falciparum -Genes at each time point are ranked from highest (red) to lowest (green) relative expression with respect to time 1. - For each list of ranked genes generated in any time point, the significant over-represented GO terms in the tail corresponding to the highest expression values are recorded. -The partitions used to decide that a given term is significantly over-represented in the upper tail of the list with respect to the lower part are used for the graphical representation....

Beyond arrays: evolutionary systems biology 20,469 known Ensembl human protein-coding genes from the Ensembl v.30.35h were used Comparison of the relative rates of synonymous (Ks) and non- synonymous (Ka) substitutions. The ratio of these values, the (=Ka/Ks) is a widely accepted measure of the selective pressure Mutations occur on single genes but natural selection acts on phenotypes by operating on whole sub-cellular systems (represented by GO). We are interested in the human linage

Fig 6 GO termp-value sensory perception of smell (GO: ) 1.3 x sensory perception of chemical stimulus (GO: ) G-protein coupled receptor protein signalling pathway (GO: ) GO terms positively selected in humans FatiScan is applied to the list of human genes ordered according  values If genes positively selected are firstly detected and then analysed for significant enrichment of GO (two- steps approach), no results are found Log 

Comparison of methods for testing directly GO (or other terms) at a glance Terms from distinc repositories, reported by different methods in the diabetes dataset (Mootha et al., 2003) GSEA 2003 FatiScan 2005 PAGE 2005 Tian 2005

The babelomics suite for functional annotation of experiments Biological information from: GO Interpro motifs KEGG pathways Swissprot keywords Tissues Text-mining Chromosomal location For Human, mouse, rat, chicken, fly, worm, yeast, A. thaliana and bacteria Tests for lists of genes or blocks of functionally related genes

DNMAD Hierarchical SOM SOTAK-means Expresso Preprocessor Affymetrix arrays Two-colour arrays Clustering Class Prediction Raw data Differential expresion GEPAS Functional Annotation FatiGO+ FatiGO Marmite TMT FatiScan GSEA CAAT KNN DLDA SVM Random forest Normalization Prophet T-Rex Two classes Multi classes Correlation Survival Blocks of genes Two sets of genes ISACGH Babelomics Arrays-CGH RIDGE analysis Herrero et al., 2003, 2004; Vaquerizas et al., 2005 NAR; Montaner et al., 2006 NAR; Al-Shahrour et al., 2005, 2006 NAR; 2005 Bioinformatics

Some numbers More than 150,000 experiments analysed during the last year. More than 500 experiments per day. 24h usage map as of June 8, 2006

Summary Methods that directly address functional hipothesis are much more sensitive for the functional interpretation of any type of large-scale experiment. Methods that do not require of the original data (such as the FatiScan) can be applied in a wider range of experimental designs in microarrays (class comparison, survival, etc.) and can be applied to any large-scale experiment or theoretical study in which a value can be assigned to any gen that allows generating a list of ranked genes. Despite the differences in the tests, distinct functional interpretation methods seem to produce comparable results (although a more detailed benchmarking is necessary).

The bioinformatics department at Centro de Investigación Príncipe Felipe (Valencia, Spain)... Joaquín Dopazo Eva Alloza Leonardo Arbiza Fátima Al-Shahrour Jordi Burguet Lucía Conde Hernán Dopazo Toni Gabaldon Jaime Huerta Marc Martí Ignacio Medina Pablo Minguez David Montaner Joaquín Tárraga Juan Manuel Vaquerizas...and the INB, Instituto Nacional de Bioinformática (Functional Genomics Node)