1. Pathway analysis using BioConductor The global test revisited

2. R User group 6 dec 2005 Overview Introduction Annotation Pathway analysis Demonstration

3. R User group 6 dec 2005 Introduction Pathway Set of related genes Functional Structural Described as lists of gene identifiers Micro array 1000s of tests Description Location on chip/slide Sequence ID On chip replication

4. R User group 6 dec 2005 Feature description Proprietary ID (Affymetrix/Agilent) GenBank, RefSeq, EnsembleID Symbol, LocusLink /Entrez Gene,Unigene SwissProt Chromosomal location EC number, GO, KEGG

5. R User group 6 dec 2005

7. Annotation sources Batch Gene Finder: http://cgap.nci.nih.gov/Genes http://cgap.nci.nih.gov/Genes BioMart: http://www.ebi.ac.uk/BioMart/martview http://www.ebi.ac.uk/BioMart/martview Resourcerer: http://www.tigr.org/tigr-scripts/magic/r1.pl http://www.tigr.org/tigr-scripts/magic/r1.pl Bioconductor metadata http://www.bioconductor.orghttp://www.bioconductor.org NettAffx http://www.affymetrix.com/analysis/index.affx http://www.affymetrix.com/analysis/index.affx

8. R User group 6 dec 2005 Create Annotation for Array Select / create unique identifier for probes on array i.e. Use positional information b01r03c14 Use this identifier as rownames of data and annotation Use annotation sources to connect sequence ids to gene ids

9. R User group 6 dec 2005 Connecting sequence ids to gene ids # 2 matrices, myAnnot and GBFAnnot (from Gene Batch Finder) # Create temporary annotation with correct dimensions tmpAnnot<- matrix("", nrow=nrow(veerannot), ncol=ncol(GBFAnnot), dimnames=list(rownames(veerannot), colnames(GBFAnnot))) ind<-match(myAnnot[,1],rownames(GBFAnnot)) tmpAnnot[!is.na(ind),]<-GBFAnnot[ind[!is.na(ind)],] myAnnot<-cbind(myAnnot,tmpAnnot)

10. R User group 6 dec 2005 Selecting probes by pathway Using BioConductor metadata package Using BioConductor GO and Mapping >library(hgu95av2) >get("GO:0005868",envir=hgu95av2GO2PROBE) NAS TAS TAS ISS "37300_at" "40318_at" "40319_at" "40949_at" "40950_at" "946_at" > library(GO) > ll<-get("GO:0005868",envir=GOLOCUSID) > rownames(myAnnot)[myAnnot[,”LocusLink”] %in% ll] [1] "Contig51966_RC" "NM_004411" "Contig47291_RC" "NM_006141" [5] "NM_014183" "AB002323" "NM_006519"

11. R User group 6 dec 2005 Pathway analysis List based methods Order based methods Statistical combination of results

12. R User group 6 dec 2005 List based Pathway analysis Compare the proportion if differentially expressed genes in a pathway to the proportion on the array R: phyper(), GOHyperG() in GOstats package (BioConductor) phyper(PWde,ARde,ARall-ARde,PWall,lower.tail=FALSE) >phyper(15,1000,9000,100,lower.tail=FALSE) [1] 0.03910265

13. R User group 6 dec 2005 Order based analysis Genes are ordered by difference, from up- to non- to downregulated. Interesting pathways form clusters along this order In R: Gene Set Enrichment Analysis (GSEA) package http://www.broad.mit.edu/gsea/software/software_index.html http://www.broad.mit.edu/gsea/software/software_index.html

14. R User group 6 dec 2005 Statistical combination of results All genes in a pathway contribute their statistical influence In R: globaltest package (BioConductor)

15. R User group 6 dec 2005 Demonstration