1. Demonstration Trupti Joshi Computer Science Department 317 Engineering Building North E-mail: joshitr@missouri.edu 573-884-3528(O)

2. Examples l Microarray Data l GeneSpring l Functional Analysis l Pathway Analysis

3. Microarray Data

4. DATA l Affymetrix Chips -Experiments: 4mer,8mer,Chitin- Mix,Mock (Raw data, Expt Details, Gene-Chip Analysis, Processed data.txt) -3 Replicates each

5. Affymetrix : Raw Data.CEL

6. Affymetrix : Report.RPT

7. Affymetrix : Processed Data.TXT

8. Post- Normalization Calculations: Log Transformations and Fold Change Control

9. GeneSpring Software l GeneSpring (Silicon Genetics) å Broadly used å Nice user interface å Data Normalization (Lowess, etc.) å Powerful ANOVA statistical analysis X t-test/1-way ANOVA test X 2-way ANOVA tests X 1-way post-hoc tests for reliably identifying differentially expressed genes å Incorporation of different analysis tools X Clustering X Visual filtering X Pathway viewing X Scripting

11. Normalization in GeneSpring

16. 2 109 2 10 555 22 1 4mer 8mer Mix UP UP (Functions) 1 60 0 5 308 11 0 4mer 8mer Mix Affymetrix Chitin Expts : GeneSpring Results

17. Function Analysis : GO l Aim: To study functional categories distribution based on Gene Ontology Annotations in order to understand the genes and pathways involved in experimental conditions. l Three key parts: å gene name/id å GO term(s) å evidence for association

18. 3 Ontologies l A gene product has one or more molecular functions and is used in one or more biological processes; it might be associated with one or more cellular components. l For example, the gene product cytochrome c can be described by the - molecular function term electron transporter activity, -biological process terms oxidative phosphorylation and induction of cell death, -cellular component terms mitochondrial matrix and mitochondrial inner membrane.

19. Example Ontology

20. 1. Downloads Ontologies – (various – GO, OBO, XML, OWL MySQL) Annotations – gene association files Ontologies and Annotations – MySQL and XML 2. Web-based access AmiGO (http://www.godatabase.org) QuickGO (http://www.ebi.ac.uk/ego) How to access the GO and its annotations

21. Access gene product functional information Provide a link between biological knowledge and … gene expression profiles proteomics data Find how much of a proteome is involved in a process/ function/ component in the cell using a GO-Slim (a slimmed down version of GO to summarize biological attributes of a proteome) Map GO terms and incorporate manual GOA annotation into own databases to enhance your dataset or to validate automated ways of deriving information about gene function (text-mining). What can scientists do with GO?

22. GO for microarray analysis l Annotations give ‘function’ label to genes l Ask meaningful questions of microarray data e.g. å genes involved in the same process, same/different expression patterns?

23. Microarray analysis Whole genome analysis (J. D. Munkvold et al., 2004)

24. Function Distribution of All Annotated Arabidopsis Genes

25. GO Biological Process

26. MIPS Function

27. GO FUNCTIONS WS 5hr Sample 1 : 0-3 mm Sample 2 : 3-11 mm * Numbers of genes observed are shown in brackets

28. GO for microarray analysis experimental condition Gene component process function

29. attacked time control Puparial adhesion Molting cycle hemocyanin Defense response Immune response Response to stimulus Toll regulated genes JAK-STAT regulated genes Immune response Toll regulated genes Amino acid catabolism Lipid metobolism Peptidase activity Protein catabloism Immune response Bregje Wertheim at the Centre for Evolutionary Genomics, Department of Biology, UCL and Eugene Schuster Group, EBI. MicroArray data analysis

30. Color indicates up/down regulation GoMiner Tool, John Weinstein et al, NCI: Genome Biol. 4 (R28) 2003 Apotosis Regulator Red: up by 1.5 fold Blue: down 1.5 fold

31. KEGG Pathways Analysis l List of Arabidopsis genes assigned to KEGG Pathways acquired l UP or DOWN regulated genes mapped to Pathways

32. AT1G65930; AT5G14590; AT5G08300 AT5G08300 ; AT2G05710; AT2G05710; AT4G35830; AT2G05710; AT2G47510 AT5G43330; AT3G47520 ; AT5G09660; AT3G15020 AT5G55070 AT3G55410; AT3G55410; AT3G55410 AT2G42790 Red : 5hr 0-3mm Blue : 5hr 3-11 mm Purple : 48hr 0-3mm Green : 48 hr 3-11mm

33. AT3G47340 Red : 5hr 0-3mm Blue : 5hr 3-11 mm Purple : 48hr 0-3mm Green : 48 hr 3-11mm AT1G72330 AT4G24830 AT5G65010

34. Examples l Microarray Data l GeneSpring l Functional Analysis l Pathway Analysis