Demonstration Trupti Joshi Computer Science Department 317 Engineering

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

Examples l Microarray Data l Gene. Spring l Functional l Pathway Analysis

Microarray Data

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

Affymetrix : Raw Data. CEL

Affymetrix : Report. RPT

Affymetrix : Processed Data. TXT

Post- Normalization Calculations: Log Transformations and Fold Change Control

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

Normalization in Gene. Spring

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

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

3 Ontologies l 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. 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.

Example Ontology

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

What can scientists do with GO? • 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).

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?

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

Function Distribution of All Annotated Arabidopsis Genes

GO Biological Process

MIPS Function

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

ne po m co tio n nc fu oc pr Ge experimental condition ne es s nt GO for microarray analysis

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

Color indicates up/down regulation Apotosis Regulator Red: up by 1. 5 fold Blue: down 1. 5 fold Go. Miner Tool, John Weinstein et al, NCI: Genome Biol. 4 (R 28) 2003

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

AT 5 G 08300 ; AT 2 G 05710; AT 4 G 35830; AT 2 G 05710; AT 2 G 42790 AT 5 G 43330; AT 3 G 47520 ; AT 5 G 09660; AT 3 G 15020 AT 1 G 65930; AT 5 G 14590; AT 2 G 47510 AT 5 G 08300 AT 5 G 55070 Red : 5 hr 0 -3 mm Blue : 5 hr 3 -11 mm AT 3 G 55410; AT 3 G 55410 Purple : 48 hr 0 -3 mm Green : 48 hr 3 -11 mm

Red : 5 hr 0 -3 mm Blue : 5 hr 3 -11 mm Purple : 48 hr 0 -3 mm Green : 48 hr 3 -11 mm AT 1 G 72330 AT 4 G 24830 AT 3 G 47340 AT 5 G 65010

Examples l Microarray Data l Gene. Spring l Functional l Pathway Analysis