Demonstration

Trupti Joshi

Computer Science Department317 Engineering Building North

E-mail: joshitr@missouri.edu573-884-3528(O)

Examples

Microarray Data GeneSpring Functional Analysis Pathway Analysis

Microarray Data

DATA

Affymetrix Chips-Experiments: 4mer,8mer,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

GeneSpring Software

GeneSpring (Silicon Genetics) Broadly used Nice user interface Data Normalization (Lowess, etc.) Powerful ANOVA statistical analysis

t-test/1-way ANOVA test 2-way ANOVA tests 1-way post-hoc tests for reliably identifying differentially expressed genes

Incorporation of different analysis tools Clustering Visual filtering Pathway viewing Scripting

Normalization in GeneSpring

2

109

210

555

22

1

4mer8mer

Mix

UP

UP (Functions)

1

60

05

308

11

0

4mer8mer

Mix

Affymetrix Chitin Expts : GeneSpring Results

Function Analysis : GO

Aim:

To study functional categories distribution based on Gene Ontology Annotations in order to understand the genes and pathways involved in experimental conditions.

Three key parts: gene name/id

GO term(s)

evidence for association

3 Ontologies 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

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

• 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?

GO for microarray analysis

Annotations give ‘function’ label to genes

Ask meaningful questions of microarray data e.g.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

Up regulated - All in 8Mer (373 genes)

0%

0%0%0%0%0%1%

0%2%

response to stress

4%

cell communication

8%response to

external stimulus12%

cell growth and/or maintenance

19%

metabolism47%

cell death7%

cell communication

cell death

cell differentiation

cell growth and/ormaintenancemembrane fusion

morphogenesis

regulation of geneexpression\, epigeneticaging

embryonic development

homeostasis

metabolism

nodulation

response to endogenousstimulusresponse to externalstimulus response to stress

Down regulated - All in 8Mer (35 genes)

2%

response to external stimulus

4%

cell communication

4%

response to stress

4%

response to endogenous

stimulus12%

cell growth and/or

maintenance20%

metabolism54%

cell communication

cell growth and/or maintenance

morphogenesis

metabolism

response to endogenous stimulus

response to external stimulus

response to stress

GO Biological Process

UP 8M-ALL

14%

9%

0%

22%

0%13%

8%

14%

3%

1%

6%

8%2%

METABOLISM

ENERGY

CELL CYCLE AND DNA PROCESSING

TRANSCRIPTION

PROTEIN SYNTHESIS

PROTEIN FATE

CELLULAR TRANSPORT, TRANSPORTFACILITATION AND TRANSPORT ROUTES

CELLULAR COMMUNICATION/SIGNALTRANSDUCTION MECHANISM

CELL RESCUE, DEFENSE AND VIRULENCE

INTERACTION WITH THE CELLULARENVIRONMENT

INTERACTION WITH THE ENVIRONMENT

CELL FATE

DEVELOPMENT

DOWN 8M-ALL

14%

12%

8%

30%

12%

8%

8%

4%4%

METABOLISM

ENERGY

CELL CYCLE AND DNA PROCESSING

TRANSCRIPTION

PROTEIN FATE

CELLULAR TRANSPORT, TRANSPORTFACILITATION AND TRANSPORT ROUTES

CELLULAR COMMUNICATION/SIGNALTRANSDUCTION MECHANISM

CELL RESCUE, DEFENSE AND VIRULENCE

INTERACTION WITH THE ENVIRONMENT

MIPS Function

SAMPLE 1 : Bar Plot

0.47 (52)

2.62 (1)

2.62 (2)

2.62 (1)

1.69 (302)

0.87 (1)

0.52 (10)

0.44 (1)

0.37 (7)

0.26 (2)

0.21 (1)

0.08 (1) response to stress

morphogenesis

response to endogenous stimulus

response to external stimulus

post-embryonic development

cell growth and/or maintenance

cell communication

regulation of gene expression\, epigenetic

metabolism

pattern specification

aging

secretion

Fu

nct

ion

Cat

ego

ries

Ratio Observed/Expected

GO FUNCTIONS

WS 5hr

Sample 1 : 0-3 mm

Sample 2 : 3-11 mm

SAMPLE 2 : Bar Plot

0.23 (1)0.24 (2)0.26 (3)

0.49 (1)0.57 (2)0.61 (10)0.62 (11)0.67 (65)

0.91 (1)1.50 (241)

1.95 (1)2.44 (2)

morphogenesis response to endogenous stimulus

response to stresspost-embryonic development

cell differentiation cell communication

response to external stimuluscell growth and/or maintenance

regulation of gene expression\, epigeneticmetabolism

pattern specificationaging

Fu

nct

ion

al C

ateg

ori

es

Ratio Observed/Expected

* Numbers of genes observed are shown in brackets

GO for microarray analysis

experimental condition

Gen

e

com

pone

nt

proc

ess

func

tion

Selected Gene Tree: pearson lw n3d ...Branch color classification:Set_LW_n3d_5p_...

Colored by: Copy of Copy of C5_RMA (Defa...Gene List: all genes (14010)

attacked

time

control

Puparial adhesionMolting cyclehemocyanin

Defense responseImmune responseResponse to stimulusToll regulated genesJAK-STAT regulated genes

Immune responseToll regulated genes

Amino acid catabolismLipid metobolism

Peptidase activityProtein catabloismImmune response

Selected Gene Tree: pearson lw n3d ...Branch color classification:Set_LW_n3d_5p_...

Colored by: Copy of Copy of C5_RMA (Defa...Gene List: all genes (14010)

Bregje Wertheim at the Centre for Evolutionary Genomics, Department of Biology, UCL and Eugene Schuster Group, EBI.

MicroArray data analysis

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

KEGG Pathways Analysis

List of Arabidopsis genes assigned to KEGG Pathways acquired

UP or DOWN regulated genes mapped to Pathways

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

AT3G47340

Red : 5hr 0-3mm Blue : 5hr 3-11 mm Purple : 48hr 0-3mm Green : 48 hr 3-11mm

AT1G72330

AT4G24830

AT5G65010

Examples

Microarray Data GeneSpring Functional Analysis Pathway Analysis