Example for a JProGO analysis:

Switch from aerobic to anaerobic life conditions in E. coli

Biological and experimental background:

Kang et al. (2005) performed a microarray experiment in order to identify genes that are regulated by the global transcriptional regulator FNR (Fumarate and nitrate reduction regulatory protein) in Escherichia coli. Since FNR is one key regulator for the induction and repression of genes under anaerobic conditions the authors first compared the transcriptomes of wild-type cells cultivated under aerobic and under anaerobic conditions. For both conditions, three independent cultures were grown and the RNA was prepared. Affymetrix microarrays were used for the transcriptome analysis. The complete processed data set including the expression ratios for each triplicate and the corresponding p-values (probabilities of differential expression) and q-values can be obtained as compressed Microsoft Excel file. The microarray data subset used for the analysis at hand is provided as text file .

JProGO analysis:

We analyzed the probabilities of differential expression with our JProGO tool using all implemented statistical tests. For the threshold value-based hypergeometric test and Fisher's exact test (threshold value: 0.95) no significant GO nodes were obtained (with an significance level of 0.05 and Bonferroni correction). However, for all three threshold-value independent statistical tests we found several significant nodes which are shown below both as table and as GO subgraph.

Results and Discussion:

All three threshold independent methods identified GO nodes which represent functions and process known to be affected under anaerobic conditions. For example, they recognized processes that derivate energy from the oxidation of organic compounds such as carbohydrates as significantly affected in their gene expression profile. GO nodes which reflect these processes are "main pathways of carbohydrate metabolism" and "generation of precursor metabolites and energy". In addition, the t-Test identified "succinate dehydrogenase activity" which is mainly found in the tricarboxylic acid cycle. In deed, both, Kolmogorov-Smirnov and Mann-Whitney U-test confirmed and expanded this finding since they recognized the corresponding GO node "tricarboxylic acid cycle".

Overview on performed analyses

Student's t-Test

Kolmogorov-Smirnov test

Mann-Whitney U-Test

Results of Student's t-Test:

20 significant GO nodes

1a) Result Table

GO Category	GO Accession	GO Name	p-value
MF	GO:0000104	succinate dehydrogenase activity	4.1291E-107
MF	GO:0048038	quinone binding	1.7678E-11
MF	GO:0005506	iron ion binding	4.0647E-9
MF	GO:0016491	oxidoreductase activity	2.8895E-8
BP	GO:0006091	generation of precursor metabolites and energy	1.1961E-7
MF	GO:0043167	ion binding	6.3743E-7
MF	GO:0046872	metal ion binding	6.3743E-7
MF	GO:0016728	oxidoreductase activity, acting on CH2 groups, disulfide as acceptor	2.8317E-6
MF	GO:0016151	nickel ion binding	3.69E-6
MF	GO:0046914	transition metal ion binding	4.7072E-6
BP	GO:0006072	glycerol-3-phosphate metabolism	6.8101E-6
MF	GO:0043169	cation binding	6.9173E-6
BP	GO:0006092	main pathways of carbohydrate metabolism	1.1952E-5
BP	GO:0006118	electron transport	1.2248E-5
MF	GO:0051539	4 iron, 4 sulfur cluster binding	2.2896E-5
BP	GO:0015980	energy derivation by oxidation of organic compounds	2.4876E-5
MF	GO:0051536	iron-sulfur cluster binding	2.4964E-5
MF	GO:0051540	metal cluster binding	2.4964E-5
MF	GO:0048037	cofactor binding	2.8086E-5
MF	GO:0016651	oxidoreductase activity, acting on NADH or NADPH	4.4148E-5

1b) GO Subgraph Visualization

(Analysis Method: Student's t-test)

GO subgraph containing the nodes with a significant p-value and all their parent nodes up to the root.

Relevant GO nodes can easily be detected by their size and brightness: The lower the p-value of the GO node the larger and the brighter it is. In addition, nodes with significant p-values have a thicker border. The three sub-ontology are reflected by the color of the node (red = molecular function, green = biological process, blue = cellular component)

Click on a particalular node in order to show the genes assigned to it and their expression profile:

Results of Kolmogorov-Smirnov test:

18 significant GO nodes

2a) Result Table

GO Category	GO Accession	GO Name	p-value
BP	GO:0006092	main pathways of carbohydrate metabolism	2.3929E-12
BP	GO:0006099	tricarboxylic acid cycle	4.6896E-11
BP	GO:0009109	coenzyme catabolism	4.6896E-11
BP	GO:0046356	acetyl-CoA catabolism	4.6896E-11
BP	GO:0051187	cofactor catabolism	4.6896E-11
BP	GO:0009060	aerobic respiration	1.875E-10
BP	GO:0006084	acetyl-CoA metabolism	4.5661E-10
BP	GO:0015980	energy derivation by oxidation of organic compounds	5.622E-10
BP	GO:0006091	generation of precursor metabolites and energy	6.7633E-9
BP	GO:0045333	cellular respiration	2.2937E-8
MF	GO:0016491	oxidoreductase activity	9.5815E-7
BP	GO:0044248	cellular catabolism	4.5524E-6
MF	GO:0005506	iron ion binding	4.6734E-6
BP	GO:0009056	catabolism	1.3578E-5
BP	GO:0006118	electron transport	2.1169E-5
MF	GO:0043167	ion binding	2.2231E-5
MF	GO:0046872	metal ion binding	2.2231E-5
MF	GO:0051539	4 iron, 4 sulfur cluster binding	3.6388E-5

2b) GO Subgraph Visualization

(Analysis Method: Kolmogorov-Smirnov test)

GO subgraph containing the nodes with a significant p-value and all their parent nodes up to the root.

Click on a particalular node in order to show the genes assigned to it and their expression profile:

Results of Mann-Whitney U-test:

23 significant GO nodes

3a) Result Table

GO Category	GO Accession	GO Name	p-value
BP	GO:0006091	generation of precursor metabolites and energy	2.6169E-10
BP	GO:0006092	main pathways of carbohydrate metabolism	9.3872E-10
BP	GO:0009060	aerobic respiration	2.9706E-9
BP	GO:0006099	tricarboxylic acid cycle	3.6572E-9
BP	GO:0009109	coenzyme catabolism	3.6572E-9
BP	GO:0046356	acetyl-CoA catabolism	3.6572E-9
BP	GO:0051187	cofactor catabolism	3.6572E-9
MF	GO:0016491	oxidoreductase activity	5.911E-9
MF	GO:0005506	iron ion binding	6.1682E-9
BP	GO:0015980	energy derivation by oxidation of organic compounds	1.4319E-8
BP	GO:0006084	acetyl-CoA metabolism	1.571E-8
BP	GO:0045333	cellular respiration	9.1292E-8
MF	GO:0043167	ion binding	9.9237E-8
MF	GO:0046872	metal ion binding	9.9237E-8
BP	GO:0006118	electron transport	7.5776E-7
MF	GO:0046914	transition metal ion binding	9.2834E-7
MF	GO:0043169	cation binding	1.0464E-6
MF	GO:0051539	4 iron, 4 sulfur cluster binding	2.5537E-6
MF	GO:0051536	iron-sulfur cluster binding	1.0265E-5
MF	GO:0051540	metal cluster binding	1.0265E-5
BP	GO:0044248	cellular catabolism	1.292E-5
MF	GO:0016151	nickel ion binding	2.5244E-5
BP	GO:0009056	catabolism	2.9307E-5

3b) GO Subgraph Visualization

(Analysis Method: Mann-Whitney U-test)

GO subgraph containing the nodes with a significant p-value and all their parent nodes up to the root.

Click on a particalular node in order to show the genes assigned to it and their expression profile: