Example for a JProGO analysis:

Effect of knockout of the global transcriptional regulator Lrp in E. coli

Biological and experimental background:

Hung et al. (2002) performed a microarray experiment in order to identify genes that are regulated by the global transcriptional regulator Lrp (Leucine responsive regulatory protein) in Escherichia coli. Lrp regualted several amino acid biosynthetic pathways. Wild-type cells and cells with a knockout in the gene for Lrp were cultivated in medium containing leucine. For both conditions, four independent cultures were grown and the RNA was prepared. Affymetrix and Nylon filter DNA microarrays were employed for the transcriptome analysis. The complete processed data set including the expression ratios for the 4 replicates and the corresponding posterior probabilities of differential expression (ppde) as Microsoft Excel file. The microarray data subset used for the analysis at hand is provided as text file .

JProGO analysis:

We used the expression ratios for the functional interpretation with our JProGO tool using all implemented statistical tests. All 5 statistical tests identified significant GO nodes (with an significance level of 0.05 and Bonferroni correction). As cut-off expression ratio for the threshold-based tests we used 0.5 for genes to be considered as down-regulated and 2.0 for genes to be considered as up-regulated.

Results and Discussion:

All 5 methods identified GO nodes that represent active transport process and amino acid biosynthetic pathways. This fits well with the biological expectation for such an Lrp knockout experiment. Both threshold-based methods and all threshold independent ones except Student's t-Test found the histidine biosynthesis altered in its gene expression profile. Interestingly, in contrast only Student's t-Test recognized "leucine biosynthesis" which one also would expect to be affected if the Leucine-responsive regulatory protein is reduced in its activity.

Overview on performed analyses

Fisher's exact test

Hypergeometric test

Student's t-Test

Kolmogorov-Smirnov test

Mann-Whitney U-Test

1) Results of Fisher's exact test:

In total 11 significant GO nodes (7 for the down-regulated and 4 for the up-regulated genes)

1a) Result Table

1a I) Nodes with overrepresentation down-regulated genes

threshold expression ratio: 0.5

GO Category	GO Accession	GO Name	p-value
BP	GO:0051179	localization	1.3751E-9
BP	GO:0051234	establishment of localization	1.3751E-9
BP	GO:0006810	transport	1.8499E-9
MF	GO:0005215	transporter activity	1.9645E-7
BP	GO:0019321	pentose metabolism	1.8195E-5
BP	GO:0006412	protein biosynthesis	3.0768E-5
CC	GO:0005737	cytoplasm	5.238E-5

1a II) Nodes with overrepresentation of up-regulated genes

threshold expression ratio: 2.0

GO Category	GO Accession	GO Name	p-value
BP	GO:0000105	histidine biosynthesis	2.1918E-5
BP	GO:0006547	histidine metabolism	2.1918E-5
BP	GO:0009075	histidine family amino acid metabolism	2.1918E-5
BP	GO:0009076	histidine family amino acid biosynthesis	2.1918E-5

1b) GO Subgraph Visualization

1b I) Nodes with overrepresentation of down-regulated genes

(Analysis Method: Fisher's exact-test, treshold value: 0.5)

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:

1b II) Nodes with overrepresentation of up-regulated genes
(Analysis Method: Fisher's exact-test, treshold value: 2.0)
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:

2) Results of hypergeometric test:

In total 9 significant GO nodes (5 for the down-regulated and 4 for the up-regulated genes)

2a) Result Table

2a I) Nodes with overrepresentation down-regulated genes

threshold expression ratio: 0.5

GO Category	GO Accession	GO Name	p-value
BP	GO:0051179	localization	1.0233E-9
BP	GO:0051234	establishment of localization	1.0233E-9
BP	GO:0006810	transport	1.2206E-9
MF	GO:0005215	transporter activity	1.1344E-7
BP	GO:0019321	pentose metabolism	1.8195E-5

2a II) Nodes with overrepresentation of up-regulated genes

threshold expression ratio: 2.0

GO Category	GO Accession	GO Name	p-value
BP	GO:0000105	histidine biosynthesis	2.1918E-5
BP	GO:0006547	histidine metabolism	2.1918E-5
BP	GO:0009075	histidine family amino acid metabolism	2.1918E-5
BP	GO:0009076	histidine family amino acid biosynthesis	2.1918E-5

2b) GO Subgraph Visualization

2b I) Nodes with overrepresentation of down-regulated genes

(Analysis Method: Hypergeometric test, treshold value: 0.5)

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:

2b II) Nodes with overrepresentation of up-regulated genes

(Analysis Method: Hypergeometric test, treshold value: 2.0)

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:

3) Results of Student's t-test:

40 significant GO nodes

3a) Result Table

GO Category	GO Accession	GO Name	p-value
MF	GO:0005386	carrier activity	6.255E-12
MF	GO:0042623	ATPase activity, coupled	1.8957E-10
MF	GO:0016820	hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances	3.8069E-10
MF	GO:0042626	ATPase activity, coupled to transmembrane movement of substances	3.8069E-10
MF	GO:0043492	ATPase activity, coupled to movement of substances	3.8069E-10
MF	GO:0015290	electrochemical potential-driven transporter activity	6.1132E-10
MF	GO:0015291	porter activity	6.1132E-10
BP	GO:0019321	pentose metabolism	1.4459E-9
BP	GO:0015672	monovalent inorganic cation transport	1.6916E-9
BP	GO:0006551	leucine metabolism	7.9931E-9
BP	GO:0009098	leucine biosynthesis	7.9931E-9
MF	GO:0046873	metal ion transporter activity	9.9471E-9
MF	GO:0008509	anion transporter activity	1.0484E-7
MF	GO:0016773	phosphotransferase activity, alcohol group as acceptor	5.2619E-7
MF	GO:0008863	formate dehydrogenase activity	9.869E-7
BP	GO:0051179	localization	1.0378E-6
BP	GO:0051234	establishment of localization	1.0378E-6
BP	GO:0006810	transport	1.4237E-6
BP	GO:0006814	sodium ion transport	1.7686E-6
BP	GO:0006820	anion transport	2.6371E-6
MF	GO:0015075	ion transporter activity	3.202E-6
MF	GO:0015103	inorganic anion transporter activity	3.5284E-6
MF	GO:0046915	transition metal ion transporter activity	4.4546E-6
BP	GO:0046903	secretion	4.8129E-6
MF	GO:0008565	protein transporter activity	5.7804E-6
BP	GO:0015698	inorganic anion transport	5.9471E-6
BP	GO:0016310	phosphorylation	7.1123E-6
BP	GO:0006793	phosphorus metabolism	9.6498E-6
BP	GO:0006796	phosphate metabolism	9.6498E-6
BP	GO:0006119	oxidative phosphorylation	9.8615E-6
BP	GO:0009306	protein secretion	1.3002E-5
MF	GO:0015077	monovalent inorganic cation transporter activity	1.5155E-5
BP	GO:0008643	carbohydrate transport	3.3785E-5
BP	GO:0019323	pentose catabolism	3.718E-5
BP	GO:0019566	arabinose metabolism	3.718E-5
BP	GO:0019568	arabinose catabolism	3.718E-5
MF	GO:0042625	ATPase activity, coupled to transmembrane movement of ions	3.8656E-5
MF	GO:0008901	ferredoxin hydrogenase activity	4.4855E-5
MF	GO:0016695	oxidoreductase activity, acting on hydrogen as donor	4.4855E-5
MF	GO:0016699	oxidoreductase activity, acting on hydrogen as donor, iron-sulfur protein as acceptor	4.4855E-5

3b) 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.

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

4) Results of Kolmogorov-Smirnov test:

43 significant GO nodes

4a) Result Table

GO Category	GO Accession	GO Name	p-value
MF	GO:0005215	transporter activity	0.0
BP	GO:0006810	transport	0.0
BP	GO:0051179	localization	0.0
BP	GO:0051234	establishment of localization	0.0
CC	GO:0005840	ribosome	8.7874E-13
MF	GO:0005198	structural molecule activity	1.4305E-12
MF	GO:0003735	structural constituent of ribosome	1.9576E-12
BP	GO:0006412	protein biosynthesis	4.0734E-12
CC	GO:0030529	ribonucleoprotein complex	1.0767E-11
MF	GO:0005386	carrier activity	5.9903E-10
MF	GO:0015290	electrochemical potential-driven transporter activity	1.271E-9
MF	GO:0015291	porter activity	1.271E-9
BP	GO:0009058	biosynthesis	1.3912E-9
BP	GO:0044260	cellular macromolecule metabolism	1.7427E-9
BP	GO:0019538	protein metabolism	1.7964E-9
BP	GO:0044267	cellular protein metabolism	1.7964E-9
BP	GO:0044249	cellular biosynthesis	2.5046E-9
MF	GO:0019843	rRNA binding	7.0864E-9
BP	GO:0044238	primary metabolism	1.6122E-7
BP	GO:0009059	macromolecule biosynthesis	2.6887E-7
CC	GO:0005737	cytoplasm	2.9762E-7
MF	GO:0003723	RNA binding	2.1457E-6
BP	GO:0006633	fatty acid biosynthesis	8.1227E-6
BP	GO:0006457	protein folding	8.5712E-6
BP	GO:0006520	amino acid metabolism	1.0707E-5
CC	GO:0043228	non-membrane-bound organelle	1.0805E-5
CC	GO:0043232	intracellular non-membrane-bound organelle	1.0805E-5
BP	GO:0006519	amino acid and derivative metabolism	1.2197E-5
BP	GO:0019321	pentose metabolism	1.7311E-5
CC	GO:0043226	organelle	1.7344E-5
CC	GO:0043229	intracellular organelle	1.7344E-5
BP	GO:0006811	ion transport	2.0822E-5
BP	GO:0000105	histidine biosynthesis	2.1211E-5
BP	GO:0006547	histidine metabolism	2.1211E-5
BP	GO:0009075	histidine family amino acid metabolism	2.1211E-5
BP	GO:0009076	histidine family amino acid biosynthesis	2.1211E-5
BP	GO:0009308	amine metabolism	2.4863E-5
BP	GO:0008652	amino acid biosynthesis	3.584E-5
MF	GO:0005351	sugar porter activity	3.6292E-5
MF	GO:0015144	carbohydrate transporter activity	3.6292E-5
MF	GO:0051119	sugar transporter activity	3.6292E-5
BP	GO:0044237	cellular metabolism	3.7752E-5
BP	GO:0016053	organic acid biosynthesis	4.5651E-5
BP	GO:0046394	carboxylic acid biosynthesis	4.5651E-5
BP	GO:0019752	carboxylic acid metabolism	4.6136E-5
BP	GO:0008152	metabolism	5.0219E-5

4b) 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:

5) Results of Mann Whitney U-test:

43 significant GO nodes

5a) Result Table

GO Category	GO Accession	GO Name	p-value
BP	GO:0051179	localization	2.2127E-26
BP	GO:0051234	establishment of localization	2.2127E-26
BP	GO:0006810	transport	3.9515E-26
MF	GO:0005215	transporter activity	1.9875E-20
BP	GO:0006412	protein biosynthesis	2.7062E-12
CC	GO:0005840	ribosome	2.601E-11
MF	GO:0005198	structural molecule activity	2.8885E-11
MF	GO:0003735	structural constituent of ribosome	4.6668E-11
MF	GO:0015290	electrochemical potential-driven transporter activity	1.1822E-10
MF	GO:0015291	porter activity	1.1822E-10
CC	GO:0030529	ribonucleoprotein complex	1.2424E-10
BP	GO:0019538	protein metabolism	2.5045E-10
BP	GO:0044267	cellular protein metabolism	2.5045E-10
MF	GO:0005386	carrier activity	3.7581E-10
BP	GO:0044260	cellular macromolecule metabolism	1.102E-9
BP	GO:0044249	cellular biosynthesis	2.0129E-9
BP	GO:0009058	biosynthesis	3.1652E-9
BP	GO:0044238	primary metabolism	1.9795E-8
BP	GO:0009059	macromolecule biosynthesis	1.6108E-7
CC	GO:0005737	cytoplasm	2.1201E-7
BP	GO:0006811	ion transport	2.5258E-7
MF	GO:0019843	rRNA binding	5.8349E-7
BP	GO:0006457	protein folding	2.851E-6
CC	GO:0043228	non-membrane-bound organelle	6.0605E-6
CC	GO:0043232	intracellular non-membrane-bound organelle	6.0605E-6
BP	GO:0006812	cation transport	6.4039E-6
CC	GO:0043226	organelle	8.4559E-6
CC	GO:0043229	intracellular organelle	8.4559E-6
BP	GO:0044237	cellular metabolism	9.4416E-6
BP	GO:0030001	metal ion transport	1.4052E-5
MF	GO:0003723	RNA binding	1.8201E-5
BP	GO:0000105	histidine biosynthesis	2.0466E-5
BP	GO:0006547	histidine metabolism	2.0466E-5
BP	GO:0009075	histidine family amino acid metabolism	2.0466E-5
BP	GO:0009076	histidine family amino acid biosynthesis	2.0466E-5
MF	GO:0005351	sugar porter activity	2.0698E-5
MF	GO:0015144	carbohydrate transporter activity	2.0698E-5
MF	GO:0051119	sugar transporter activity	2.0698E-5
BP	GO:0019321	pentose metabolism	2.1495E-5
BP	GO:0019752	carboxylic acid metabolism	3.0893E-5
BP	GO:0006520	amino acid metabolism	3.6656E-5
BP	GO:0008152	metabolism	5.397E-5
BP	GO:0006082	organic acid metabolism	5.4076E-5

5b) 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:

Example for a JProGO analysis:

Effect of knockout of the global transcriptional regulator Lrp in E. coli

1) Results of Fisher's exact test:

1a) Result Table

1a I) Nodes with overrepresentation down-regulated genes

1a II) Nodes with overrepresentation of up-regulated genes

1b) GO Subgraph Visualization

1b I) Nodes with overrepresentation of down-regulated genes

1b II) Nodes with overrepresentation of up-regulated genes

2) Results of hypergeometric test:

2a) Result Table

2a I) Nodes with overrepresentation down-regulated genes

2a II) Nodes with overrepresentation of up-regulated genes

2b) GO Subgraph Visualization

2b I) Nodes with overrepresentation of down-regulated genes

2b II) Nodes with overrepresentation of up-regulated genes

3) Results of Student's t-test:

3a) Result Table

3b) GO Subgraph Visualization

4) Results of Kolmogorov-Smirnov test:

4a) Result Table

4b) GO Subgraph Visualization

5) Results of Mann Whitney U-test:

5a) Result Table

5b) GO Subgraph Visualization