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Using CoryneCenter
The usage of the CoryneCenter functionality is explained in detail from the perspectives of the systems (GenDB, Emma and CoryneRegNet) that have been integrated using web service functionality. This tutorial will provide the user of the respective system with the necessary information to utilize the novel analysis features that are provided in the CoryneCenter context. Various screenshots (please click to zoom in) for each of the three integrated systems depict these features.
Using CoryneCenter from the CoryneRegNet perspective
The following use case demonstrates how the CoryneCenter functionality can be used to analyze a complex microarray experiment in the context of gene regulatory networks.
- From the portal page www.CoryneCenter.DE select the CoryneRegNet button.
- Select the 'Login' button at the CoryneRegNet entry page.
- Select the Corynebacterium glutamicum ATCC 13032 as organism. Enter 'ram' and 'glxr' in the search fields and choose the 'or' operator. Query for the 'Gene name' and push 'Start search'. (Refer screenshot 1)
- The matching genes are presented, scroll to the end of the list and unmark 'Include genes from regulations' and press the 'GraphVis' button. (Refer screenshot 2)
- Please accept the certificate. Afterwards the visualization is prepared and after some seconds a Java applet appears.
- Anser the question to import homology data with 'No'. (optional)
- Mark all genes of the network by dragging a rectangle around them and select 'Tools', 'Gene expression data', 'Import expression data from EMMA web service'. (Refer screenshot 3)
- The EMMA web service wizard appears. Press 'Next' to import all available projects.
- Select the 'EMMA2.Coryne-Center' project and press 'Next'.
- Select the 'GlucoseAcetat' experiment and press 'Next'.
- Select 'Carbon Source' as factor and press 'Next'.
- Select the 'Acetat.Glucose' as factor value and press 'Finish'.
- Expression values for all selected genes are imported from the microarray experiment stored in EMMA. This may take a while.
- All imported expression values are presented in a separate table (refer screenshot 4).
- To obtain an optimal layout of the resulting graph, select 'Graph layouts' in the menu and choose 'Hierarchical' and press 'Ok'.
- The result is shown in screenshot 5.
- One can see directly that the target genes of ramA are consistently upregulated (nodes with dashed green borders), while the target genes of ramB are inconsistently regulated. Some genes are upregulated, other are downregulated (nodes with dashed red borders). The node sizes correspond to the change of the relative gene expression levels in the microarray experiment.
- This combined result indicates an additional yet unknown regulatory network and provides a starting point for further analyses.
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Using CoryneCenter from the GenDB perspective
The following use case demonstrates how a GenDB user can benefit from the integration of the system in the CoryneCenter infrastructure. The analysis of the regulation of the genes of one metabolomic pathway are presented for the organism Corynebacterium glutamicum ATCC 13032. Please follow the steps listed below to obtain an overview of the novel GenDB features. For an general overview of the established GenDB functionality, please refer to the GenDB homepage and the GenDB Project Wiki.
- From the portal page www.CoryneCenter.DE select the GenDB button.
- This starts a user session for the CoryneCenter GenDB web project. The contig view of GenDB is presented (Refer Screenshot 1). Select C.glutamicum from the 'Contigs' entry in the main menu.
- In the 'Edit' menu select the 'Search' entry. Please fill out the Search form that will show up according to Screenshot 2. Hit the 'Search' button.
- The matching genes are presented, please select the link to cg2103 from the result list. This selects the coding sequence cg2103 which represents the dtxR gene in C.glutamicum as active region. It will be centered in the main contig view and all other region specific frames of the webinterface show the information available for this gene/region.
- CoryneCenter specific features appear in the context specific menu at the lower right of the main contig view.
- Visualize in CoryneRegNet
- Show Regulations
- Regulation Report
- Linear Regulation Report
- The regulon of a coding sequence can be viewed as linearized gene clusters by selecting the 'Linear Regulation Report'. For dtxR this is depicted in Screenshot 3.
- In order to obtain an overview of the chromosomal location of the genes that are regulated by e.g. dtxR, the 'Regulation Report' can be generated. It shows an interactive circular representation of the genes of a chromosome and highlights the regulon of the selected regulator. Additional information can be obtained by hovering with the mouse cursor over a gene of interest. Please click the 'Regulation Report' button which will open a new browser window in GenDB or refer to Screenshot 4.
- Apart from these visualizations, the direct connection to the CoryneRegNet user interface is available for every coding sequence through the 'Visualize in CoryneRegNet' button.
- In order to observe the effect of regulations on complete metabolic pathways, GenDB now also offers the enriched KEGG Pathway visualization. CoryneRegNet information is accessed via web service and projected to an interactive KEGG Pathway map. For a demonstration, please select 'Classification', 'KEGG' from the main menu of the GenDB web interface. This will open up the KEGG perspective of GenDB in a novel window. From the pathway list to the left, please select 'Sulfur metabolism'. The pathway is generated and additional regulatory information for the enzymes is presented as red and green arrows with tooltip informations above the E.C. number boxes. (Refer to Screenshot 5)
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Using CoryneCenter from the Emma perspective
In the following the extended CoryneCenter functionality that an Emma user can access is explained and demonstrated. Please follow the step-by-step tutorial to obtain an overview of the advanced visualization and analysis features now available in EMMA.
- From the portal page www.CoryneCenter.DE select the EMMA button.
- This starts a guest session for the CoryneCenter project in EMMA. This project has been connected to the CoryneCenter functionality and features a micro array experiment of C.glutamicum grown on two different carbon sources (Glucose and Acetate).
- Select 'Browse Experiments' in the main window of EMMA. This generates a list of available micro array experiments for the current user.
- Select the 'GlucoseAcetat' Experiment by clicking on the experiment name. The experiment overview page is generated presenting the 'Basic description' and the 'Analysis Results' of the experiment. (Screenshot 2)
- Click on the KEGG button at the bottom of the experiment overview page to view the log-ratio changes of the measured transcript concentrations for a specific pathway. From the alphabetic list of KEGG pathways, please select e.g. 'Sulfur Metabolism'. (Refer Screenshot 3)
- In order to obtain a detailed regulation and expression report for a specific gene that is present in the KEGG pathway, please click on the box with the respective E.C. number. To view detailed information of the gene with identifier cg3115 (also presented in Screenshot 4) please click on the EC-Number 2.7.7.4 in the 'Sulfur Metabolism' pathway.
- At the bottom of this page the user finds a list of regulators in CoryneRegNet that habe been predicted or identified experimentally. If the gene is present in an operone structure this information from CoryneRegNet is presented as well. Furthermore, links to the respective publications in PubMed are generated. (See Screenshot 5)
- The M/A plot in EMMA provides a starting point for the interactive analysis of genes that show a different expression for varying experimental conditions. Please navigate back to the experiment overview page (Screenshot 2) and select the 'M/A-plot' button (located at the bottom of the page). The M/A plot for the 'Glucose vs. Acetate' experiment is generated (Refer Screenshot 6a). By clicking 'Load spot information' in the 'Basic Menu' to the left, additional informations for the reporters are retrieved. Afterwards, the M/A-plot is rendered interactive and will present the reporter ID for every spot touched with the mouse cursor. Please click the green spot at M ~ 4, A ~ 13. The reporter overview page with the regulation information for the according gene is generated. (Refer Screenshot 6b)
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Screenshots 6a & 6b