Tutorial 2


Extend pathway with regulatory information

CyTargetLinker provides a quick and extensive enrichment of biological networks in Cytoscape. The visualization options allow biological interpretation of complex regulatory networks in a graphical way.

In this tutorial a step-by-step explanation is given on how to extend a pathway from Wikipathways with regulatory information in Cytoscape. (Note that in this tutorial the Cytoscape version 2.8.3 is used.)


First you need to download a pathway of interest from Wikipathways. In this tutorial you will use the mouse Cholesterol Biosynthesis pathway. Go to Wikipathways and search the Cholesterol Biosynthesis pathway in homo sapiens. After opening the pathway download the pathway in gpml format via the download button on the bottom of the pathway, see Figure 1.

  • Figure 1: Download pathway as gpml in Wikipathways.

 



Install the gmpl plugin version 1.1 via the plugin manager in Cytoscape. The gpml plugin makes it possible to open pathways in gpml format into Cytoscape. Go to File Import Network (Multiple File Types). Thereafter, select the gpml files of the pathway which you stored locally and click import, see also Figure 2. Upload the pathway as a Cytoscape network (Figure 3).

However CyTargetLinker is not limited to the provided RINs. A user can also create their own RIN. A detailed description on how to create your own RIN will soon be available as a tutorial.

Before starting CyTargetLinker you need to download the RINs here and store them in a directory. The RINs provided on the CyTargetLinker website are species-specific. In this tutorial the human RINs are needed. Download all the RINs for homo sapiens, depicted in Figure 4.

  • Figure 2: Import pathway as gpml

  • Figure 3: Import as Cytoscape network

The gpml pathway is now opened as a Cytoscape network. In this network the connected lines are edges and the gene, protein and metabolite boxes are nodes, see Figure 4.

 

  • Figure 4: Cholesterol Biosynthesis pathway loaded in Cytoscape

 



If the genes and/or proteins in your network are not annotated with identifiers supported by CyTargetLinker, you need to reannotate them. For this you can use the CyThesaurus plugin in Cytoscape. A detailed description and tutorial is available at the CyThesaurus website

 



A regulatory interaction network (RIN) is a network containing regulatory interactions that are usually derived from an online interaction database. The networks are stored in XGMML (the eXtensible Graph Markup and Modeling Language)format, which is supported by Cytoscape. Each regulatory interaction consists of two nodes, a source (regulatory component) and target biomolecule, connected through one directed edge. Several RINs for different species and interaction types are provided.

However CyTargetLinker is not limited to the provided RINs. A user can also create their own RIN. A detailed description on how to create your own RIN will soon be available as a tutorial.

Before starting CyTargetLinker you need to download the RINs here and store them in a directory. The RINs provided on the CyTargetLinker website are species-specific. In this tutorial the human RINs are needed. Download all the RINs for homo sapiens, depicted in Figure 5.

  • Figure 5: Provided RINs for homo sapiens

 



Install the CyTargetLinker app via the plugin manager in Cytoscape version 2.8.3.

Open the CyTargetLinker in Cytoscape via Plugins CyTargetLinker plugin Load Regulatory Interaction Networks. Thereafter the CyTargetLinker selection panel is displayed (Figure 6).

  • Figure 6: CyTargetLinker selection panel

 

1. Select the user network. This is the biological network you want to extend with regulatory information. Here we select the Cholesterol Biosynthesis.

2. Select the network attribute. This is the column containing the identifier which is supported by CyTargetLinker. The supported identifiers are listed in overview of the RINS here. In the Cholesterol Biosynthesis network the genes are annotated with NCBI gene identifiers or Ensembl identifiers. These identifiers are present in the GeneID column.

3. Select the directory containing the species specific regulatory interaction networks (RINs) which you downloaded in step 3.

4. Specify the name of the extended biological network. By default the plugin/app adds CLT_ to the name of the initial network.

5. Specify the direction of the interaction. Interactions from source to target (adding target nodes) or from target to source (adding regulatory nodes) or both (default) can be selected.

 

Click ok en thereafter select the select the transcription factor-gene interactions from ENCODE and the drug-target gene interactions from drugbank (Figure 7).

 

  • Figure 7: RINs selection panel

 

The extended network is now visualized in a new Cytoscape network, as shown in Figure 8. To always visualize the drugbank identifiers and the gene/transcription factor names in the network go to View Show Graphical Details. In the network the nodes originating from the pathways are defined as grey circles and the added nodes as pink rounded rectangles. The color of the edges defines the origin of the interaction, as shown in the results panel on the right hand site. The color of the edges can be adapted in the results panel by click on the color.

 

  • Figure 8: Extended Cholesterol Biosynthesis network


 

In the visual style of CyTargetLinker all added nodes are displayed as pink rounded rectangles. Here we will change the color of the added nodes depending on the biological type. Per added node the biological type is present as an attribute. To see which biological node types are in your network go to Data panel, click at the select attribute button and select biologicalType. When selecting the nodes in the network you can see which types are present (Figure 9). In the extended Cholesterol Biosynthesis network, genes, transcription factors and drugs were added.

 

  • Figure 9: Biological node types in extended network

 

Next the colors of added nodes will be changed so it is possible to differentiate the node types. Go to the Vizmapper panel on the left hand site. Select the Node Color and color the nodes based on their biological type using discrete mapping. In the extended Cholesterol Biosynthesis network the genes, drugs and transcription factors are displayed in green, yellow and red, respectively, see Figure 10.

  • Figure 10: Visualization biological types