Tutorial 1b

Extend a set of microRNAs with regulatory information using the CyTargetLinker 3.0 app

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 list of microRNAs with predicted and validated microRNA-target interactions (MTIs) in Cytoscape versions 3.x using the new CyTargetLinker 3.0 app

First you will need to create a biological network in Cytoscape 3.x containing your microRNAs of interest. The datafile should contain at least two columns, (i) the microRNA name and (ii) the corresponding miRBase accession number. An example datafile, in which two microRNAs known to be involved in obesity are listed, can be found here. Open the datafile containing the microRNAs in Cytoscape via File Import Network File. First you select the network file that you want to load into Cytoscape, see Figure 1. Thereafter a panel becomes visable were you should select the column with microRNA names as the source interaction and transfer the header of the column as attribute names, as shown in Figure 2.

  • Figure 1: Select file containing microRNAs

  • Figure 2: Import microRNAs into Cytoscape 3.x

Thereafter the miRBase accession number and possibly other characteristics of the microRNAs present as columns in the datafile used in step 1 should be added as attributes. Go to File Import Table File. In the panel you should select the correct Network collection, indicate at Import Data as that the information should be added to the nodes, so as Node Table Columns and transfer the header of the column as attribute names. The first column is automatically taken as the node identifier, as shown in Figure 3.


  • Figure 3: Add attributes to the microRNA network


The resulting biological network of two microRNAs from the example data, as shown in Figure 4.

  • Figure 4: microRNA network in Cytoscape


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 app manager in Cytoscape version 3.0

Open the CyTargetLinker in Cytoscape via Apps CyTargetLinker plugin Extend network. 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 human microRNAs.

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. For the microRNAs we recommend to use the miRBase accession numbers.

3. Select the directory containing the species specific regulatory interaction networks (RINs) which you downloaded in step 2.

4. Select the direction of the interaction. The user can decide how he wants to extend the network, i.e., 1) add regulators (interactions from target to source),  2) add targets ( interactions from source to target)  or  3) both (default). Here we add only the targets of the microRNAs.


Click ok en thereafter select the RINs containing microRNA-target information (Figure 7). In this case we have three resources containing microRNA-target interactions, i.e., microCosm, miRTarBase and TargetScan.


  • Figure 7: RINs selection panel


The extended network is now visualized in a new Cytoscape network, as shown in Figure 8. To visualize the microRNA names and gene names in the network go to View Show Graphical Details. In the network microRNAs and target genes are defined as grey circles and pink hexagons, respectively. The predicted MTIs are visualized in blue (microCosm: 1543) and purple (TargetScan: 2550) and the validated MTIs in blue (miRTarBase: 17), as shown in the CyTargetLink-panel on the left hand site. The color of the edges can be adapted in the results panel by clicking on the color. The hide&show and threshold functionalitites will be explained in step 4.


  • Figure 8: Extended microRNA network with MTIs


The visualization options in CyTartgetLinker make is possible to investigate the regulatory network in more detail.

Hide and show functionality

It is possible to hide or show specific RINs.

Here we are interested in only the validated MTIs. Go to the results panel and hide the RINs containing predicted MTI, i.e., TargetScan and MicroCosm, by clicking on the dropdown menu. Now only the RINs from miRTarbase are shown in the network and the layout is immediately adapted (Figure 9).

  • Figure 9: Validated MTIs using hide&show functionality

Threshold functionality

It is possible to show interactions that are present in at least a specific number of RINs.

Here we are interested in MTIs that are present in at least 2 RINs. Go to the CyTargeLinker-panel, make sure all the RINs are shown, and set the overlap thershold to 2. Now the overlap between the RINs is shown and the layout is immediately adapted (Figure 10). Per RIN it is indicated first how many interactions are still present when applying a threshold of 2 and the second number respresent the total number of interactions were added to the original network. For example, after setting the threshold to 2, 11 microRNA-target interactions from miRTarBase are still present, whereas the total amount of added interactions from miRTarBase was 17.

  • Figure 10: Overlap MTIs using threshold functionality