Tutorial 1

Extend a set of microRNAs with target information

CyTargetLinker provides a quick and extensive enrichment of 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). The tutorial was created for the following versions:

Step 1: Import a set of microRNAs into Cytoscape

First you will need to create a biological network in Cytoscape 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 downloaded from here (microRNA.xlsx).

Figure 1
Figure 1

The resulting biological network of two microRNAs from the example data, as shown in Figure 2.

Figure 2
Figure 2

Step 2: Download MTI linksets

A linkset is a network containing additional information about and links between nodes in the network. This could be regulatory interactions like microRNA-gene interactions. The networks are stored in XGMML (the eXtensible Graph Markup and Modeling Language) format, which is supported by Cytoscape. Each linkset contains interactions consisting of two nodes, source and target, connected through one directed edge. Several linksets for different use cases, species and interaction types are provided on our website, see Figure 3. CyTargetLinker is not limited to the provided linksets. A user can also easily create their own linksets.

Before starting CyTargetLinker you need to download the linksets of interest and store them in a directory. In this example, we will use microRNA-gene interaction linksets from miRTarBase (validated interactions) and TargetScan (predicted interactions). For this tutorial, you can download a zip file containing both linksets from here.

Figure 3
Figure 3

Step 3: Built regulatory network in Cytoscape

Figure 4
Figure 4

After clicking ‘OK’, you can select which of the linksets in the folder should be used - in this tutorial, we will use both available linksets in one extension step (Figure 5).

Figure 5
Figure 5

After the extension (this might take a little while depending on the size of the original network and linksets), the extended network is visualized in a new Cytoscape network, as shown in Figure 6. Go to ‘Apps -> CyTargetLinker -> Show result panel’ to see how many interactions are added from which linkset. A pop-up box appears that says Show/hide Result Panel. Make sure the box is checked and press ok. In the Results Panel, select CTL_microRNAs (SUID:94) from the drop-down box. For the current extension 942 edges from miRTarBase and 2,997 from TargetScan were added. The edge color indicates from which linkset the interaction was added (red for miRTarBase and blue for TargetScan).

Figure 6
Figure 6

Since the default layout (forced-directed) is not ideal for this specific extended network, you can try other layouts (e.g. the yFiles organic layout, see Figure 7) by clicking Layout -> yFiles Organic Layout. This gives you a better idea about how many shared targets the two microRNAs have.

Figure 7
Figure 7

Step 4: Adapt visualization options

At any point you can hide certain link sets by using the ‘Show/Hide’ dropdown box in the results panel. You can also change the edge color used for a specific link set in this panel.

A very useful feature for large networks with multiple link sets containing the same interaction type is the ‘Overlap threshold’ functionality. If you select ‘2’, you will only see those interactions that are present in at least two link sets. If you try this in the tutorial network, you will only see 454 microRNA-gene relationships (two edges between each of them – one from each database, see Figure 8). Those interactions are the only ones present in both databases.

Figure 8
Figure 8