This tutorial demonstrates the new functions in VisANT 4.0, we will try to test a simple hypothesis of drug repositioning: drugs targeting to the known disease genes may be repositioning candidates if they are not drugs already used for the disease. Using breast cancer as an example, we take following steps to illustrate how we can achieve this in VisANT 4.08:
1.1 Start VisANT by either double-clicking the downloaded VisAnt.jar file, or click the Start button on the VisANT web page (http://visant.bu.edu)

1.2 Clear the network by click the Clear button on VisANT ToolBox, and set the current species to Home sapients (Fig. S1)

1.3 Open the method table using the menu View→ Method Table. Scroll down the method table to make the method M6001 visible, and click the button All near the method to load all drug-target interaction available in VisANT system, as shown in Fig. S1


Fig. S1, Construct drug-target network in VisANT using Method Table.


1.4 Do layout using the menu Layout→ Spring Embedded Relax. Stop the layout when the network looks similar to the one shown in Fig. S2.

Fig. S2 Drug-target network after layout. The topology structure is very clear.


1.5 The node is too small in Fig. S2. Click the button Zoom Out on VisANT toolbox, and then click button Reset button to reset the node to the default size. Then click the button Zoom Out several times so that the node size will be similar to the one shown in Fig. S3, then click the button Fit to Page.

1.6 Rearrange the network by moving small subnetwork into the spare space (Fig. S2) to make the network more compact, and then click the button Fit to Page. The network shall look similar to the one shown in Fig. S3.


Fig. S3. Drug-target network after manual rearrangement.


1.7 Select all nodes by either using mouse to drag a rectangle to cover all nodes, or through the key combination CTRL-A, or through the menu Edit→ Select All.

1.8 Click on the button Properties on right side bar in VisANT so that properties window will be popped up (Fig. S4), and click the Dock Button (as shown in Fig. S4) to dock the property window, and change the width of the property window if necessary. Make sure that two node properties: Expansion symbol and KEGG symbol are unchecked.


Fig. S4.


1.9 Click the Dock button again to hide the property window. Click on the empty space in the network to clear the selection. The network shall look similar to the one shown in Fig. S5



Fig. S5

2.1 Click the Hierarchy Explorer on VisANT ToolBox, so that disease classification is ready to explore. Make sure that the More button (represented by an icon of up arrow near Search button) is visible. Change the width of the Hierarchy Explorer if necessary, as shown in Fig. S6.


Fig.S6


2.2 Click the Morebutton, and select the hierarchy to be search is Disease as shown in Fig. S7. For the operation of drag&drop, select the Metanode of existing components only, as also shown in Fig. S7. Click the More button again to hide the configuration panel.


Fig. S7


2.3 Type in breast cancer as the key words to be searched, and click the button Search. The results are shown in Fig. S8.


Fig. S8


2.4 As shown in Fig. S8, the number of genes associated with the disease will be shown when the expansion symbol of the tree node is clicked. In this example, GAD (Genetic Association Database, Ref. 25 in the main manuscript) predicted that there are 879 genes associated with breast cancer. Drag and drop this tree node to the drug-target network, a metanode of 73 genes are created and the nodes of the 73 genes are selected, as shown in Fig. S8.

2.5 To distinguish these breast cancer genes predicted by GAD database, lets change these nodes of disease genes into to blue color. Click the button Properties on right side bar, and change the color to blue as shown in Fig. S9.


Fig. S9


2.6 Because we only need to know the genes associated with breast cancer, we will therefore remove the metanode of breast cancer. Select the metanode by click on it, and ungroup it using menu MetaGraph→ Grouping→ Ungroup Selected Nodes. The resulting network will look similar to the one shown in Fig. S10. The drugs that connecting to the genes in the blue color shall be the repositioning candidates for breast cancer, given the hypothesis we made in this case study. 


Fig. S10


The hypothesis we made obviously is primary and simple. As a quick test of this hypothesis, we compared our prediction against the predictions of an independent repositioning study (Ref. 5 in main manuscript) , which list following drugs as repositioning candidates for breast cancer with supporting evidence:
In the case of UC/DB:
Amiloride, Dizocilpine, Estradiol, Irinotecan, Metergoline, Nocodaole, Sirolimus, Thioridazine, Valproic acid
In the case of DC/UB:
Artemisinn, Bupropion, Dexamethasone, Dizocilpine, Dydrogesterone, Etoposide, Gabapentin, Irinotecan, Mestranol, Methotrexate, Nimesulide, Nomegestrol, Novobiocin, Prochlorperazine, Sirolimus, Testosterone, Valproic acid, Trifluoperazine, Troglitazone   
The following steps illustrate how we can examine whether above repositioning candidates overlap with the results in this case study.
3.1 Make sure drug hierarchy will be searched as shown in Fig. S11:


Fig. S11


3.2 Copy/paste drug Amiloride into the search box, and click the button Search. If the drug can not be found in the ATC hierarchy, continue with next step. Otherwise, the result will be shown in the hierarchy true, as shown in Fig. S12:


Fig. S12


3.3 As shown in Fig. 12, the ATC hierarchy indicates there are two drugs under the classification. Drag & drop the tree node to the network panel, unlike step 2.3, a collapsed therapy is created, which indicates that the drug-target network do not have drug Amiloride. Select and delete the therapy node using the menu Edit→ Delete Selected Nodes.

3.4 Continue with drugs Dizocilpine, Estradiol by repeating step 3.2, Dizocilpine can be bypassed because nothing can be found in the drug hierarchy for it.

3.5 When dragging & dropping Estradio tree node into the network, it does find several drugs in the drug-target network, and these drugs are targeting to the blue nodes, and that is what we are looking for, as shown in Fig. S13:


Fig. S13.


3.6 Lets change the color of these drugs so they can be distinguished from the rest. Use the same instructions as step 2.5-2.6, and change the color to purple (RGB; 102, 0, 204), and ungroup the therapy metanode:


Fig. S14


3.7 Repeat steps 3.2-3.6 for all the rest drug, the resulting network will look similar to the network shown in Fig. S15 (modified from the same network used for Fig. 3 in the manuscript, which ahs exactly same contents in comparison to the network shown in Fig. S14 with slightly management of nodes positions). During the process of searching for the drugs, users can always use pop-up menu Collapse All to collapse all expanded trees.


Fig. S15


3.8 So exactly how many drugs have supporting evidence as the repositioning candidates for the breast cancer? To answer this question, right mouse click over the purpose nodes, and use the pop-up menu Nodes→ Select Nodes of Same Properties, as shown in Fig. S16. The number 32 (located at left-bottom corner in Fig. S16) is the total number of selected nodes (SN).


Fig. S16


3.9 Add the network legend by menu View→ Network Legend. Customize the label of the nodes embedded in the Legend nodes so they look similar to the one shown in Fig. S17. To save your time, the VisML file of the network shown in Fig. S21 is available at:  http://visant.bu.edu/sample/visml_files/NAR2013_fig3_full.xml and can be loaded into VisANT directly using the menu Files→ Open URL. It can also be loaded into VisANT through Java Web Start by clicking on the following URL:
http://visant.bu.edu:8080/vserver/DAI?command=link&location=http://visant.bu.edu/sample/visml_files/ NAR2013_fig3_full.xml


Fig. S17. The final network for the case study (the same network used for Fig. 3 in the manuscript).


Alternatively we can start with the genes that are associated with breast cancer, and then query for the drugs that are targeting to them.

4.1 Clear the network by clicking on the button Clear on VisANT ToolBox, as shown in Fig. S18


Fig. S18


4.2 Change the option of the drag&drop operation to the one shown in Fig. S18, and search the disease hierarchy for breast cancer. Then drag & drop the breast cancer tree node of GAD hierarchy to network panel. A disease node with 870 embedded will be created as shown in Fig. S18.

4.3 Open the Method Table in VisANT using menu View→ Method Table, deselect all method, and then select the method M6001 as shown in Fig. S19


Fig. S19.


4.4 Ungroup the disease node and then select all nodes and query for the interactions, which will take about 6 minutes to finish. The resulting network will looks similar to the one shown in Fig. S20:


Fig. S20


4.5 Repeat step 1.4-1.6 to layout the network similar to the one shown in Fig. S21

Note: Steps 4.4-4.5 may take about 10 more minutes. To save your time, the VisML file of the network shown in Fig. S21 is available at:

http://visant.bu.edu/sample/visml_files/NAR_2013_FigS21.xml

and can be loaded into VisANT directly using the menu Files→ Open URL.
It can also be loaded into VisANT through Java Web Start by clicking on the following URL:

http://visant.bu.edu:8080/vserver/DAI?command=link&location=http://visant.bu.edu/sample/visml_files/NAR_2013_FigS21.xml


Fig. S21. 


4.6 Filter these genes that have not been targeted by any drugs. Select menu Filters→ Select Nodes Based on Their Degrees. And type in 1- as shown in Fig. S21. All nodes with at least one connection will be selected. Then apply menu Edit→ Invert Node Selection, and all nodes have no connection are selected. Delete them by applying menu Edit→ Delete Selected Nodes. The network after filtering is shown in Fig. S22.


Fig. S22


4.7 Apply menu Edit→ Delete Invisible Nodes to further clean the network.

4.8 Select any gene node, and apply menu Nodes→ Select Nodes of Same Properties, 73 nodes will be selected, exact the same number of genes in step 2.4

4.9 Clear selection by mouse clicking on empty space in the network. Select any drug nodes, and apply menu Nodes→ Select Nodes of Same Properties, 774 drug nodes are selected. Combine with the number 32 in step 3.8, there are at least 4.1% (32/774) drugs having supporting evidence as the repositioning candidates for breast cancer.

4.10 The network shown in Fig. S22 can be used to compare the results with other study, using same steps specified in the session 3.