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USER MANUAL GRN plugin The GRN query plugin is designed to retrieve the transcriptional regulatory information within undirected coexpression network. It utilizes regulatory knowledge from the databases Transcriptional Regulatory Element Database (TRED)(Jiang, Xuan, Zhao, & Zhang, 2007), Molecular Signatures Database (MSigDB) (Liberzon et al., 2011) and JASPAR database (Sandelin, Alkema, Engström, Wasserman, & Lenhard, 2004). Invoking the GRN Plugin The GRN query plugin can be initiated from the menu bar Window > GRN Input data The GRN query plugin expects to receive input data in tabular form, with each line in the file representing a regulation link (from_node, direction, to_node). For example, a sample file with 14 regulation links and 11 genes with this format might look as follows: Sp2 Sp2 Sp2 Sp2 Sp2 Sp2 Sp2 Sp2 Sp2 Wt1 Wt1 Wt1 Wt1 Wt1 undirected undirected undirected undirected undirected undirected undirected undirected undirected undirected undirected undirected undirected undirected Th Tnfrsf23 Tsix Usp29 Wt1 Xlr4c Zim1 Zim2 Zim3 Xlr4c Zfp264 Zim1 Zim2 Zim3 The user can load the input co-expression network using the button “choose network file” Then, the plugin will calculate automatically the number of edges and nodes in the loaded network Plugin options The options panel can be invoked from preferences > plugins > GRN plugin tab. The application gives the user the options to select the databases of interest to retrieve the regulatory information from and also identify some parameters related to each one such as the species, and automatic update with every run. Additionally, the user can set the binding motif parameters such as the length of considered promotor region, and whether taking the motif complement into consideration or not. Moreover, the user has the option to expand the network by (a) adding further transcription factors that are annotated as known regulators of the input gene network, and /or by (b) including additional target genes that are annotated to be regulated by the TFs in the input network, and /or (c) searching for regulation interactions between the additional target genes and the additional TFs Create a task After setting all parameters, user needs to create a task by pressing the button “create task” to register this plugin run into the task queue. Finally, to run the plugin, click menu bar “Task” > “Run Tasks” Output After finishing the task, the resulting GRN network will be displayed in the network panel (network tab). This graphical display is user interactive and indicates whether the edge is directed or not. The application uses force directed algorithms to layout the nodes and make the network aesthetically displayed as much as possible. Nevertheless, the layout is sometimes heuristic and is not always good. Therefore, we advice the user to use the mouse to move the nodes and edges around to manually create a more readable layout. The output GRN network displayed in the network panel. Transcription factors involved in the input network are identified and marked in yellow, while the remaining genes are in blue. Marked in orange the additional transcription factors; while the additional target genes are marked in green. The edge labels depicts the confidence level of the corresponding regulation link. Also, one can export the network as an image or as a network file (.sif) to be imported, and visualized by other network analysis tools like Cytoscape (Smoot, Ono, Ruscheinski, Wang, & Ideker, 2011) or Visant (Hu et al., 2013).