HowToRunGalaxyNGSPlot
Figure 1. The Galaxy ngs.plot interface. Numbers in red will be referenced in the text below as Figure 1-number.
- Go to your local or institutional Galaxy web page that has ngs.plot installed. Click the Analyze Data tab at the top of the page (Figure 1-1). This would load a page that is separated into three columns. The left column displays all the tools and plug-ins available on your Galaxy server. The middle column is the main display area, and will display the form interface for your selected tool or the output files generated. The right column will list all the files that you upload to the system, or are generated by your analysis runs.
- On the left column, click Get Data, followed by Upload File (Figure 1-2). A pop-up window will appear and follow the instructions to select one or more files to upload. The list of uploaded files will then appear on the right column. Figure 1-3 shows that we had uploaded the two BAM and one BED input files.
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Back to the left column, click NGS.PLOT V2.47.1, followed by ngs.plot (Figure 1-4). This will load the ngs.plot interface onto the center column (Figure 1-5). This interface consists of a fillable form showing the arguments to be set on ngs.plot, filled with the default settings that can be changed by the user.
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For example, we can choose to set the Genomic region field to 'Genebody' (Figure 1-6), the Flanking region size field to '3000' (Figure 1-7), etc., and for the Sample 1: Input BAM file field, users are to choose from among the dropdown list showing the same files as they are listed on the right column (eg. choose '1:p196_tet1.bam'). Finally click Execute at the bottom of the page.
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A message will then appear informing that the job has been submitted, and will also list three output files from the run. The three output file consist of an enrichment profile plot, a heatmap, and a zipped file containing the all the necessary information require for future replotting. These output files are stored on the Galaxy system, and are thus added onto the right column list. During the progress of program run, the list of output files will be displayed on a yellow background, that will change to green once the analysis run is complete (Figure 1-8). Users can then click on the respective file to view or download the output data.
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Note: this single ngs.plot form interface in the Galaxy plug-in has been designed to be fully interactive and will adapt to request further required run information based on user input. Therefore, this same interface can also be used to execute the more advanced functionalities of ngs.plot, such as multi-sample analyses, pairing of samples to calculate fold changes, using custom genelists or BED files (must be uploaded as instructed above), etc. Additional arguments will be added and non-relevant ones removed automatically from the form interface as the user progressively adds more program execution information. Therefore, the user just needs to fill in the entire form provided by the Galaxy interface, and is no longer required to create any configuration files that is required by the command line method, as it will now be generated automatically. Users are however advised to fill the form interface information systematically from the top to the bottom of the page, in order not to miss out on fields that might change along the process.
- To replot the average profile or heatmap graphs based on the outputs from an earlier ngs.plot run, click NGS.PLOT V2.47.1 then replot to load the replot interface (Figure 1-9). Since the zipped output file of the earlier run is already stored on the Galaxy system (Figure 1-10), simply select it for the Input zip file created by ngsplot field in the replot form interface. Proceed to fill in the rest of the form interface information and click Execute to run. As before, the replotting output file will also remain on the Galaxy system and appear in the right column (Figure 1-11), where it can be clicked on to view or download.
This latest implementation of ngs.plot on Galaxy has been designed to be easy-to-use and intuitive. For a better understanding of the different functionalities of ngs.plot, please refer to the Examples section of our main homepage or our ngs.plot publication.