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All executable programs have a help section. Use -help to view usage instructions.
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1. Open a terminal or a command line
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2. Create a new directory where your search should be done, change into this directory.
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3. Copy the definition file xquest.def and xmm.def to this folder.
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The xmm.def file specifies parameters to search for isotopic scan pairs: view template
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The xquest.def file specifies parameters for the xQuest search: view template
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To generate template definition files execute the following command in the folder:
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`runXquest.pl -getdef`
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4. Edit the definition files.
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For searches with non labeled cross-linkers adapt the parameters described in this document: Light only searches with xQuest (link)
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Please note: If you want to run with a decoy database used the string \"decoy_ProteinX\" in your fasta sequence headers.
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5. Prepare for search.
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* Create a file with the filenames to search. (e.g. name the file "files", only use the basenames, one per line). This is used to do a batch search, where multiple MS files are searched with the same parameters (but can also be used with only 1 file).
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* Then execute pQuest.pl: this program prepares the folder structure for the search.
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Please note: For conversion of your RAW files to mzXML files use 32-bit binary encoding precision.
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`pQuest.pl -list files -path /path/to/mzXML_files/`
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6. Search
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Now the xQuest search can be executed. Since several tasks have to be fulfilled, several programs are executed sequentially:
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xmm.pl: Search for isotopic pairs.
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compare_peaks3.pl: Comparing light and heavy scans, sorting of peaks into common-ions and cross-links ions.
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xquest.pl: xQuest search.
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To run these tools, several options are availaible. The program runXquest.pl is a wrapper for the whole pipeline.
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a. Directly execute the search(es): - This will directly execute the search on the command line.
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`runXquest.pl -list files -xmlmode -pseudosh`
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b. Generate .sh or .bat files for the search(es), and submit the commands then to a queuing system.
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`runXquest.pl -list files -xmlmode -pseudosh -sh`
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7. Merge search results
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When the searches are finished, the result xml files (xquest.xml) of each experiment (MS run) can optionally be merged into one single result xml file. Merging is done with the script mergexml.pl which creates a new folder, merges the xml files, and copies the *.spec.xml files into the new folder. The spec.xml files are used to save the MS/MS spectra in a central file. In the new folder (here: Test_results) you will find the merged xml file (merged_xquest.xml), the spec.xml files, xquest.def file and the sequence databases.
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`mergexml.pl -list resultdirectories_fullpath -resdir Test_results -v`
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8. xProphet analysis
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In this example, the xProphet analysis is done by using the merged xml file (merged_xquest.xml). First, a definition file is needed (xproph.def) which specifies the default parameters for xProphet. This file is created by simply executing xProphet without parameters.
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Change to the xProphet analysis folder (Test_results) that contains the merged result files and execute xProphet to write the definition file:
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```
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cd Test_results
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xprophet.pl
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```
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Edit the xprop.def file.
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Then the xProphet analysis is executed by:
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`xprophet.pl -in merged_xquest.xml -out xquest.xml`
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The output of xProphet is an xml file (in this example: xquest.xml).
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9. Copy results to the results folder
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The results folder is copied to the web server directory and file permissions are set:
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```
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cp -R Test_results /home/xquest/results
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chmod -R 777 /home/xquest/results/Test_results
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```
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To view the results, open the Web Browser and browse to the link of the results manager.
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Click on the Link Test_results to view the results.
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Please note: When the results are displayed for the first time, a new index is created. That might take a few minutes.
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Also, if the results are displayed as plain text in the web browser you have to refresh the browser window. |
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\ No newline at end of file |