xiSEARCH: step by step instructions
- Ensure java version 8 (64 bit) is installed on your computer (https://www.java.com/ note that when downloading with a 32-bit browser an explicit selection of a 64-bit download is required).
- For large scale analysis a computer with 16 GB memory is recommended.
- Operating system: Windows (Windows Server 2008 and 2012 and Windows 8 and 10) and Linux (Debian 9, Ubuntu 14.04, Ubuntu 16.04) were tested successfully – but expect that any system running java 8 64 bit should suffice
- Download the ZIP file from http://xi3.bio.ed.ac.uk/downloads/xiSEARCH/.
- Extract the compressed files to your computer (e.g. C:\Users\John). You will have a new folder (C:\Users\John\xiSEARCH).
A short rundown of the usage can be found here.
- If you are using Windows start the startXiWindows file. If you are using Unix start the startXiUnix file. This will start Xi with a simple graphical user interface (GUI), and the actual java library (xiSEARCH.jar).
- The GUI has 5 tabs. In the first tab Run, choose the location where you want to save your results under the box result. Optional: In peak annotations you can define a file for writing out full annotations of the spectra.
- In the second tab Peak Lists select the peak lists to be searched (Supported formats: mgf-files as generated from msconvert or apl-files as produced by MaxQuant. For mgf-files of other sources the config may need to be adapted to define how to read out a run-name and a scan-number from the TITLE= tags in the files).
- In the third tab Fasta Files select the FASTA files to be searched. Load opens a file selection dialog. Multiple files can be selected there from the computer and one can be defined as custom decoy database by checking the appropriate box. If no decoy database is given, the program automatically generates one based on the target database. Xi does not run by default without decoy database.
- In the fourth tab Config the search is configured. All parameters are defined in form of a text file that can be edited in the GUI. The tab opens displaying a default config as an example that defines a search with BS3 as cross-linker, carboxyamidomethylation on cysteines as fixed modification, oxidation of methionine as variable modification and trypsin as the protease of choice. Additional options are still included with a short description of their function, example and the syntax. When you have your config file defined you can save it for further searches under the box Save. Load allows you to open saved config files from your computer.
- Go back to the first tab Run and press the Start button to start your search.
Ones the results are written out we suggest to filter the data to a defined confidence with xiFDR.
- Depending on the size of the sequence-database and the number of search threads the start-script might need to be adapted to permit xi to use a larger amount of memory (-Xmx option). This should not exceed the amount of free memory available without running Xi. For example, if a computer has 8 GB of RAM but per default 5 are used by other programs then Xi should get at most 3 GB as otherwise part of the program is likely to be swapped out to disk and in effect will run extremely slow.
- Additionally to the GUI a command line interface is provided that can be used via “java –Xmx4g -cp Xlink.jar rappsilber.applications.Xi --config=[config-file] --peaks=[path to peaklist] --fasta=[path to fasta file] --conf=[some xi-parameters] --output=[csv-file] --peaksout=[csv-file] --exampleconfig=[path]” Description of the arguments can be gained by running java -cp Xlink.jar rappsilber.applications.Xi --help
- xiSEARCH does not assess the FDR. This is done by a separate application xiFDR 35. xiSEARCH result files can be directly read into xiFDR.
- A more detailed step by step presentation of raw-file conversion, xiSEARCH and confidence filtering with xiFDR can be found here