Complete workflow to run OpenSWATH.

This implements the OpenSWATH workflow as described in Rost and Rosenberger et al. (Nature Biotechnology, 2014) and provides a complete, integrated analysis tool without the need to run multiple tools consecutively. See also http://openswath.org/ for additional documentation.

It executes the following steps in order, which is implemented in OpenSwathWorkflow:

Reading of input files, which can be provided as one single mzML or multiple "split" mzMLs (one per SWATH)
Computing the retention time transformation, mass-to-charge and ion mobility correction using calibrant peptides
Reading of the transition list
Extracting the specified transitions
Scoring the peak groups in the extracted ion chromatograms (XIC)
Reporting the peak groups and the chromatograms

See below or have a look at the INI file (via "OpenSwathWorkflow -write_ini myini.ini") for available parameters and more functionality.

Input: SWATH maps and assay library (transition list)

SWATH maps can be provided as mzML files, either as single file directly from the machine (this assumes that the SWATH method has 1 MS1 and then n MS2 spectra which are ordered the same way for each cycle). E.g. a valid method would be MS1, MS2 [400-425], MS2 [425-450], MS1, MS2 [400-425], MS2 [425-450] while an invalid method would be MS1, MS2 [400-425], MS2 [425-450], MS1, MS2 [425-450], MS2 [400-425] where MS2 [xx-yy] indicates an MS2 scan with an isolation window starting at xx and ending at yy. OpenSwathWorkflow will try to read the SWATH windows from the data, if this is not possible please provide a tab-separated list with the correct windows using the -swath_windows_file parameter (this is recommended). Note that the software expects extraction windows (e.g. which peptides to extract from which window) which cannot have overlaps, otherwise peptides will be extracted from two different windows.

Alternatively, a set of split files (n+1 mzML files) can be provided, each containing one SWATH map (or MS1 map).

Since the file size can become rather large, it is recommended to not load the whole file into memory but rather cache it somewhere on the disk using a fast-access data format. This can be specified using the -readOptions cacheWorkingInMemory parameter (this is recommended!).

The assay library (transition list) is provided through the -tr parameter and can be in one of the following formats:

TraML
OpenSWATH TSV transition lists
OpenSWATH PQP SQLite files
SpectraST MRM transition lists
Skyline transition lists
Spectronaut transition lists

Parameters

The current parameters are optimized for 2 hour gradients on SCIEX 5600 / 6600 TripleTOF instruments with a peak width of around 30 seconds using iRT peptides. If your chromatography differs, please consider adjusting -Scoring:TransitionGroupPicker:min_peak_width to allow for smaller or larger peaks and adjust the -rt_extraction_window to use a different extraction window for the retention time. In m/z domain, consider adjusting -mz_extraction_window to your instrument resolution, which can be in Th or ppm.

Furthermore, if you wish to use MS1 information, use the -enable_ms1 flag and provide an MS1 map in addition to the SWATH data.

If you encounter issues with peak picking, try to disable peak filtering by setting -Scoring:TransitionGroupPicker:compute_peak_quality false which will disable the filtering of peaks by chromatographic quality. Furthermore, you can adjust the smoothing parameters for the peak picking, by adjusting -Scoring:TransitionGroupPicker:PeakPickerChromatogram:sgolay_frame_length or using a Gaussian smoothing based on your estimated peak width. Adjusting the signal to noise threshold will make the peaks wider or smaller.

Output: Feature list and chromatograms

The output of the OpenSwathWorkflow is a feature list, either as FeatureXML or a OpenSWATH SQLite file (use -out_features) while the latter is more memory friendly and can be directly used as input to other tools such as pyProphet (a Python re-implementation of mProphet) software tool, see Reiter et al (2011, Nature Methods). If you analyze large datasets, it is recommended to only use the OSWFile format. For downstream analysis (e.g. using pyProphet) the OSWFile format is recommended.

In addition, the extracted chromatograms can be written out using the -out_chrom parameter.

Feature list output format

For more information on the feature tables in the OpenSWATH SQLite file output, see the OpenSwathOSWWriter class.

Execution flow:

The overall execution flow for this tool is implemented in OpenSwathWorkflow.

The command line parameters of this tool are:

INI file documentation of this tool: