With the increasing size and number of runs acquired by data-independent acquisition (DIA)-based methods, data analysis algorithms like OpenSWATH  are challenged by the size and data formats of the input data. Additionally, recent extensions like TRIC , IPF  and error-rate control on different levels and in different contexts  produce additional layers of data that could not be ideally represented by the original data structures.
For this reason, we are currently adapting the tools for new SQLite  based data formats that represent the data in a non-redundant fashion, require less storage and processing time. The new data formats have been implemented in OpenSWATH and PyProphet and the final results can be exported to the legacy TSV reports.
PQP files represent the data stored in TraML files. OSW files copy the exact data structure of the PQP files and append
feature tables generated by OpenSWATH. Finally, PyProphet appends
score tables linked to the
feature tables. OpenSWATH stores the results of one run in a single OSW file. However, PyProphet can merge OSW files in a non-redundant, non-destructive fashion.
Contact and Support¶
The new data formats are currently in development and must NOT be used in production environments. We would however be very grateful for testing of the new workflows and reporting of problems and bugs.
You can contact the author George Rosenberger.
To use the new data formats, please use the following versions of our tools:
Full support for PQP and OSW files is provided in
OpenMS/develop since revision
5e81a69 (2017-11-30). Please follow the instructions in the OpenSWATH tutorial to install OpenMS.
We have developed a new, substantially changed version of PyProphet that integrates the new functionality of both IPF and can conduct error-rate control in different contexts and on different levels. If Python and PIP are configured correctly, the following command can be used to install the development version:
pip install git+https://github.com/PyProphet/pyprophet
The general workflow is very similar to the original OpenSWATH workflow with a few minor changes:
1. Peptide Query Parameter Generation¶
Peptide query parameters should be generated identically as described previously, including appended decoys. Optionally,
OpenSwathAssayGenerator can append peptide query parameters for IPF. The final TraML should then be converted to a peptide query parameter (PQP) file with
TargetedFileConverter from OpenMS:
TargetedFileConverter -in assays_ipf_decoys.TraML -out assays_ipf_decoys.pqp
2. Targeted data extraction using OpenSWATH¶
The next step is conducted using OpenSWATH.
OpenSwathWorkflow -in MSDATA.mzXML.gz \ -tr assays_ipf_decoys.pqp \ -out_osw MSDATA_RESULTS.osw [OTHER PARAMETERS]
The workflow is executed identically as before, with the only change being that the PQP file is used
-tr assays_ipf_decoys.pqp and an OSW file is exported
If you execute
OpenSwathWorkflow with the flag
-enable_uis_scoring, OpenSWATH will store identification instead of detection transition quantities. If IPF should NOT be applied, this will introduce some limitations for TRIC or other downstream applications. It is thus currently recommended to only use
-enable_uis_scoring if IPF scoring is required.
3. Statistical validation using PyProphet¶
PyProphet is then applied to the OSW files. Importantly, the updated version has changed substantially internally and in terms of the command line interface. Several different commands can be run to consecutively to do the analysis:
pyprophet merge --out=merged.osw \ --subsample_ratio=1 *.osw
This command will merge and optionally subsample multiple files. If a set of runs should be analyzed in an experiment-wide fashion, we recommend to conduct this step. If semi-supervised learning is too slow, create an additional merged file with a smaller
subsample_ratio. The model will be stored in the output and can be applied to the full file.
pyprophet score --in=merged.osw --level=ms2
The main command will conduct semi-supervised learning and error-rate estimation in a fully automated fashion.
--help will show the full selection of parameters to adjust the process. The default parameters are recommended for SCIEX TripleTOF 5600/6600 instrument data, but can be adjusted in other scenarios.
When using the IPF extension, the parameter
--level can be set to
transition. If MS1 or transition-level data should be scored, the command is executed three times, e.g.:
pyprophet score --in=merged.osw --level=ms1 \ score --in=merged.osw --level=ms2 \ score --in=merged.osw --level=transition
Importantly, PyProphet will store all results in the input OSW files. This can be changed by specifying
--out. However, since all steps are non-destructive, this is not necessary.
If IPF should be applied after scoring, the following command can be used:
pyprophet ipf --in=merged.osw
To adjust the IPF-specific parameters, please consult
pyprophet ipf --help.
To conduct peptide inference in run-specific, experiment-wide and global contexts, the following command can be applied:
pyprophet peptide --in=merged.osw --context=run-specific \ peptide --in=merged.osw --context=experiment-wide \ peptide --in=merged.osw --context=global
This will generate individual PDF reports and store the scores in a non-redundant fashion in the OSW file.
Analogously, this can be conducted on protein-level as well:
pyprophet protein --in=merged.osw --context=run-specific \ protein --in=merged.osw --context=experiment-wide \ protein --in=merged.osw --context=global
Finally, we can export the results to legacy OpenSWATH TSV report:
pyprophet export --in=merged.osw --out=legacy.tsv
By default, both peptide- and transition-level quantification is reported, which is necessary for requantification or
SWATH2stats. If peptide and protein inference in the global context was conducted, the results will be filtered to 1% FDR by default. Further details can be found by
pyprophet export --help.
By default, IPF results will be used if available. This can be disabled by setting
--no-ipf. The IPF results require different properties for TRIC. Please ensure that you want to analyze the results in the context of IPF, else, use the
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