 |
OpenMS
|
High-level DIA workflow front-end that prepares peptide query parameters, performs targeted extraction, rescoring, inference, and export in one invocation.
Four library modes:
auto: detect FASTA input as predicted mode; otherwise probe the input library for decoy transitions and automatically choose between the prepared and transition-list pathsprepared_pqp: accept an already prepared peptide query parameter library (tsv, pqp, oswpq, or TraML) and normalize it to an internal prepared_library.pqptransition_list: accept an empirical transition library, run assay preparation and decoy generation, normalize the result to prepared_library.pqp, then continuepredicted: digest a FASTA database, enumerate configured peptide modifications and precursor charges, predict RT/MS2 and optional CCS with the native PeptDeep ONNX models, then reuse the same assay preparation, decoy generation, PQP normalization, extraction, rescoring, inference, and export path as empirical librariesThe downstream run then executes:
workflow.osw) or Parquet archive (workflow.oswpq) containerout_dirBy default intermediate files are removed after success. Use workflow:keep_intermediate_files to retain them, or workflow:intermediate_dir to control their location. When cleanup is enabled, OpenDIA creates a unique run-owned child directory beneath the selected intermediate location and recursively removes only that child after success. User-supplied directories and unrelated pre-existing contents are never recursively removed.
The command line parameters of this tool are:
OpenDIA -- High-level DIA workflow front-end built on the OpenSWATH/OpenMS stack.
Full documentation: http://www.openms.de/doxygen/nightly/html/TOPP_OpenDIA.html
Version: 3.7.0-pre-nightly-2026-10-24 Oct 25 2026, 01:35:50, Revision: 3e47bc7
To cite OpenMS:
+ Pfeuffer, J., Bielow, C., Wein, S. et al.. OpenMS 3 enables reproducible analysis of large-scale mass spec
trometry data. Nat Methods (2024). doi:10.1038/s41592-024-02197-7.
Usage:
OpenDIA <options>
This tool has algorithm parameters that are not shown here! Please check the ini file for a detailed descript
ion or use the --helphelp option
Options (mandatory options marked with '*'):
-in <files>* Input DIA files separated by
blank. (valid formats: 'mzML'
, 'mzXML', 'sqMass', 'd',
'raw')
-tr <file>* Library input file, or FASTA
database for predicted mode.
(valid formats: 'traML', 'tsv
', 'pqp', 'oswpq', 'fasta',
'fa', 'faa')
-tr_type <type> Library input file type --
default: determined from file
extension or content. (valid
: 'traML', 'tsv', 'pqp', 'osw
pq', 'fasta')
-out_dir <dir> Directory for final exported
OpenDIA outputs.
Workflow options.:
-workflow:library_mode <choice> How to enter the workflow:
auto-detect FASTA/predicted
or transition-library prepara
tion, force prepared peptide-
query normalization, force
transition-list assay/decoy
preparation, or predict a
library from FASTA. (default:
'auto') (valid: 'auto', 'pre
pared_pqp', 'transition_list'
, 'predicted')
-workflow:working_format <choice> Internal workflow container
used after extraction/scoring
: 'sqlite' writes a .osw work
flow, 'parquet' writes a .osw
pq archive. (default: 'sqlite
') (valid: 'sqlite', 'parquet
')
-workflow:keep_intermediate_files <true|false> Whether to retain prepared_li
brary.pqp and the single work
ing workflow.osw (.sqlite
workflow) or workflow.oswpq
(.parquet archive workflow)
after success. (default: 'fal
se') (valid: 'true', 'false')
-workflow:intermediate_dir <dir> Optional parent directory
for intermediate workflow
files. Preserved automaticall
y on failure. When cleanup
is enabled, OpenDIA creates
a unique run-owned child bene
ath this directory and remove
s only that child after succe
...
out_dir.
Assay-generation parameters used when workflow:library_mode=transition_list.:
-PeptideQueryParameters:AssayGenerator:min_transitions <int> Minimal number of transitions
. (default: '6')
-PeptideQueryParameters:AssayGenerator:max_transitions <int> Maximal number of transitions
. (default: '6')
-PeptideQueryParameters:AssayGenerator:allowed_fragment_types <type> Allowed fragment types. (defa
ult: 'b,y')
-PeptideQueryParameters:AssayGenerator:allowed_fragment_charges <type> Allowed fragment charge state
s. (default: '1,2,3,4')
-PeptideQueryParameters:AssayGenerator:enable_detection_specific_losses Allow specific neutral losses
for detecting transitions.
-PeptideQueryParameters:AssayGenerator:enable_detection_unspecific_losses Allow unspecific neutral loss
es for detecting transitions.
-PeptideQueryParameters:AssayGenerator:precursor_mz_threshold <double> Precursor m/z threshold in
Thomson for precursor annotat
ion. (default: '0.025')
-PeptideQueryParameters:AssayGenerator:precursor_lower_mz_limit <double> Lower precursor m/z limit.
(default: '400.0')
-PeptideQueryParameters:AssayGenerator:precursor_upper_mz_limit <double> Upper precursor m/z limit.
(default: '1200.0')
-PeptideQueryParameters:AssayGenerator:product_mz_threshold <double> Fragment m/z threshold in
Thomson for annotation. (defa
ult: '0.025')
-PeptideQueryParameters:AssayGenerator:product_lower_mz_limit <double> Lower fragment m/z limit.
(default: '350.0')
-PeptideQueryParameters:AssayGenerator:product_upper_mz_limit <double> Upper fragment m/z limit.
(default: '2000.0')
-PeptideQueryParameters:AssayGenerator:swath_windows_file <file> Optional SWATH window file
for exclusion of fragment
ions in the precursor isolati
on window. (valid formats:
'txt')
-PeptideQueryParameters:AssayGenerator:unimod_file <file> Optional Unimod XML file used
for IPF transition generatio
n. (valid formats: 'xml')
-PeptideQueryParameters:AssayGenerator:enable_ipf Generate identifying transiti
ons for IPF during transition
-list library preparation.
FASTA digestion and native PeptDeep prediction parameters used when workflow:library_mode=predicted.:
-PeptideQueryParameters:LibraryPrediction:enzyme <name> Protease used for in-silico
FASTA digestion. (default:
'Trypsin') (valid: 'Trypsin',
'Arg-C', 'no cleavage', 'uns
pecific cleavage', 'glutamyl
endopeptidase', 'Alpha-lytic
protease', '2-iodobenzoate',
'Arg-C/P', 'Asp-N', 'Asp-N/B'
, 'Asp-N_ambic', 'Chymotrypsi
n', 'Chymotrypsin/P', 'CNBr',
...
in')
-PeptideQueryParameters:LibraryPrediction:missed_cleavages <int> Maximum number of missed clea
vages. (default: '2') (min:
'0')
-PeptideQueryParameters:LibraryPrediction:min_peptide_length <int> Minimum peptide length retain
ed for prediction. (default:
'7') (min: '2')
-PeptideQueryParameters:LibraryPrediction:max_peptide_length <int> Maximum peptide length retain
ed for prediction. (default:
'30') (min: '2')
-PeptideQueryParameters:LibraryPrediction:precursor_charges <list> Precursor charge states to
predict. (default: '[2 3]')
(min: '1')
-PeptideQueryParameters:LibraryPrediction:fixed_modifications <mods> Fixed OpenMS/UniMod modificat
ion names applied before pred
iction. (default: '[Carbamido
methyl (C)]')
-PeptideQueryParameters:LibraryPrediction:variable_modifications <mods> Variable OpenMS/UniMod modifi
cation names enumerated befor
e prediction.
-PeptideQueryParameters:LibraryPrediction:max_variable_modifications <int> Maximum number of variable
modifications per peptide.
(default: '1') (min: '0')
-PeptideQueryParameters:LibraryPrediction:clip_nterm_methionine <true|false> Also digest the mature protei
n sequence after initiator-me
thionine removal. (default:
'true') (valid: 'true', 'fals
e')
-PeptideQueryParameters:LibraryPrediction:prediction_batch_size <int> Number of precursors predicte
d per materialization batch.
(default: '500') (min: '1')
-PeptideQueryParameters:LibraryPrediction:inference_threads <int> ONNX Runtime intra-op thread
count for each PeptDeep predi
ctor. (default: '4') (min:
'1')
-PeptideQueryParameters:LibraryPrediction:nce <double> Normalized collision energy
supplied to PeptDeep MS2 pred
iction. (default: '30.0')
(min: '0.0' max: '100.0')
-PeptideQueryParameters:LibraryPrediction:instrument_index <int> PeptDeep instrument category
(0=QE, 1=Lumos, 2=timsTOF,
3=SciexTOF, 4=ThermoTOF, 7=ot
her/unknown). (default: '0')
(min: '0' max: '7')
-PeptideQueryParameters:LibraryPrediction:predict_ccs <true|false> Predict CCS and store convert
ed 1/K0 values in the materia
lized library. (default: 'tru
e') (valid: 'true', 'false')
-PeptideQueryParameters:LibraryPrediction:out <file> Optional reusable prepared
target/decoy PQP generated
from the FASTA input. (valid
formats: 'pqp')
Decoy-generation parameters used when workflow:library_mode=transition_list or predicted.:
-PeptideQueryParameters:DecoyGenerator:method <type> Decoy generation method. (def
ault: 'shuffle') (valid: 'shu
ffle', 'pseudo-reverse', 'rev
erse', 'shift')
-PeptideQueryParameters:DecoyGenerator:shuffle_seed <int> Shuffle random seed (default
1 for reproducible decoys,
-1 for time-based seeding).
(default: '1') (min: '-1')
-PeptideQueryParameters:DecoyGenerator:decoy_tag <type> Decoy tag. (default: 'DECOY_'
)
-PeptideQueryParameters:DecoyGenerator:switchKR <true/false> Whether to switch terminal K
and R to achieve different
precursor masses. (default:
'true') (valid: 'true', 'fals
e')
Targeted data extraction options.:
-TargetedDataExtraction:swath_windows_file <file> Optional tab-separated SWATH
window file used during extra
ction.
-TargetedDataExtraction:write_debug_files <true|false> Automatically write per-run
calibration/debug files into
out_dir using <basename>.debu
g.* names when explicit debug
output paths are not set.
(default: 'false') (valid:
'true', 'false')
-TargetedDataExtraction:write_chromatograms <true|false> Automatically write per-run
extracted chromatograms into
out_dir using <basename>.chro
m.sqMass for sqlite workflows
or <basename>.chrom.xic for
parquet workflows when Target
edDataExtraction:out_chrom
is not set. (default: 'false'
) (valid: 'true', 'false')
-TargetedDataExtraction:write_mobilograms <true|false> Automatically write per-run
extracted ion mobilograms
into out_dir using <basename>
.mobi.xim when TargetedDataEx
traction:out_mobilogram is
not set. (default: 'false')
(valid: 'true', 'false')
-TargetedDataExtraction:pasef Treat the input as PASEF /
diaPASEF data.
-TargetedDataExtraction:ion_mobility_mode <choice> How to use the ion mobility
dimension. 'auto' detects
ion mobility SWATH windows
and enables PASEF-style extra
ction/scoring automatically,
filling negative IM extractio
n windows with 0.06 1/K0 defa
ults. 'disabled' disables
...
ty. (default: 'auto') (valid:
'auto', 'disabled')
-TargetedDataExtraction:rt_extraction_window <double> Only extract RT around this
value (-1 means extract the
whole range; 600 means +/-
300 s around the expected
elution). (default: '600.0')
-TargetedDataExtraction:ion_mobility_window <double> Extraction window in ion mobi
lity dimension (full width;
negative values extract the
whole range unless detected
diaPASEF/timsTOF data trigger
automatic 0.06 1/K0 defaults
). (default: '-1.0')
-TargetedDataExtraction:mz_extraction_window <double> MS2 extraction window in Thom
son or ppm (see TargetedDataE
xtraction:mz_extraction_windo
w_unit). (default: '50.0')
(min: '0.0')
-TargetedDataExtraction:mz_extraction_window_ms1 <double> MS1 extraction window in Thom
son or ppm (see TargetedDataE
xtraction:mz_extraction_windo
w_ms1_unit). (default: '50.0'
) (min: '0.0')
-TargetedDataExtraction:im_extraction_window_ms1 <double> MS1 ion mobility extraction
window. Negative values disab
le MS1 ion mobility extractio
n unless detected diaPASEF/ti
msTOF data trigger automatic
0.06 1/K0 defaults. (default:
'-1.0')
-TargetedDataExtraction:keep_cached_files If set, do not remove cached
input files created in Target
edDataExtraction:tempDirector
y.
-TargetedDataExtraction:irt_mzml <file> Optional chromatogram mzML
containing the iRT peptides.
If multiple runs are analyzed
, explicit file names are
expanded per run as <basename
>_<requested-name>. (valid
formats: 'mzML')
-TargetedDataExtraction:irt_trafo <file> Optional transformation file
for RT normalization. If mult
iple runs are analyzed, expli
cit file names are expanded
per run as <basename>_<reques
ted-name>. (valid formats:
'trafoXML')
In-process Percolator rescoring parameters.:
-Rescoring:level <levels> One or more rescoring levels
to run sequentially. Transiti
on rescoring is appended auto
matically when peptidoform
inference is enabled. (defaul
t: '[ms1ms2]') (valid: 'ms1',
'ms2', 'ms1ms2', 'transition
')
Peptidoform inference options.:
-Inference:peptidoform:run <true|false> Enable peptidoform inference.
(default: 'false') (valid:
'true', 'false')
-Inference:peptidoform:ipf_ms1_scoring <true|false> Use MS1 precursor evidence
for peptidoform inference.
(default: 'false') (valid:
'true', 'false')
-Inference:peptidoform:ipf_ms2_scoring <true|false> Use MS2 precursor evidence
for peptidoform inference.
(default: 'false') (valid:
'true', 'false')
-Inference:peptidoform:propagate_signal_across_runs <true|false> Propagate confident transitio
n evidence across aligned
runs. (default: 'false') (val
id: 'true', 'false')
Peptide inference options.:
-Inference:peptide:global <true|false> Enable peptide inference in
the global context. (default:
'true') (valid: 'true', 'fal
se')
-Inference:peptide:experiment_wide <true|false> Enable peptide inference in
the experiment-wide context.
(default: 'false') (valid:
'true', 'false')
-Inference:peptide:run_specific <true|false> Enable peptide inference in
the run-specific context.
(default: 'false') (valid:
'true', 'false')
Protein inference options.:
-Inference:protein:global <true|false> Enable protein inference in
the global context. (default:
'true') (valid: 'true', 'fal
se')
-Inference:protein:experiment_wide <true|false> Enable protein inference in
the experiment-wide context.
(default: 'false') (valid:
'true', 'false')
-Inference:protein:run_specific <true|false> Enable protein inference in
the run-specific context.
(default: 'false') (valid:
'true', 'false')
Gene inference options.:
-Inference:gene:global <true|false> Enable gene inference in the
global context. (default:
'false') (valid: 'true', 'fal
se')
-Inference:gene:experiment_wide <true|false> Enable gene inference in the
experiment-wide context. (def
ault: 'false') (valid: 'true'
, 'false')
-Inference:gene:run_specific <true|false> Enable gene inference in the
run-specific context. (defaul
t: 'false') (valid: 'true',
'false')
Options for scored feature parquet export.:
-Export:parquet:run <true|false> Whether to export scored feat
ure parquet tables. (default:
'false') (valid: 'true',
'false')
-Export:parquet:include_transition_data <true|false> Whether to also export transi
tion-level parquet tables.
(default: 'true') (valid:
'true', 'false')
-Export:parquet:exclude_decoys <true|false> Whether to exclude decoys
from parquet feature exports.
(default: 'false') (valid:
'true', 'false')
Options for user-facing filtered result export.:
-Export:results:run <true|false> Whether to export the filtere
d results table. (default:
'true') (valid: 'true', 'fals
e')
-Export:results:format <choice> Output format for the filtere
d results table. (default:
'tsv') (valid: 'tsv', 'parque
t')
-Export:results:ipf <choice> How IPF results should be
represented when SCORE_IPF
is present. (default: 'peptid
oform') (valid: 'peptidoform'
, 'augmented', 'disable')
-Export:results:transition_quantification <true|false> Whether to aggregate transiti
on-level quantification colum
ns. (default: 'true') (valid:
'true', 'false')
-Export:results:max_transition_pep <num> Maximum transition PEP used
for aggregated transition
quantification. (default:
'0.7')
-Export:results:ipf_max_peptidoform_pep <num> Maximum IPF PEP used for pept
idoform export filtering.
(default: '0.4')
-Export:results:max_rs_peakgroup_qvalue <num> Maximum run-specific peakgrou
p q-value retained. (default:
'0.05')
-Export:results:use_peptide_scores <true|false> Whether to append peptide-lev
el scores if SCORE_PEPTIDE
is present and to require
passing the peptide-level
threshold. (default: 'true')
(valid: 'true', 'false')
-Export:results:max_global_peptide_qvalue <num> Maximum global peptide-level
q-value retained when peptide
filtering is enabled. (defau
lt: '0.01')
-Export:results:use_protein_scores <true|false> Whether to append protein-lev
el scores if SCORE_PROTEIN
is present and to require
passing the protein-level
threshold. (default: 'true')
(valid: 'true', 'false')
-Export:results:max_global_protein_qvalue <num> Maximum global protein-level
q-value retained when protein
filtering is enabled. (defau
lt: '0.01')
-Export:results:use_gene_scores <true|false> Whether to append gene-level
scores if SCORE_GENE is prese
nt and to require passing
the gene-level threshold.
(default: 'false') (valid:
'true', 'false')
-Export:results:max_global_gene_qvalue <num> Maximum global gene-level
q-value retained when gene
filtering is enabled. (defaul
t: '0.01')
-Export:results:use_alignment <true|false> Whether to recover aligned
features when legacy alignmen
t tables are present. (defaul
t: 'false') (valid: 'true',
'false')
-Export:results:max_alignment_pep <num> Maximum alignment PEP retaine
d when alignment recovery is
enabled. (default: '0.7')
-Export:results:exclude_decoys <true|false> Whether to exclude decoys
from the filtered results
export. (default: 'true')
(valid: 'true', 'false')
Options for quantification matrix exports.:
-Export:matrix:precursor <true|false> Whether to export the precurs
or-level quantification matri
x. (default: 'false') (valid:
'true', 'false')
-Export:matrix:peptide <true|false> Whether to export the peptide
-level quantification matrix.
(default: 'true') (valid:
'true', 'false')
-Export:matrix:protein <true|false> Whether to export the protein
-level quantification matrix.
(default: 'true') (valid:
'true', 'false')
-Export:matrix:gene <true|false> Whether to export the gene-le
vel quantification matrix.
(default: 'false') (valid:
'true', 'false')
-Export:matrix:format <choice> Output format for quantificat
ion matrices. (default: 'tsv'
) (valid: 'tsv', 'parquet')
-Export:matrix:ipf <choice> How IPF results should be
represented when SCORE_IPF
is present. (default: 'peptid
oform') (valid: 'peptidoform'
, 'augmented', 'disable')
-Export:matrix:transition_quantification <true|false> Whether to aggregate transiti
on-level quantification colum
ns onto the filtered feature
rows. (default: 'true') (vali
d: 'true', 'false')
-Export:matrix:max_transition_pep <num> Maximum transition PEP used
for aggregated transition
quantification. (default:
'0.7')
-Export:matrix:ipf_max_peptidoform_pep <num> Maximum IPF PEP used for pept
idoform export filtering.
(default: '0.4')
-Export:matrix:max_rs_peakgroup_qvalue <num> Maximum run-specific peakgrou
p q-value retained. (default:
'0.05')
-Export:matrix:use_peptide_scores <true|false> Whether peptide-level scores
should be required when pepti
de filtering is enabled. (def
ault: 'true') (valid: 'true',
'false')
-Export:matrix:max_global_peptide_qvalue <num> Maximum global peptide-level
q-value retained when peptide
filtering is enabled. (defau
lt: '0.01')
-Export:matrix:use_protein_scores <true|false> Whether protein-level scores
should be required when prote
in filtering is enabled. (def
ault: 'true') (valid: 'true',
'false')
-Export:matrix:max_global_protein_qvalue <num> Maximum global protein-level
q-value retained when protein
filtering is enabled. (defau
lt: '0.01')
-Export:matrix:use_gene_scores <true|false> Whether gene-level scores
should be required when gene
filtering is enabled. (defaul
t: 'false') (valid: 'true',
'false')
-Export:matrix:max_global_gene_qvalue <num> Maximum global gene-level
q-value retained when gene
filtering is enabled. (defaul
t: '0.01')
-Export:matrix:use_alignment <true|false> Whether to recover aligned
features when legacy alignmen
t tables are present. (defaul
t: 'false') (valid: 'true',
'false')
-Export:matrix:max_alignment_pep <num> Maximum alignment PEP retaine
d when alignment recovery is
enabled. (default: '0.7')
-Export:matrix:exclude_decoys <true|false> Whether to exclude decoys
from the filtered matrix inpu
t rows. (default: 'true')
(valid: 'true', 'false')
-Export:matrix:top_n <num> Number of top precursors /
peptides retained during matr
ix summarization. (default:
'3')
-Export:matrix:consistent_top <true|false> Whether to use the same top
precursors / peptides across
all runs. (default: 'true')
(valid: 'true', 'false')
-Export:matrix:normalization <choice> Normalization applied to matr
ix sample columns after summa
rization. (default: 'none')
(valid: 'none', 'median',
'medianmedian')
Common TOPP options:
-ini <file> Use the given TOPP INI file
-threads <n> Sets the number of threads
allowed to be used by the
TOPP tool (0 = all available
cores) (default: '1')
-write_ini <file> Writes the default configurat
ion file
--help Shows options
--helphelp Shows all options (including
advanced)
The following configuration subsections are valid:
- Rescoring In-process Percolator rescoring parameters.
- TargetedDataExtraction:Calibration Parameters for calibrant iRT peptides for RT normali
zation and mass / ion mobility correction.
- TargetedDataExtraction:Calibration:MassIMCorrection m/z and ion mobility calibration.
- TargetedDataExtraction:Calibration:RTNormalization RT normalization model and outlier handling.
- TargetedDataExtraction:Library Transition-library reader parameters section.
- TargetedDataExtraction:MRMMapping Parameters for mapping chromatograms to transitions
in SRM/MRM data.
- TargetedDataExtraction:Scoring Scoring parameters section.
You can write an example INI file using the '-write_ini' option.
Documentation of subsection parameters can be found in the doxygen documentation or the INIFileEditor.
For more information, please consult the online documentation for this tool:
- http://www.openms.de/doxygen/nightly/html/TOPP_OpenDIA.html
INI file documentation of this tool:
This section lists all parameters supported by the tool. Parameters are organized into hierarchical subsections that group related settings together. Subsections may contain further subsections or individual parameters.
Each parameter entry contains the following information:
Parameter tags provide additional information about how a parameter is used. Some tags indicate whether a parameter is required or intended for advanced configuration, while others may be used internally by OpenMS or workflow tools.
Parameters highlighted as required must be specified for the tool to run successfully. Parameters marked as advanced allow fine-tuning of algorithm behavior and are typically not needed for standard workflows.
1.9.8