AssayGeneratorMetabo

Generates an assay library using DDA data (Metabolomics)

potential predecessor tools	→ AssayGeneratorMetabo →	potential successor tools
FeatureFinderMetabo		OpenSWATH pipeline
AccurateMassSearch

Build an assay library from DDA data (MS and MS/MS) (mzML). Please provide a list of features found in the data (featureXML).

Features can be detected using the FeatureFinderMetabo (FFM) and identifcation information can be added using the AccurateMassSearch featureXML output.

If the FeatureFinderMetabo featureXML does not contain any identifications the "use_known_unknowns" flag is used automatically.

Internal procedure AssayGeneratorMetabo:

1. Input mzML and featureXML
   
2. Reannotate precursor mz and intensity
   
3. Filter feature by number of masstraces
   
4. Assign precursors to specific feature (FeatureMapping)
   
5. Extract feature meta information (if possible)
   
6. Find MS2 spectrum with highest intensity precursor for one feature
   
7. Annotation is performed either the MS2 with the highest intensity precursor or a consensus spectrum can be used for the transition extraction.
   
8. Calculate thresholds (maximum and minimum intensity for transition peak)
   
9. Extract and write transitions (tsv, traml)

The command line parameters of this tool are:

AssayGeneratorMetabo -- Assay library generation from DDA data (Metabolomics)
Full documentation: http://www.openms.de/doxygen/release/3.4.1/html/TOPP_AssayGeneratorMetabo.html
Version: 3.4.1 May 19 2025, 14:24:34, Revision: 8aec8ec
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:
  AssayGeneratorMetabo <options>

Options (mandatory options marked with '*'):
  -in <file(s)>*                                    MzML input file(s) used for assay library generation (val
                                                    id formats: 'mzML')
  -in_featureinfo <file(s)>*                        FeatureXML input file(s) containing identification inform
                                                    ation (e.g. AccurateMassSearch) (valid formats: 'featureX
                                                    ML')
  -out <file>*                                      Assay library output file (valid formats: 'tsv', 'traML',
                                                     'pqp')
  -ambiguity_resolution_mz_tolerance <num>          Mz tolerance for the resolution of identification ambigui
                                                    ty over multiple files (default: '10.0')
  -ambiguity_resolution_rt_tolerance <num>          RT tolerance in seconds for the resolution of identificat
                                                    ion ambiguity over multiple files (default: '10.0')
  -total_occurrence_filter <num>                    Filter compound based on total occurrence in analysed 
                                                    samples (default: '0.1') (min: '0.0' max: '1.0')
  -method <choice>                                  Spectrum with the highest precursor intensity or a consen
                                                    sus spectrum is used for assay library construction (if 
                                                    no fragment annotation is used). (default: 'highest_inten
                                                    sity') (valid: 'highest_intensity', 'consensus_spectrum')

  -exclude_ms2_precursor                            Excludes precursor in ms2 from transition list
  -use_known_unknowns                               Use features without identification information
  -min_transitions <int>                            Minimal number of transitions (default: '3')
  -max_transitions <int>                            Maximal number of transitions (default: '6')
  -cosine_similarity_threshold <num>                Threshold for cosine similarity of MS2 spectra from the 
                                                    same precursor used in consensus spectrum creation (defau
                                                    lt: '0.98')
  -transition_threshold <num>                       Further transitions need at least x% of the maximum inten
                                                    sity (default 5%) (default: '5.0')
                                                    
  -precursor_mz_distance <num>                      Max m/z distance of the precursor entries of two spectra 
                                                    to be merged in [Da]. (default: '1.0e-04')
  -precursor_consensus_spectrum_rt_tolerance <num>  Tolerance window (left and right) for precursor selection
                                                     [seconds], for consensus spectrum generation (only avail
                                                    able without fragment annotation) (default: '5.0')
                                                    
  -deisotoping_use_deisotoper                       Use Deisotoper (if no fragment annotation is used)
  -deisotoping_fragment_tolerance <num>             Tolerance used to match isotopic peaks (default: '1.0')
  -deisotoping_fragment_unit <choice>               Unit of the fragment tolerance (default: 'ppm') (valid: 
                                                    'ppm', 'Da')
  -deisotoping_min_charge <num>                     The minimum charge considered (default: '1') (min: '1')
  -deisotoping_max_charge <num>                     The maximum charge considered (default: '1') (min: '1')
  -deisotoping_min_isopeaks <num>                   The minimum number of isotopic peaks (at least 2) require
                                                    d for an isotopic cluster (default: '2') (min: '2')
  -deisotoping_max_isopeaks <num>                   The maximum number of isotopic peaks (at least 2) conside
                                                    red for an isotopic cluster (default: '3') (min: '3')
  -deisotoping_keep_only_deisotoped                 Only monoisotopic peaks of fragments with isotopic patter
                                                    n are retained
  -deisotoping_annotate_charge                      Annotate the charge to the peaks
                                                    
  -filter_by_num_masstraces <number>                Number of mass traces each feature has to have to be incl
                                                    uded. To use this parameter, setting the feature_only 
                                                    flag is necessary (default: '1') (min: '1')
  -precursor_mz_tolerance <value>                   Tolerance window for precursor selection (Feature selecti
                                                    on in regard to the precursor) (default: '10.0')
  -precursor_mz_tolerance_unit <choice>             Unit of the preprocessing_precursor_mz_tolerance (default
                                                    : 'ppm') (valid: 'ppm', 'Da')
  -precursor_rt_tolerance <value>                   Tolerance window (left and right) for precursor selection
                                                     [seconds] (default: '5.0')
  -feature_only <choice>                            Uses the feature information from in_featureinfo to reduc
                                                    e the search space to MS2 associated with a feature (defa
                                                    ult: 'false') (valid: 'false', 'true')
                                                    
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 (default: '1')
  -write_ini <file>                                 Writes the default configuration file
  --help                                            Shows options
  --helphelp                                        Shows all options (including advanced)

INI file documentation of this tool:

Legend:

required parameter

advanced parameter

+AssayGeneratorMetaboAssay library generation from DDA data (Metabolomics)

version3.4.1 Version of the tool that generated this parameters file.

++1Instance '1' section for 'AssayGeneratorMetabo'

in[] MzML input file(s) used for assay library generationinput file*.mzML

in_featureinfo[] FeatureXML input file(s) containing identification information (e.g. AccurateMassSearch)input file*.featureXML

out Assay library output fileoutput file*.tsv, *.traML, *.pqp

ambiguity_resolution_mz_tolerance10.0 Mz tolerance for the resolution of identification ambiguity over multiple files

ambiguity_resolution_mz_tolerance_unitppm Unit of the ambiguity_resolution_mz_toleranceppm, Da

ambiguity_resolution_rt_tolerance10.0 RT tolerance in seconds for the resolution of identification ambiguity over multiple files

total_occurrence_filter0.1 Filter compound based on total occurrence in analysed samples0.0:1.0

methodhighest_intensity Spectrum with the highest precursor intensity or a consensus spectrum is used for assay library construction (if no fragment annotation is used).highest_intensity, consensus_spectrum

exclude_ms2_precursorfalse Excludes precursor in ms2 from transition listtrue, false

use_known_unknownsfalse Use features without identification informationtrue, false

min_transitions3 Minimal number of transitions

max_transitions6 Maximal number of transitions

cosine_similarity_threshold0.98 Threshold for cosine similarity of MS2 spectra from the same precursor used in consensus spectrum creation

transition_threshold5.0 Further transitions need at least x% of the maximum intensity (default 5%)

min_fragment_mz0.0 Minimal m/z of a fragment ion choosen as a transition

max_fragment_mz2000.0 Maximal m/z of a fragment ion choosen as a transition

precursor_mz_distance1.0e-04 Max m/z distance of the precursor entries of two spectra to be merged in [Da].

precursor_recalibration_window0.01 Tolerance window for precursor selection (Annotation of precursor mz and intensity)

precursor_recalibration_window_unitDa Unit of the precursor_mz_tolerance_annotationDa, ppm

precursor_consensus_spectrum_rt_tolerance5.0 Tolerance window (left and right) for precursor selection [seconds], for consensus spectrum generation (only available without fragment annotation)

deisotoping_use_deisotoperfalse Use Deisotoper (if no fragment annotation is used)true, false

deisotoping_fragment_tolerance1.0 Tolerance used to match isotopic peaks

deisotoping_fragment_unitppm Unit of the fragment toleranceppm, Da

deisotoping_min_charge1 The minimum charge considered1:∞

deisotoping_max_charge1 The maximum charge considered1:∞

deisotoping_min_isopeaks2 The minimum number of isotopic peaks (at least 2) required for an isotopic cluster2:∞

deisotoping_max_isopeaks3 The maximum number of isotopic peaks (at least 2) considered for an isotopic cluster3:∞

deisotoping_keep_only_deisotopedfalse Only monoisotopic peaks of fragments with isotopic pattern are retainedtrue, false

deisotoping_annotate_chargefalse Annotate the charge to the peakstrue, false

filter_by_num_masstraces1 Number of mass traces each feature has to have to be included. To use this parameter, setting the feature_only flag is necessary1:∞

precursor_mz_tolerance10.0 Tolerance window for precursor selection (Feature selection in regard to the precursor)

precursor_mz_tolerance_unitppm Unit of the preprocessing_precursor_mz_toleranceppm, Da

precursor_rt_tolerance5.0 Tolerance window (left and right) for precursor selection [seconds]

feature_onlyfalse Uses the feature information from in_featureinfo to reduce the search space to MS2 associated with a featurefalse, true

log Name of log file (created only when specified)

debug0 Sets the debug level

threads1 Sets the number of threads allowed to be used by the TOPP tool

no_progressfalse Disables progress logging to command linetrue, false

forcefalse Overrides tool-specific checkstrue, false

testfalse Enables the test mode (needed for internal use only)true, false