sarah-haynes
commented
3 years ago Hi Frank, can you share the log (console output) from the pipeline run so we can see the error message? Thanks, Sarah
Closed fstein closed 3 years ago
sarah-haynes
commented
3 years ago Hi Frank, can you share the log (console output) from the pipeline run so we can see the error message? Thanks, Sarah
fstein
commented
3 years ago Sure:
INFO[15:00:38] Executing Pipeline v4.1.0
INFO[15:00:38] Creating workspace
INFO[15:00:38] Initiating the workspace on C:/MS_TestRun
INFO[15:00:38] Creating workspace
WARN[15:00:38] A meta data folder was found and will not be overwritten.
INFO[15:00:38] Annotating the database
INFO[15:00:39] Running the Database Search
MSFragger version MSFragger-3.4
Batmass-IO version 1.23.6
timsdata library version timsdata-2-8-7-1
(c) University of Michigan
RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved.
System OS: Windows 10, Architecture: AMD64
Java Info: 1.8.0_201, Java HotSpot(TM) 64-Bit Server VM, Oracle Corporation
JVM started with 53 GB memory
Checking database...
Checking spectral files...
C:\MS_TestRun\Ernie_200910_P0000_Ernie_TMTyeast_25ng_60min_R1.mzML: Scans = 15246
***FIRST SEARCH****
Parameters:
num_threads = 8
database_name = C:\MS_TestRun\2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas
decoyprefix = rev
precursor_mass_lower = -20.0
precursor_mass_upper = 20.0
precursor_mass_units = 1
data_type = 0
precursor_true_tolerance = 20.0
precursor_true_units = 1
fragment_mass_tolerance = 20.0
fragment_mass_units = 1
calibrate_mass = 2
use_all_mods_in_first_search = false
write_calibrated_mgf = 0
isotope_error = 0/1
mass_offsets = 0
labile_search_mode = OFF
restrict_deltamass_to = all
precursor_mass_mode = SELECTED
localize_delta_mass = false
delta_mass_exclude_ranges = (-1.5,3.5)
fragment_ion_series = b,y
ion_series_definitions =
search_enzyme_name = stricttrypsin
search_enzyme_sense_1 = C
search_enzyme_cut_1 = KR
search_enzyme_nocut_1 =
allowed_missed_cleavage_1 = 2
num_enzyme_termini = 2
clip_nTerm_M = true
allow_multiple_variable_mods_on_residue = false
max_variable_mods_per_peptide = 3
max_variable_mods_combinations = 5000
output_format = pepxml
output_report_topN = 1
output_max_expect = 50.0
report_alternative_proteins = false
override_charge = false
precursor_charge_low = 1
precursor_charge_high = 6
digest_min_length = 7
digest_max_length = 50
digest_mass_range_low = 200.0
digest_mass_range_high = 5000.0
max_fragment_charge = 2
deisotope = 1
deneutralloss = true
track_zero_topN = 0
zero_bin_accept_expect = 0.0
zero_bin_mult_expect = 1.0
add_topN_complementary = 0
minimum_peaks = 15
use_topN_peaks = 150
minIonsScoring = 2
min_matched_fragments = 4
minimum_ratio = 0.01
intensity_transform = 0
remove_precursor_peak = 0
remove_precursor_range = -1.500000,1.500000
clear_mz_range_low = 125.5
clear_mz_range_high = 131.5
excluded_scan_list_file =
mass_diff_to_variable_mod = 0
min_sequence_matches = 2
check_spectral_files = true
variable_mod_01 = 15.994900 M 3
variable_mod_02 = 42.010600 [^ 1
variable_mod_06 = 229.162930 n^ 1
variable_mod_07 = 229.162930 S 1
add_A_alanine = 0.000000
add_C_cysteine = 57.021460
add_Cterm_peptide = 0.0
add_Cterm_protein = 0.0
add_D_aspartic_acid = 0.000000
add_E_glutamic_acid = 0.000000
add_F_phenylalanine = 0.000000
add_G_glycine = 0.000000
add_H_histidine = 0.000000
add_I_isoleucine = 0.000000
add_K_lysine = 229.162930
add_L_leucine = 0.000000
add_M_methionine = 0.000000
add_N_asparagine = 0.000000
add_Nterm_peptide = 0.0
add_Nterm_protein = 0.0
add_P_proline = 0.000000
add_Q_glutamine = 0.000000
add_R_arginine = 0.000000
add_S_serine = 0.000000
add_T_threonine = 0.000000
add_V_valine = 0.000000
add_W_tryptophan = 0.000000
add_Y_tyrosine = 0.000000
Number of unique peptides
of length 7: 84703
of length 8: 81329
of length 9: 77405
of length 10: 72902
of length 11: 71900
of length 12: 66591
of length 13: 63744
of length 14: 60464
of length 15: 56608
of length 16: 54471
of length 17: 51326
of length 18: 48168
of length 19: 44809
of length 20: 43036
of length 21: 40923
of length 22: 37881
of length 23: 36015
of length 24: 33916
of length 25: 31304
of length 26: 29495
of length 27: 27834
of length 28: 26493
of length 29: 24903
of length 30: 22759
of length 31: 21176
of length 32: 19741
of length 33: 18323
of length 34: 17128
of length 35: 15792
of length 36: 14514
of length 37: 13725
of length 38: 12801
of length 39: 11748
of length 40: 10738
of length 41: 9971
of length 42: 8783
of length 43: 7439
of length 44: 5553
of length 45: 3682
of length 46: 2135
of length 47: 1133
of length 48: 525
of length 49: 264
of length 50: 118
In total 1384268 peptides.
Generated 10920963 modified peptides.
Number of peptides with more than 5000 modification patterns: 0
Selected fragment index width 0.10 Da.
507847942 fragments to be searched in 1 slices (7.57 GB total)
Operating on slice 1 of 1:
Fragment index slice generated in 9.04 s
| **MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- | --------------- | --------------- | --------------- | --------------- | MS1 (Old) | MS1 (New) | MS2 (Old) | MS2 (New) |
|---|---|---|---|---|---|---|---|---|
| Run | Median MAD | Median MAD | Median MAD | Median MAD | ||||
| 001 | 3.48 1.45 | 0.02 0.91 | 0.30 1.32 | 0.10 1.26 | ||||
| ----- | --------------- | --------------- | --------------- | --------------- |
| Finding the optimal parameters: ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Count | 6409 | 6426 | 6398 | skip rest | ||||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| Peaks | 300_0 | 200_0 | 175_0 | 150_1 | 125_1 | 100_1 | ||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| Count | 6472 | 6472 | 6472 | 6426 | skip rest | |||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| ------- | ------- | |||||||||||||||
| Int. | 1 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Count | 6453 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Rm P. | 1 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Count | 6448 | |||||||||||||||
| ------- | ------- |
New fragment_mass_tolerance = 7 PPM New use_topN_peaks = 175 New minimum_ratio = 0.000000 New intensity_transform = 0 New remove_precursor_peak = 0 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 1.892 MIN*****
****MAIN SEARCH**** Checking database... variable_mod_03 has an empty value. variable_mod_04 has an empty value. variable_mod_05 has an empty value. Parameters: num_threads = 8 database_name = C:\MS_TestRun\2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 7.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = -1/0/1/2/3 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = pepxml output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = true override_charge = false precursor_charge_low = 1 precursor_charge_high = 6 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 2 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 175 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 0 remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_06 = 229.162930 n^ 1 variable_mod_07 = 229.162930 S 1 add_A_alanine = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_K_lysine = 229.162930 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_Y_tyrosine = 0.000000 Number of unique peptides of length 7: 84703 of length 8: 81329 of length 9: 77405 of length 10: 72902 of length 11: 71900 of length 12: 66591 of length 13: 63744 of length 14: 60464 of length 15: 56608 of length 16: 54471 of length 17: 51326 of length 18: 48168 of length 19: 44809 of length 20: 43036 of length 21: 40923 of length 22: 37881 of length 23: 36015 of length 24: 33916 of length 25: 31304 of length 26: 29495 of length 27: 27834 of length 28: 26493 of length 29: 24903 of length 30: 22759 of length 31: 21176 of length 32: 19741 of length 33: 18323 of length 34: 17128 of length 35: 15792 of length 36: 14514 of length 37: 13725 of length 38: 12801 of length 39: 11748 of length 40: 10738 of length 41: 9971 of length 42: 8783 of length 43: 7439 of length 44: 5553 of length 45: 3682 of length 46: 2135 of length 47: 1133 of length 48: 525 of length 49: 264 of length 50: 118 In total 1384268 peptides. Generated 10920963 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.03 Da. 507847942 fragments to be searched in 1 slices (7.57 GB total) Operating on slice 1 of 1: Fragment index slice generated in 10.02 s
***TOTAL TIME 2.470 MIN****
INFO[15:03:10] Running the validation and inference on C:/MS_TestRun
INFO[15:03:10] Executing PeptideProphet on C:/MS_TestRun
file 1: C:\MS_TestRun\Ernie_200910_P0000_Ernie_TMTyeast_25ng_60min_R1.pepXML
processed altogether 10667 results
INFO: Results written to file: C:\MS_TestRun\interact.pep.xml
C:\MS_TestRun\interact.pep.xml
Searching the tree...
Linking duplicate entries...
Printing results...
Building Commentz-Walter keyword tree...using Accurate Mass Bins using PPM mass difference Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) adding ACCMASS mixture distribution using search_offsets in ACCMASS mixture distr: 0 init with X! Tandem stricttrypsin MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN
INFO: Processing standard MixtureModel ...
PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB
read in 0 1+, 8857 2+, 1396 3+, 414 4+, 0 5+, 0 6+, and 0 7+ spectra.
Initialising statistical models ...
Found 1963 Decoys, and 8704 Non-Decoys
Iterations: .........10.........20......
WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (5+).
WARNING: Mixture model quality test failed for charge (6+).
WARNING: Mixture model quality test failed for charge (7+).
model complete after 27 iterations
INFO[15:03:38] Running the validation and inference on C:/MS_TestRun
INFO[15:03:38] Executing ProteinProphet on C:/MS_TestRun
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64))
(no FPKM) (using degen pep info)
Reading in C:/MS_TestRun/interact.pep.xml...
...read in 0 1+, 6020 2+, 840 3+, 44 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.05
Initializing 6646 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating protein lengths and molecular weights from database c:/MS_TestRun/2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000
.........:.........:.........:.........:.........:.........:.........:.. Total: 13728
Computing degenerate peptides for 1870 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing 1706 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating sensitivity...and error tables...
Computing MU for 1966 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
INFO: mu=2.16234e-05, db_size=12304840
INFO[15:03:44] Integrating peptide validation
Running FPKM NSS NRS NSE NSI NSM Model EM:
Computing NSS values ...
Creating 6 threads Wait for threads to finish ... ...... done Computing NRS values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSE values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSI values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSM values ...
Creating 6 threads
Wait for threads to finish ...
0--------------------------------------------------50------------------------------------------------100%
..................................................................................................... done
FPKM values are unavailable ...
Iterations: ......done
INFO[15:03:50] Creating combined protein inference
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64))
(no FPKM) (using degen pep info)
Reading in C:/MS_TestRun/interact.pep.xml...
...read in 0 1+, 5465 2+, 793 3+, 41 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.9
Initializing 6123 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database c:/MS_TestRun/2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.. Total: 13728 Computing degenerate peptides for 1546 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing 1395 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating sensitivity...and error tables... Computing MU for 1629 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% INFO: mu=2.28031e-06, db_size=12304840 INFO[15:03:56] Protein inference results decoy=85 target=1310 INFO[15:03:56] Converged to 1.05 % FDR with 1144 Proteins decoy=12 threshold=0.965 total=1156 2021/11/17 15:03:56 RemoveAll .: invalid argument
fstein
commented
3 years ago I updated to the new philosopher version 4.1.1 and also used the updated parameter.yml file. I still get the same error.
fstein
commented
3 years ago Here is the new console output:
INFO[11:24:22] Executing Pipeline v4.1.1
INFO[11:24:22] Creating workspace
INFO[11:24:22] Initiating the workspace on C:/MS_TestRun
INFO[11:24:22] Creating workspace
WARN[11:24:22] A meta data folder was found and will not be overwritten.
INFO[11:24:22] Annotating the database
INFO[11:24:23] Running the Database Search
MSFragger version MSFragger-3.4
Batmass-IO version 1.23.6
timsdata library version timsdata-2-8-7-1
(c) University of Michigan
RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved.
System OS: Windows 10, Architecture: AMD64
Java Info: 1.8.0_201, Java HotSpot(TM) 64-Bit Server VM, Oracle Corporation
JVM started with 53 GB memory
Checking database...
Checking spectral files...
C:\MS_TestRun\Ernie_200910_P0000_Ernie_TMTyeast_25ng_60min_R1.mzML: Scans = 15246
***FIRST SEARCH****
Parameters:
num_threads = 8
database_name = C:\MS_TestRun\2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas
decoyprefix = rev
precursor_mass_lower = -20.0
precursor_mass_upper = 20.0
precursor_mass_units = 1
data_type = 0
precursor_true_tolerance = 20.0
precursor_true_units = 1
fragment_mass_tolerance = 20.0
fragment_mass_units = 1
calibrate_mass = 2
use_all_mods_in_first_search = false
write_calibrated_mgf = 0
isotope_error = 0/1
mass_offsets = 0
labile_search_mode = OFF
restrict_deltamass_to = all
precursor_mass_mode = SELECTED
localize_delta_mass = false
delta_mass_exclude_ranges = (-1.5,3.5)
fragment_ion_series = b,y
ion_series_definitions =
search_enzyme_name = stricttrypsin
search_enzyme_sense_1 = C
search_enzyme_cut_1 = KR
search_enzyme_nocut_1 =
allowed_missed_cleavage_1 = 2
num_enzyme_termini = 2
clip_nTerm_M = true
allow_multiple_variable_mods_on_residue = false
max_variable_mods_per_peptide = 3
max_variable_mods_combinations = 5000
output_format = tsv_pepxml_pin
output_report_topN = 1
output_max_expect = 50.0
report_alternative_proteins = false
override_charge = false
precursor_charge_low = 1
precursor_charge_high = 4
digest_min_length = 7
digest_max_length = 50
digest_mass_range_low = 200.0
digest_mass_range_high = 5000.0
max_fragment_charge = 2
deisotope = 1
deneutralloss = true
track_zero_topN = 0
zero_bin_accept_expect = 0.0
zero_bin_mult_expect = 1.0
add_topN_complementary = 0
minimum_peaks = 15
use_topN_peaks = 150
minIonsScoring = 2
min_matched_fragments = 4
minimum_ratio = 0.01
intensity_transform = 0
remove_precursor_peak = 0
remove_precursor_range = -1.500000,1.500000
clear_mz_range_low = 125.5
clear_mz_range_high = 131.5
excluded_scan_list_file =
mass_diff_to_variable_mod = 0
min_sequence_matches = 2
check_spectral_files = true
variable_mod_01 = 15.994900 M 3
variable_mod_02 = 42.010600 [^ 1
variable_mod_06 = 229.162930 n^ 1
variable_mod_07 = 229.162930 S 1
add_A_alanine = 0.000000
add_C_cysteine = 57.021460
add_Cterm_peptide = 0.0
add_Cterm_protein = 0.0
add_D_aspartic_acid = 0.000000
add_E_glutamic_acid = 0.000000
add_F_phenylalanine = 0.000000
add_G_glycine = 0.000000
add_H_histidine = 0.000000
add_I_isoleucine = 0.000000
add_K_lysine = 229.162930
add_L_leucine = 0.000000
add_M_methionine = 0.000000
add_N_asparagine = 0.000000
add_Nterm_peptide = 0.0
add_Nterm_protein = 0.0
add_P_proline = 0.000000
add_Q_glutamine = 0.000000
add_R_arginine = 0.000000
add_S_serine = 0.000000
add_T_threonine = 0.000000
add_V_valine = 0.000000
add_W_tryptophan = 0.000000
add_Y_tyrosine = 0.000000
Number of unique peptides
of length 7: 84703
of length 8: 81329
of length 9: 77405
of length 10: 72902
of length 11: 71900
of length 12: 66591
of length 13: 63744
of length 14: 60464
of length 15: 56608
of length 16: 54471
of length 17: 51326
of length 18: 48168
of length 19: 44809
of length 20: 43036
of length 21: 40923
of length 22: 37881
of length 23: 36015
of length 24: 33916
of length 25: 31304
of length 26: 29495
of length 27: 27834
of length 28: 26493
of length 29: 24903
of length 30: 22759
of length 31: 21176
of length 32: 19741
of length 33: 18323
of length 34: 17128
of length 35: 15792
of length 36: 14514
of length 37: 13725
of length 38: 12801
of length 39: 11748
of length 40: 10738
of length 41: 9971
of length 42: 8783
of length 43: 7439
of length 44: 5553
of length 45: 3682
of length 46: 2135
of length 47: 1133
of length 48: 525
of length 49: 264
of length 50: 118
In total 1384268 peptides.
Generated 10920963 modified peptides.
Number of peptides with more than 5000 modification patterns: 0
Selected fragment index width 0.10 Da.
507847942 fragments to be searched in 1 slices (7.57 GB total)
Operating on slice 1 of 1:
Fragment index slice generated in 6.58 s
| **MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- | --------------- | --------------- | --------------- | --------------- | MS1 (Old) | MS1 (New) | MS2 (Old) | MS2 (New) |
|---|---|---|---|---|---|---|---|---|
| Run | Median MAD | Median MAD | Median MAD | Median MAD | ||||
| 001 | 3.48 1.45 | 0.02 0.91 | 0.30 1.32 | 0.10 1.26 | ||||
| ----- | --------------- | --------------- | --------------- | --------------- |
| Finding the optimal parameters: ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Count | 6409 | 6426 | 6398 | skip rest | ||||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| Peaks | 300_0 | 200_0 | 175_0 | 150_1 | 125_1 | 100_1 | ||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| Count | 6472 | 6472 | 6472 | 6426 | skip rest | |||||||||||
| ------- | ------- | ------- | ------- | ------- | ------- | ------- | ||||||||||
| ------- | ------- | |||||||||||||||
| Int. | 1 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Count | 6453 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Rm P. | 1 | |||||||||||||||
| ------- | ------- | |||||||||||||||
| Count | 6448 | |||||||||||||||
| ------- | ------- |
New fragment_mass_tolerance = 7 PPM New use_topN_peaks = 175 New minimum_ratio = 0.000000 New intensity_transform = 0 New remove_precursor_peak = 0 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 1.300 MIN*****
****MAIN SEARCH**** Checking database... variable_mod_03 has an empty value. variable_mod_04 has an empty value. variable_mod_05 has an empty value. Parameters: num_threads = 8 database_name = C:\MS_TestRun\2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 7.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = -1/0/1/2/3 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = tsv_pepxml_pin output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = true override_charge = false precursor_charge_low = 1 precursor_charge_high = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 2 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 175 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 0 remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_06 = 229.162930 n^ 1 variable_mod_07 = 229.162930 S 1 add_A_alanine = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_K_lysine = 229.162930 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_Y_tyrosine = 0.000000 Number of unique peptides of length 7: 84703 of length 8: 81329 of length 9: 77405 of length 10: 72902 of length 11: 71900 of length 12: 66591 of length 13: 63744 of length 14: 60464 of length 15: 56608 of length 16: 54471 of length 17: 51326 of length 18: 48168 of length 19: 44809 of length 20: 43036 of length 21: 40923 of length 22: 37881 of length 23: 36015 of length 24: 33916 of length 25: 31304 of length 26: 29495 of length 27: 27834 of length 28: 26493 of length 29: 24903 of length 30: 22759 of length 31: 21176 of length 32: 19741 of length 33: 18323 of length 34: 17128 of length 35: 15792 of length 36: 14514 of length 37: 13725 of length 38: 12801 of length 39: 11748 of length 40: 10738 of length 41: 9971 of length 42: 8783 of length 43: 7439 of length 44: 5553 of length 45: 3682 of length 46: 2135 of length 47: 1133 of length 48: 525 of length 49: 264 of length 50: 118 In total 1384268 peptides. Generated 10920963 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.03 Da. 507847942 fragments to be searched in 1 slices (7.57 GB total) Operating on slice 1 of 1: Fragment index slice generated in 6.17 s
***TOTAL TIME 1.725 MIN****
INFO[11:26:09] Running the validation and inference on C:/MS_TestRun
INFO[11:26:09] Executing PeptideProphet on C:/MS_TestRun
file 1: C:\MS_TestRun\Ernie_200910_P0000_Ernie_TMTyeast_25ng_60min_R1.pepXML
processed altogether 10667 results
INFO: Results written to file: C:\MS_TestRun\interact.pep.xml
C:\MS_TestRun\interact.pep.xml
Searching the tree...
Linking duplicate entries...
Printing results...
Building Commentz-Walter keyword tree...using Accurate Mass Bins using PPM mass difference Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) adding ACCMASS mixture distribution using search_offsets in ACCMASS mixture distr: 0 init with X! Tandem stricttrypsin MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN
INFO: Processing standard MixtureModel ...
PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB
read in 0 1+, 8857 2+, 1396 3+, 414 4+, 0 5+, 0 6+, and 0 7+ spectra.
Initialising statistical models ...
Found 1963 Decoys, and 8704 Non-Decoys
Iterations: .........10.........20......
WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (5+).
WARNING: Mixture model quality test failed for charge (6+).
WARNING: Mixture model quality test failed for charge (7+).
model complete after 27 iterations
INFO[11:26:28] Running the validation and inference on C:/MS_TestRun
INFO[11:26:28] Executing ProteinProphet on C:/MS_TestRun
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64))
(no FPKM) (using degen pep info)
Reading in C:/MS_TestRun/interact.pep.xml...
...read in 0 1+, 6020 2+, 840 3+, 44 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.05
Initializing 6646 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating protein lengths and molecular weights from database c:/MS_TestRun/2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000
.........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000
.........:.........:.........:.........:.........:.........:.........:.. Total: 13728
Computing degenerate peptides for 1870 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing probabilities for 1966 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100%
Computing 1706 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating sensitivity...and error tables...
Computing MU for 1966 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
INFO: mu=2.16234e-05, db_size=12304840
INFO[11:26:32] Integrating peptide validation
Running FPKM NSS NRS NSE NSI NSM Model EM:
Computing NSS values ...
Creating 6 threads Wait for threads to finish ... ...... done Computing NRS values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSE values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSI values ...
Creating 6 threads Wait for threads to finish ... 0--------------------------------------------------50------------------------------------------------100% ..................................................................................................... done Computing NSM values ...
Creating 6 threads
Wait for threads to finish ...
0--------------------------------------------------50------------------------------------------------100%
..................................................................................................... done
FPKM values are unavailable ...
Iterations: ......done
INFO[11:26:36] Creating combined protein inference
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64))
(no FPKM) (using degen pep info)
Reading in C:/MS_TestRun/interact.pep.xml...
...read in 0 1+, 5465 2+, 793 3+, 41 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.9
Initializing 6123 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database c:/MS_TestRun/2021-11-15-decoys-contam-Saccharomyces_cerrevisiae_UP000002311_05202016_6749entries.fasta.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.. Total: 13728 Computing degenerate peptides for 1546 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 1629 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing 1395 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating sensitivity...and error tables... Computing MU for 1629 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% INFO: mu=2.28031e-06, db_size=12304840 INFO[11:26:41] Protein inference results decoy=85 target=1310 INFO[11:26:41] Converged to 1.05 % FDR with 1144 Proteins decoy=12 threshold=0.965 total=1156 2021/11/19 11:26:41 RemoveAll .: invalid argument
fstein
commented
3 years ago And here is the new parameter.yml file: philosopher.yml.txt
fstein
commented
3 years ago Any idea what I might do wrong?
fstein
commented
3 years ago This is the output of the folder after running the pipeline command:
fstein
commented
3 years ago I just realized, that the files combined.prot and combined.pep are the output of the "Integrated Reports:" step and all files of the intermediate steps are missing. So with the philosopher.yml file it runs the "Integrated Reports:" directly after the "Protein Inference:" step. Is there a way to set the order of the steps? Or why is it skipping FDR Filtering, Label-Free Quantification, Isobaric Quantification and Individual Reports? Thanks for your help.
Cheers,
Frank
fstein
commented
3 years ago I guess I found the solution myself. Looks like Integrated Reports with Abacus only work if one has multiple folders and not just a single one.
prvst
commented
3 years ago Hi @fstein As you noticed we recently pushed an update, and Sarah was investigating your issue. We're glad that you found the reason. Thanks for reaching out.
fstein
commented
3 years ago Dear @prvst, maybe it could be an option to have a bit more descriptive error message in this case. One could check if multiple folders were given as an argument and only then allow execution of the Integrated Reports steps by Abacus.
Hi,
I tried to set up the pipeline for running msfragger and philosopher. When I do this, I stops after the Protein Inference step. If I then modify the parameter.yml file and set only one step to yes to be executed, the analysis does finish if I execute it in the following order: FDR Filtering: Label-Free Quantification Isobaric Quantification Individual Reports Integrated Reports
I have a TMT11 plex experiment (1 raw file which I converted to an mzML file with the following command: 'msconvert" *.raw --mzML --64 --zlib --filter "peak Picking true 1-"'
I attached my parameter file. I also have another issue: When trying to print the philosopher.yml file using the "philosopher pipeline --print" command, it would print me a philosopher.yml file from version Version 3.6.0. The latest version is 4.0.1. If I use the Version 3.6.0, the pipeline complains alot about missing parameter.
Thanks for your help.
Cheers,
Frank philosopher.yml.txt