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Nesvilab / FragPipe

A cross-platform Graphical User Interface (GUI) for running MSFragger and Philosopher - powered pipeline for comprehensive analysis of shotgun proteomics data
http://fragpipe.nesvilab.org
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Issue With Analysis of Results Using Philosopher #6

Closed ghost closed 7 years ago

ghost commented 7 years ago

Hello,

I am having an issue using Philosopher to analyze the results of MSFragger. Here is the command line output of running the program:

Will execute 12 commands: java -jar -Xmx8G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.mzXML

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.pepXML

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 


Executing command:
$> java -jar -Xmx8G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.mzXML 
Process started
Peptide index read in 219ms
Selected fragment tolerance 0.02 Da and maximum fragment slice size of 4981.53MB

205026136 fragments to be searched in 1 slices (1.53GB total)
Operating on slice 1 of 1: 
6415ms
    b1906_293T_proteinID_01A_QE3_122212.mzXML 
95173ms

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 1535/41820 (3.67%) - 303.66 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 3103/41820 (7.42%) - 310.25 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 4708/41820 (11.26%) - 313.66 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 6267/41820 (14.99%) - 310.25 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 7847/41820 (18.76%) - 313.74 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 9405/41820 (22.49%) - 310.17 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 11017/41820 (26.34%) - 318.89 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 12561/41820 (30.04%) - 303.64 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 14120/41820 (33.76%) - 306.53 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 15691/41820 (37.52%) - 307.74 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 17216/41820 (41.17%) - 303.48 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 18757/41820 (44.85%) - 302.04 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 20296/41820 (48.53%) - 304.51 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 21853/41820 (52.25%) - 310.90 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 23449/41820 (56.07%) - 315.79 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 25014/41820 (59.81%) - 311.44 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 26569/41820 (63.53%) - 308.35 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 28138/41820 (67.28%) - 308.49 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 29743/41820 (71.12%) - 312.68 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 31368/41820 (75.01%) - 317.57 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 32957/41820 (78.81%) - 316.34 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 34589/41820 (82.71%) - 321.77 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 36193/41820 (86.54%) - 313.34 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 37875/41820 (90.57%) - 327.62 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 39645/41820 (94.80%) - 345.77 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 41399/41820 (98.99%) - 350.24 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 95173ms [progress: 41820/41820 (100.00%) - 214.25 spectra/s]
 - completed 134540ms

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[08:47:04] Creating workspace                           
WARN[08:47:04] existing workspace detected, will not overwrite 
INFO[08:47:04] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.pepXML 
Process started
 file 1: C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.pepXML

 processed altogether 34084 results

INFO: Results written to file: C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml

  - C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml

  - Building Commentz-Walter keyword tree...

  - Searching the tree...
  - Linking duplicate entries...
  - Printing results...

Using Decoy Label "rev".
Decoy Probabilities will be reported.
Using non-parametric distributions
 (X! Tandem) (using Tandem's expectation score for modeling)

init with X! Tandem trypsin 

 PeptideProphet  (TPP v5.0.1 Post-Typhoon dev, Build 201705191533-7588 (Windows_NT-x86_64)) AKeller@ISB
 read in 0 1+, 18190 2+, 12391 3+, 3101 4+, 324 5+, 73 6+, and 3 7+ spectra.
Found 0 Decoys, and 34082 Non-Decoys
WARNING: No decoys with label rev were found in this dataset. reverting to fully unsupervised method.
negmean = 0.0533258

MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... 
Initialising statistical models ...
INFO[08:48:17] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[08:48:17] Removing workspace                           
WARN[08:48:17] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[08:48:17] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[08:48:17] Creating workspace                           
WARN[08:48:17] existing workspace detected, will not overwrite 
INFO[08:48:17] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml 
Process started
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.0.1 Post-Typhoon dev, Build 201705191533-7588 (Windows_NT-x86_64))
 (no FPKM) (using degen pep info)
Reading in C:/Users/acadigan/Desktop/AlexStuff/MSFragger/TestData/interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml...

did not find any PeptideProphet results in input data!  Did you forget to run PeptideProphet?
...read in 0 1+, 0 2+, 0 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0

WARNING: no data - output file will be empty

INFO[08:48:19] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[08:48:20] Removing workspace                           
WARN[08:48:20] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[08:48:20] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[08:48:20] Creating workspace                           
WARN[08:48:20] existing workspace detected, will not overwrite 
INFO[08:48:20] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta 
Process started
INFO[08:48:20] Processing database                          

INFO[08:48:34] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml 
Process started
INFO[08:48:34] Processing peptide identification files      

INFO[08:48:37] 1+ Charge profile                             decoy=0 target=0
INFO[08:48:37] 2+ Charge profile                             decoy=0 target=18190
INFO[08:48:37] 3+ Charge profile                             decoy=0 target=12391
INFO[08:48:37] 4+ Charge profile                             decoy=0 target=3101
INFO[08:48:37] 5+ Charge profile                             decoy=0 target=324
INFO[08:48:37] 6+ Charge profile                             decoy=0 target=73
INFO[08:48:37] Database search results                       ions=24050 peptides=20473 psms=34084

INFO[08:48:37] Converged to 0.00 % FDR with 34084 PSMs       decoy=0 threshold=0 total=34084

INFO[08:48:38] Converged to 0.00 % FDR with 20473 Peptides   decoy=0 threshold=0 total=20473

INFO[08:48:38] Converged to 0.00 % FDR with 24050 Ions       decoy=0 threshold=0 total=24050
FATA[08:48:38] No Protein groups detected, check your file and try again 

Process finished, exit value: 1

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report 
Process started
INFO[08:48:38] Creating PSM report                          
INFO[08:48:38] Creating peptide Ion report                  
INFO[08:48:38] Creating peptide report                      

INFO[08:48:38] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[08:48:38] Removing workspace                           
WARN[08:48:38] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[08:48:38] Done                                         

Process finished, exit value: 0

=========================
===
===        Done
===
=========================

Here is another run using a different mzXML file:

Will execute 12 commands:
java -jar -Xmx14G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1922_293T_proteinID_02A_QE3_122212.mzXML 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1922_293T_proteinID_02A_QE3_122212.pepXML 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1922_293T_proteinID_02A_QE3_122212.pep.xml 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report 

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean

Executing command: $> java -jar -Xmx14G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1922_293T_proteinID_02A_QE3_122212.mzXML Process started Peptide index read in 203ms Selected fragment tolerance 0.02 Da and maximum fragment slice size of 9350.73MB

205026136 fragments to be searched in 1 slices (1.53GB total) Operating on slice 1 of 1: 6426ms b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 1221/46856 (2.61%) - 240.07 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 2682/46856 (5.72%) - 291.79 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 4167/46856 (8.89%) - 293.77 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 5615/46856 (11.98%) - 283.87 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 7069/46856 (15.09%) - 285.04 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 8494/46856 (18.13%) - 279.36 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 9875/46856 (21.08%) - 274.06 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 11288/46856 (24.09%) - 280.30 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 12729/46856 (27.17%) - 285.97 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 14184/46856 (30.27%) - 283.35 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 15607/46856 (33.31%) - 282.40 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 17022/46856 (36.33%) - 277.40 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 18461/46856 (39.40%) - 285.57 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 19909/46856 (42.49%) - 285.49 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 21353/46856 (45.57%) - 287.36 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 22801/46856 (48.66%) - 288.16 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 24272/46856 (51.80%) - 292.68 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 25758/46856 (54.97%) - 291.32 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 27261/46856 (58.18%) - 292.87 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 28720/46856 (61.29%) - 291.22 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 30186/46856 (64.42%) - 291.74 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 31671/46856 (67.59%) - 294.70 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 33136/46856 (70.72%) - 292.59 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 34644/46856 (73.94%) - 295.51 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 36177/46856 (77.21%) - 306.11 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 37713/46856 (80.49%) - 299.18 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 39250/46856 (83.77%) - 300.25 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 40773/46856 (87.02%) - 304.11 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 42394/46856 (90.48%) - 319.60 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 44034/46856 (93.98%) - 326.37 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 45652/46856 (97.43%) - 323.08 spectra/s]

b1922_293T_proteinID_02A_QE3_122212.mzXML 147102ms [progress: 46856/46856 (100.00%) - 248.09 spectra/s]

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init Process started INFO[09:17:45] Creating workspace
WARN[09:17:45] existing workspace detected, will not overwrite INFO[09:17:45] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1922_293T_proteinID_02A_QE3_122212.pepXML Process started file 1: C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1922_293T_proteinID_02A_QE3_122212.pepXML

processed altogether 38597 results

INFO: Results written to file: C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact-b1922_293T_proteinID_02A_QE3_122212.pep.xml

Using Decoy Label "rev". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling)

init with X! Tandem trypsin

PeptideProphet (TPP v5.0.1 Post-Typhoon dev, Build 201705191533-7588 (Windows_NT-x86_64)) AKeller@ISB read in 0 1+, 21822 2+, 13646 3+, 2786 4+, 259 5+, 80 6+, and 1 7+ spectra. Found 0 Decoys, and 38594 Non-Decoys WARNING: No decoys with label rev were found in this dataset. reverting to fully unsupervised method. negmean = 0.0533258

MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... Initialising statistical models ... INFO[09:19:15] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean Process started INFO[09:19:15] Removing workspace
WARN[09:19:15] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. INFO[09:19:15] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init Process started INFO[09:19:16] Creating workspace
WARN[09:19:16] existing workspace detected, will not overwrite INFO[09:19:16] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1922_293T_proteinID_02A_QE3_122212.pep.xml Process started ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.0.1 Post-Typhoon dev, Build 201705191533-7588 (Windows_NT-x86_64)) (no FPKM) (using degen pep info) Reading in C:/Users/acadigan/Desktop/AlexStuff/MSFragger/TestData/interact-b1922_293T_proteinID_02A_QE3_122212.pep.xml...

did not find any PeptideProphet results in input data! Did you forget to run PeptideProphet? ...read in 0 1+, 0 2+, 0 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0

WARNING: no data - output file will be empty

INFO[09:19:18] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean Process started INFO[09:19:18] Removing workspace
WARN[09:19:18] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. INFO[09:19:18] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init Process started INFO[09:19:18] Creating workspace
WARN[09:19:18] existing workspace detected, will not overwrite INFO[09:19:18] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta Process started INFO[09:19:18] Processing database

INFO[09:19:32] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml Process started INFO[09:19:32] Processing peptide identification files

INFO[09:19:43] 1+ Charge profile decoy=0 target=0 INFO[09:19:43] 2+ Charge profile decoy=0 target=40012 INFO[09:19:43] 3+ Charge profile decoy=0 target=26037 INFO[09:19:43] 4+ Charge profile decoy=0 target=5887 INFO[09:19:43] 5+ Charge profile decoy=0 target=583 INFO[09:19:43] 6+ Charge profile decoy=0 target=153

INFO[09:19:43] Database search results ions=49651 peptides=41576 psms=72681

INFO[09:19:43] Converged to 0.00 % FDR with 72681 PSMs decoy=0 threshold=0 total=72681

INFO[09:19:44] Converged to 0.00 % FDR with 41576 Peptides decoy=0 threshold=0 total=41576

INFO[09:19:44] Converged to 0.00 % FDR with 49651 Ions decoy=0 threshold=0 total=49651 FATA[09:19:44] No Protein groups detected, check your file and try again

Process finished, exit value: 1

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report Process started INFO[09:19:44] Creating PSM report
INFO[09:19:44] Creating peptide Ion report
INFO[09:19:44] Creating peptide report
INFO[09:19:44] Done

Process finished, exit value: 0

Executing command: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean Process started INFO[09:19:44] Removing workspace
WARN[09:19:44] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. INFO[09:19:44] Done

Process finished, exit value: 0

=========================

=== Done

=========================

I believe that MSFragger is running properly, but Philosopher is just producing output files with no results in them. I am using two different mzXML files I downloaded from this link: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2015/06/PXD001468. I have also attached a zip file of the protein data set I was using: CompleteHumanProteome.zip. Any advice on what I'm doing wrong and how to fix the issue would be greatly appreciated!

Thanks so much! -Alex

chhh commented 7 years ago

Hi Alex, The problem is, most likely, in the database that you used.

PeptideProphet was set up to use rev as the decoy identifier and didn't find that prefix in the search results, thus couldn't find decoy peptide hits and modelling failed.

If your DB has decoys with a different prefix, then you can change that in the input parameters for PeptideProphet. Go to PeptideProphet tab and in Cmd Line Options text field change --decoy rev to --decoy <your-prefix>.

Alternativelyy, you can use philospher to download a new db and append decoys (here's some info https://prvst.github.io/philosopher/database.html).

You also don't need to re-run the whole analysis again. You can provide the same input raw files, specify the same output directory, uncheck the Run MSFraggercheckbox on its tab (and maybe uncheck the Run ... checkboxes on other tabs as well) and just see if PeptideProphet will run by itself, it will pick up pepXML files from the last run. This can save you time debugging the workflow.

liangjin0912 commented 7 years ago

Hi,

I tried to use MSFragger but had the same issue as Alex. I used a new db created by philosopher and set decoy prefix as rev (here is the command: philosopher_windows_amd64.exe database --id UP000005640 --reviewed --contam --prefix rev), but still no protein groups detected and report files were empty. Is there any other way I can possibly solve the problem?

Thanks a lot!! Liang

ghost commented 7 years ago

Hello,

Thank you so much for the response and suggestions! I tried using the same command as Liang, but that did not seem to fix the problem. Here is a screen shot of exactly what I typed into PeptideProphet: peptideprophetcommandHere is the command line output:

Will execute 12 commands: java -jar -Xmx14G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.mzXML

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet philosopher_windows_amd64.exe database --id UP000005640 --reviewed --contam --prefix rev --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.pepXML

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report

C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 



Executing command:
$> java -jar -Xmx14G C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\MSFragger-Jar\MSFragger.jar C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\fragger.params C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.mzXML 
Process started
Peptide index read in 218ms
Selected fragment tolerance 0.02 Da and maximum fragment slice size of 9350.73MB
205026136 fragments to be searched in 1 slices (1.53GB total)
Operating on slice 1 of 1: 
6490ms
    b1906_293T_proteinID_01A_QE3_122212.mzXML 
56858ms

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 1320/41820 (3.16%) - 262.79 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 2891/41820 (6.91%) - 307.98 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 4458/41820 (10.66%) - 308.59 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 6012/41820 (14.38%) - 308.39 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 7596/41820 (18.16%) - 310.53 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 9126/41820 (21.82%) - 304.48 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 10739/41820 (25.68%) - 316.21 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 12284/41820 (29.37%) - 300.93 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 13811/41820 (33.02%) - 305.16 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 15342/41820 (36.69%) - 298.32 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 16894/41820 (40.40%) - 302.36 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 18384/41820 (43.96%) - 297.70 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 19926/41820 (47.65%) - 300.47 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 21475/41820 (51.35%) - 303.55 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 23040/41820 (55.09%) - 312.50 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 24598/41820 (58.82%) - 306.51 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 26154/41820 (62.54%) - 306.84 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 27704/41820 (66.25%) - 304.40 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 29267/41820 (69.98%) - 309.26 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 30835/41820 (73.73%) - 310.86 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 32432/41820 (77.55%) - 313.88 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 34025/41820 (81.36%) - 317.14 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 35611/41820 (85.15%) - 312.82 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 37224/41820 (89.01%) - 316.21 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 38907/41820 (93.03%) - 336.06 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 40595/41820 (97.07%) - 337.47 spectra/s]

    b1906_293T_proteinID_01A_QE3_122212.mzXML 56858ms [progress: 41820/41820 (100.00%) - 297.47 spectra/s]
 - completed 136107ms

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[09:12:23] Creating workspace                           
WARN[09:12:23] existing workspace detected, will not overwrite 
INFO[09:12:23] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet philosopher_windows_amd64.exe database --id UP000005640 --reviewed --contam --prefix rev --nonparam --expectscore --decoy rev --decoyprobs --masswidth 1000.0 --clevel -2 --database C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\b1906_293T_proteinID_01A_QE3_122212.pepXML 
Process started
Error: unknown flag: --id
Usage:
  philosopher peptideprophet [flags]

Flags:
      --accmass            use Accurate Mass model binning
      --clevel string      set Conservative Level in neg_stdev from the neg_mean, low numbers are less conservative, high numbers are more conservative
      --combine            combine the results from PeptideProphet into a single result file
      --database string    path to the database
      --decoy string       semi-supervised mode, protein name prefix to identify Decoy entries
      --decoyprobs         compute possible non-zero probabilities for Decoy entries on the last iteration
      --exclude            exclude deltaCn*, Mascot*, and Comet* results from results (default Penalize * results)
      --expectscore        use expectation value as the only contributor to the f-value for modeling
      --forcedistr         bypass quality control checks, report model despite bad modelling
      --glyc               enable peptide Glyco motif model
  -h, --help               help for peptideprophet
      --icat               apply ICAT model (default Autodetect ICAT)
      --ignorechg string   can be used multiple times to specify all charge states to exclude from modeling
      --instrwarn          warn and continue if combined data was generated by different instrument models
      --leave              leave alone deltaCn*, Mascot*, and Comet* results from results (default Penalize * results)
      --maldi              enable MALDI mode
      --masswidth string   model mass width (default "5.0")
      --minpeplen string   minimum peptide length not rejected (default "7")
      --minpintt string    minimum number of NTT in a peptide used for positive pI model (default 2)
      --minpiprob string   minimum probability after first pass of a peptide used for positive pI model (default 0.9)
      --minprob string     report results with minimum probability <number> (default 0.05)
      --minrtntt string    minimum number of NTT in a peptide used for positive RT model
      --minrtprob string   minimum probability after first pass of a peptide used for positive RT model
      --neggamma           use Gamma distribution to model the negative hits
      --noicat             do no apply ICAT model (default Autodetect ICAT)
      --nomass             disable mass model
      --nonmc              disable NMC missed cleavage model
      --nonparam           use semi-parametric modeling, must be used in conjunction with --decoy option
      --nontt              disable NTT enzymatic termini model
      --optimizefval       (SpectraST only) optimize f-value function f(dot,delta) using PCA
      --output string      Output name prefix (default "interact")
      --perfectlib         multiply by SpectraST library probability
      --phospho            enable peptide Phospho motif model
      --pi                 enable peptide pI model
      --ppm                use ppm mass error instead of Daltons for mass modeling
      --rt                 enable peptide RT model
      --rtcat string       enable peptide RT model, use <rtcatalog_file> peptide RTs when available as the theoretical value
      --zero               report results with minimum probability 0 (default 0.05)

FATA[09:12:23] unknown flag: --id                           

Process finished, exit value: 1

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[09:12:23] Removing workspace                           
WARN[09:12:23] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[09:12:23] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[09:12:23] Creating workspace                           
WARN[09:12:23] existing workspace detected, will not overwrite 
INFO[09:12:23] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe proteinprophet --output interact --maxppmdiff 20.0 interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml 
Process started
2017/06/26 09:12:24 open C:\Users\acadigan\AppData\Local\Temp\4e534eef-b50b-47ae-8443-c745f7c2b215\interact.prot.xml: The system cannot find the file specified.

ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.0.1 Post-Typhoon dev, Build 201705191533-7588 (Windows_NT-x86_64))
Error!  Input file not found: C:/Users/acadigan/Desktop/AlexStuff/MSFragger/TestData/interact-b1906_293T_proteinID_01A_QE3_122212.pep.xml
Exiting

Process finished, exit value: 1

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[09:12:24] Removing workspace                           
WARN[09:12:24] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[09:12:24] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --init 
Process started
INFO[09:12:25] Creating workspace                           
WARN[09:12:25] existing workspace detected, will not overwrite 
INFO[09:12:25] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe database --annotate C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\CompleteHumanProteome.fasta 
Process started
INFO[09:12:25] Processing database                          

INFO[09:12:38] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe filter --sequential --pepxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData --protxml C:\Users\acadigan\Desktop\AlexStuff\MSFragger\TestData\interact.prot.xml 
Process started
INFO[09:12:39] Processing peptide identification files      
FATA[09:12:39] No pepXML files found, check your files and try again 

Process finished, exit value: 1

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe report 
Process started
INFO[09:12:39] Creating PSM report                          
INFO[09:12:39] Creating peptide Ion report                  
INFO[09:12:39] Creating peptide report                      
INFO[09:12:39] Done                                         

Process finished, exit value: 0

Executing command:
$> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe workspace --clean 
Process started
INFO[09:12:39] Removing workspace                           
WARN[09:12:39] cannot remove the meta data: remove .meta\meta.bin: The process cannot access the file because it is being used by another process. 
INFO[09:12:39] Done                                         

Process finished, exit value: 0

=========================
===
===        Done
===
=========================

I'm guessing that I am not using the command properly.  Any advice on what I am doing wrong and how to fix the problem would be greatly appreciated!

-Alex
chhh commented 7 years ago

Hi Alex, Look at the command being run: $> C:\Users\acadigan\Desktop\AlexStuff\MSFragger\MSFraggerApp\philosopher_windows_amd64.exe peptideprophet philosopher_windows_amd64.exe database --id UP000005640 --reviewed --contam ...

You've put the name of the executable there and the database command. You don't need to do that. The tab for PeptideProphet simply generates the command line arguemnts to run. It adds Philosopher executable and the command name by itself. The database command of Philosopher can be used to download a fresh protein database and add decoys to it. You will need to run it yourself, here are some examples: https://prvst.github.io/philosopher/database.html. On the command line run something like:
philosopher_windows_amd64.exe database --id UP000005640 --contam
And it should get you the fasta file.

ghost commented 7 years ago

I got it to work! Sorry for the confusion. Thanks so much for your help!

chhh commented 7 years ago

You're welcome!