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Nesvilab / FragPipe

A cross-platform proteomics data analysis suite
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FragPipe error due to storage issue and incorrect enzyme setting #439

Closed hanna6703 closed 3 years ago

hanna6703 commented 3 years ago

Hello,

I've recently been introduced to LC-MS in my master thesis project. My supervisor is very enthusiastic about Fragpipe and recommended me to use it - although he is also relatively new to it. We have processed earlier data without problem and I have tried to repeat the process on newly acquired data.

However, I run into an error in IonQuant just after arriving to MBR FDR estimation. The error claims the first run cannot be found ("Cannot find run XXX"). As I'm quite new to both the programme and the type of data, I find it hard to find a solution. I did check the manuals as well as the issues on your page but did not find a similar error there. I am hoping you could show me the right direction.

The current configuration includes MSfragger, PeptideProphet, IonQuant and EasyPQP (see log) and is an adjustment to the MBR_LFQ workflow. I have done an earlier run and have manually entered the optimized parameters from the output to save time. My data is MS2 from a Q exactive (coupled to a Dionex system). I can share generated files if you need to see them.

I was also surprised to see in the log that the mixture model quality tests often failed and that there are little matches between runs, especially since my minimum correlation threshold is 0. My samples are very similar and even contain some biological replicates. Is that something to worry about?

Thanks a lot in advance, Hanna

log_2021-08-24_15-03-30.txt

hanna6703 commented 3 years ago

I intended to post this to the Fragpipe section. Can it be moved?

fcyu commented 3 years ago

Hi Hanna,

Thanks for your interest in our tools.

Something was wrong at the earlier stage of the job. Following is one of the error message (almost each run has this kind of error)

Rewrite pepxml [Work dir: T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\S_YD_39]
C:\MyPrograms\FragPipe\jre\bin\java.exe -cp C:\MyPrograms\FragPipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\S_YD_39\interact.pep.xml T:\ProteomicsData\2021-04-22_Hanna\Dionex\Data_Dionex_HH_IPA_DTT\H_IPA_S_YD_B_DDA01.raw
13:51:42,917 |-INFO in ch.qos.logback.classic.LoggerContext[default] - Could NOT find resource [logback-test.xml]
13:51:42,917 |-INFO in ch.qos.logback.classic.LoggerContext[default] - Could NOT find resource [logback.groovy]
13:51:42,918 |-INFO in ch.qos.logback.classic.LoggerContext[default] - Found resource [logback.xml] at [jar:file:/C:/MyPrograms/FragPipe/lib/fragpipe-15.0.jar!/logback.xml]
13:51:42,918 |-WARN in ch.qos.logback.classic.LoggerContext[default] - Resource [logback.xml] occurs multiple times on the classpath.
13:51:42,918 |-WARN in ch.qos.logback.classic.LoggerContext[default] - Resource [logback.xml] occurs at [jar:file:/C:/MyPrograms/FragPipe/lib/fragpipe-16.0.jar!/logback.xml]
13:51:42,918 |-WARN in ch.qos.logback.classic.LoggerContext[default] - Resource [logback.xml] occurs at [jar:file:/C:/MyPrograms/FragPipe/lib/fragpipe-15.0.jar!/logback.xml]
13:51:42,933 |-INFO in ch.qos.logback.core.joran.spi.ConfigurationWatchList@d706f19 - URL [jar:file:/C:/MyPrograms/FragPipe/lib/fragpipe-15.0.jar!/logback.xml] is not of type file
13:51:43,042 |-INFO in ch.qos.logback.classic.joran.action.ConfigurationAction - debug attribute not set
13:51:43,042 |-INFO in ch.qos.logback.core.joran.action.AppenderAction - About to instantiate appender of type [ch.qos.logback.core.ConsoleAppender]
13:51:43,049 |-INFO in ch.qos.logback.core.joran.action.AppenderAction - Naming appender as [STDOUT]
13:51:43,055 |-INFO in ch.qos.logback.core.joran.action.NestedComplexPropertyIA - Assuming default type [ch.qos.logback.classic.encoder.PatternLayoutEncoder] for [encoder] property
13:51:43,134 |-INFO in ch.qos.logback.classic.joran.action.LoggerAction - Setting level of logger [com.dmtavt.fragpipe.FragpipeLoader] to INFO
13:51:43,134 |-INFO in ch.qos.logback.classic.joran.action.LoggerAction - Setting level of logger [com.dmtavt.fragpipe.api.Bus] to INFO
13:51:43,134 |-INFO in ch.qos.logback.classic.joran.action.LoggerAction - Setting level of logger [com.dmtavt.fragpipe.FragpipeUpdater] to INFO
13:51:43,134 |-INFO in ch.qos.logback.classic.joran.action.RootLoggerAction - Setting level of ROOT logger to INFO
13:51:43,134 |-INFO in ch.qos.logback.core.joran.action.AppenderRefAction - Attaching appender named [STDOUT] to Logger[ROOT]
13:51:43,134 |-INFO in ch.qos.logback.classic.joran.action.ConfigurationAction - End of configuration.
13:51:43,136 |-INFO in ch.qos.logback.classic.joran.JoranConfigurator@30b7c004 - Registering current configuration as safe fallback point

These errors result in few IDs in the later FDR filtering. For example

PhilosopherFilter [Work dir: T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_20]
C:\MyPrograms\FragPipe\tools\philosopher\philosopher.exe filter --sequential --razor --prot 0.01 --tag rev_ --pepxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_20 --protxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\combined.prot.xml --razorbin T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_20\.meta\razor.bin
INFO[14:14:19] Executing Filter  v4.0.0                     
INFO[14:14:19] Processing peptide identification files      
WARN[14:14:19] No PSM was found in data set. interact.pep.xml 
INFO[14:14:19] 1+ Charge profile                             decoy=0 target=0
INFO[14:14:19] 2+ Charge profile                             decoy=0 target=0
INFO[14:14:19] 3+ Charge profile                             decoy=0 target=0
INFO[14:14:19] 4+ Charge profile                             decoy=0 target=0
INFO[14:14:19] 5+ Charge profile                             decoy=0 target=0
INFO[14:14:19] 6+ Charge profile                             decoy=0 target=0
INFO[14:14:19] Database search results                       ions=0 peptides=0 psms=0
INFO[14:14:19] Converged to 0.00 % FDR with 0 PSMs           decoy=0 threshold=10 total=0
INFO[14:14:19] Converged to 0.00 % FDR with 0 Peptides       decoy=0 threshold=10 total=0
INFO[14:14:19] Converged to 0.00 % FDR with 0 Ions           decoy=0 threshold=10 total=0
INFO[14:14:19] Protein inference results                     decoy=144 target=242
INFO[14:14:19] Converged to 4.55 % FDR with 22 Proteins      decoy=1 threshold=0.9979 total=23
INFO[14:14:20] Applying sequential FDR estimation            ions=0 peptides=0 psms=0
INFO[14:14:20] Converged to 0.00 % FDR with 0 PSMs           decoy=0 threshold=10 total=0
INFO[14:14:20] Converged to 0.00 % FDR with 0 Peptides       decoy=0 threshold=10 total=0
INFO[14:14:20] Converged to 0.00 % FDR with 0 Ions           decoy=0 threshold=10 total=0
INFO[14:14:24] Post processing identifications              
INFO[14:14:26] Assigning protein identifications to layers  
INFO[14:14:27] Processing protein inference                 
Process 'PhilosopherFilter' finished, exit code: 0
INFO[14:14:30] Synchronizing PSMs and proteins              
INFO[14:14:30] Saving                                       
INFO[14:14:30] Done                                         
PhilosopherFilter [Work dir: T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_21]
C:\MyPrograms\FragPipe\tools\philosopher\philosopher.exe filter --sequential --razor --prot 0.01 --tag rev_ --pepxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_21 --protxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\combined.prot.xml --razorbin T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_20\.meta\razor.bin
INFO[14:14:30] Executing Filter  v4.0.0                     
INFO[14:14:30] Processing peptide identification files      
WARN[14:14:30] No PSM was found in data set. interact.pep.xml 
INFO[14:14:30] 1+ Charge profile                             decoy=0 target=0
INFO[14:14:30] 2+ Charge profile                             decoy=0 target=0
INFO[14:14:30] 3+ Charge profile                             decoy=0 target=0
INFO[14:14:30] 4+ Charge profile                             decoy=0 target=0
INFO[14:14:30] 5+ Charge profile                             decoy=0 target=0
INFO[14:14:30] 6+ Charge profile                             decoy=0 target=0
INFO[14:14:30] Database search results                       ions=0 peptides=0 psms=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 PSMs           decoy=0 threshold=10 total=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 Peptides       decoy=0 threshold=10 total=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 Ions           decoy=0 threshold=10 total=0
INFO[14:14:30] Protein inference results                     decoy=144 target=242
INFO[14:14:30] Converged to 4.55 % FDR with 22 Proteins      decoy=1 threshold=0.9979 total=23
INFO[14:14:30] Applying sequential FDR estimation            ions=0 peptides=0 psms=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 PSMs           decoy=0 threshold=10 total=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 Peptides       decoy=0 threshold=10 total=0
INFO[14:14:30] Converged to 0.00 % FDR with 0 Ions           decoy=0 threshold=10 total=0
INFO[14:14:34] Post processing identifications              
INFO[14:14:36] Assigning protein identifications to layers  
INFO[14:14:37] Processing protein inference                 
INFO[14:14:39] Synchronizing PSMs and proteins              
INFO[14:14:39] Saving                                       
INFO[14:14:40] Done                                         
Process 'PhilosopherFilter' finished, exit code: 0
PhilosopherFilter [Work dir: T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_22]
C:\MyPrograms\FragPipe\tools\philosopher\philosopher.exe filter --sequential --razor --prot 0.01 --tag rev_ --pepxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_22 --protxml T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\combined.prot.xml --razorbin T:\ProteomicsData\2021-04-22_Hanna\MSfragger\HH_IPA_Unipr4565_PepProph_2\E_FL_20\.meta\razor.bin
INFO[14:14:40] Executing Filter  v4.0.0                     
INFO[14:14:40] Processing peptide identification files      
INFO[14:14:40] 1+ Charge profile                             decoy=0 target=0
INFO[14:14:40] 2+ Charge profile                             decoy=0 target=0
INFO[14:14:40] 3+ Charge profile                             decoy=0 target=0
INFO[14:14:40] 4+ Charge profile                             decoy=1 target=1
INFO[14:14:40] 5+ Charge profile                             decoy=0 target=0
INFO[14:14:40] 6+ Charge profile                             decoy=0 target=0
INFO[14:14:40] Database search results                       ions=2 peptides=2 psms=2
INFO[14:14:40] Converged to 0.00 % FDR with 1 PSMs           decoy=0 threshold=0.7338 total=1
INFO[14:14:40] Converged to 0.00 % FDR with 1 Peptides       decoy=0 threshold=0.7338 total=1
INFO[14:14:40] Converged to 0.00 % FDR with 1 Ions           decoy=0 threshold=0.7338 total=1
INFO[14:14:40] Protein inference results                     decoy=144 target=242
INFO[14:14:40] Converged to 4.55 % FDR with 22 Proteins      decoy=1 threshold=0.9979 total=23
INFO[14:14:40] Applying sequential FDR estimation            ions=1 peptides=1 psms=1
INFO[14:14:40] Converged to 0.00 % FDR with 1 PSMs           decoy=0 threshold=0.7338 total=1
INFO[14:14:40] Converged to 0.00 % FDR with 1 Peptides       decoy=0 threshold=0.7338 total=1
INFO[14:14:40] Converged to 0.00 % FDR with 1 Ions           decoy=0 threshold=0.7338 total=1
INFO[14:14:44] Post processing identifications              
INFO[14:14:46] Assigning protein identifications to layers  
INFO[14:14:48] Processing protein inference                 
INFO[14:14:50] Synchronizing PSMs and proteins              
INFO[14:14:50] Saving                                       
INFO[14:14:50] Done

It finally crashes IonQuant. I am not sure of the reason of the error at the beginning but it should be due to some disk issues. May I ask what kind of disk or storage you are using?

Thanks,

Fengchao

hanna6703 commented 3 years ago

Hi Fengchao,

Thank you for your quick reply! I have been using the T disc, which is actually a server. Could that be the problem?

Kind regards, Hanna

fcyu commented 3 years ago

Actually, there are not many peptides in your sample even without the previous mentioned error. Looking at PeptideProphet's message like

 PeptideProphet  (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB
 read in 69 1+, 515 2+, 527 3+, 304 4+, 0 5+, 0 6+, and 0 7+ spectra.
Found 719 Decoys, and 696 Non-Decoys
MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... 
Initialising statistical models ...
.....Iterations: ..20.......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 trypsin 
.INFO[13:49:44] Done                                         
INFO[13:49:44] Done                                         
Process 'PeptideProphet' finished, exit code: 0
Process 'PeptideProphet' finished, exit code: 0
WARNING: Mixture model quality test failed for charge (2+).
WARNING: Mixture model quality test failed for charge (3+).
WARNING: Mixture model quality test failed for charge (4+).
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+).

Iterations: .10....WARNING: Mixture model quality test failed for charge (2+).
..
.......WARNING: Mixture model quality test failed for charge (3+).
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 30 iterations
......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 trypsin 
...... PeptideProphet  (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB
 read in 68 1+, 424 2+, 517 3+, 385 4+, 0 5+, 0 6+, and 0 7+ spectra.
Found 683 Decoys, and 711 Non-Decoys
MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... 
Initialising statistical models ...
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 trypsin 
.10.20..10.Iterations: ... PeptideProphet  (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB
 read in 70 1+, 479 2+, 577 3+, 392 4+, 0 5+, 0 6+, and 0 7+ spectra.
Found 794 Decoys, and 724 Non-Decoys
MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... 
Initialising statistical models ...
........Iterations: .....WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (2+).
...
INFO[13:49:46] Done                                         
Process 'PeptideProphet' finished, exit code: 0
.........WARNING: Mixture model quality test failed for charge (3+).
WARNING: Mixture model quality test failed for charge (4+).
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 28 iterations
....10...20..........20.  - Building Commentz-Walter keyword tree...
  - Searching the tree...
  - Linking duplicate entries...
  - Printing results...

...10...........WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (2+).
WARNING: Mixture model quality test failed for charge (3+).
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 28 iterations
.........INFO[13:49:47] Done                                         
Process 'PeptideProphet' finished, exit code: 0
20....WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (2+).
WARNING: Mixture model quality test failed for charge (3+).
WARNING: Mixture model quality test failed for charge (4+).
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 30 iterations
using Accurate Mass Bins

So, I guess after address the storage issue, there is a bigger issue: your sample does not have many peptides according to your current analysis. I am not sure if you set the parameters correctly. Is there any chemical labelling or PTM enrichment in your sample? Is your sample from trypsin digestion?

Thanks,

Fengchao

fcyu commented 3 years ago

Yes, it could be the problem because there could be a big latency between the server and your computer.

If your computer has enough space, it would be better to copy the data to the local storage before running the analysis.

Best,

Fengchao

Hi Fengchao,

Thank you for your quick reply! I have been using the T disc, which is actually a server. Could that be the problem?

Kind regards, Hanna

hanna6703 commented 3 years ago

I will try running it again from the computer, that should be possible.

Your other questions had me double-check the enzyme settings and I found the most likely reason for my lack of peptides there. In the process of saving workflows I have made a mistake so the enzyme was reversed to trypsin from chymotrypsin. That should make a difference! For a second I really worried about my samples but that disappeared like snow in the sun (as we say here).

Thanks a lot for your help, Hanna