Hi Carolina, It appears the file you've sent me does contain mixture models, so I am really not sure why you are seeing this message at all. If you can send the input file for both one that shows the warning and one that doesn't I might be able to figure it out.
Cheers, David On Mon, Jun 19, 2017 at 9:56 AM, Carolina < [email protected]> wrote: > Hello David, > I would like you to check the results if you have time. I thought it was > weird because I have two other replicates and they are fine with high res, > it is QE data, but again, you might be right and this sample is having some > issue with high res. They are DDA data with three replicates, I searched > them on Comet, and then I applied PeptideProphet. I did all of them with > low resolution, but then I saw that I could apply high res to these > samples, so I did. But only in one of the replicates I got that problem > with high resolution. > > This is the link to the results: > https://we.tl/RKKys1U24y > > This is the log: > > C:/TPP/bin/xinteract (TPP v5.0.0 Typhoon, Build 201610111222-7423 > (Windows_NT-x86_64)) > PPM mode in Accurate Mass Model ... > running: "C:/TPP/bin/InteractParser "/tmp/a07480/params/Rep5_2.pep.xml" > "041117_DDA_Rep5.pep.xml" -L"7"" file 1: 041117_DDA_Rep5.pep.xml > SUCCESS: CORRECTED data file > c:\TPP\data\3lib_newComet_params_0.05mzTolerance\041117_DDA_Rep5.mzXML in > msms_run_summary tag ... > SUCCESS: CORRECTED data file > c:\TPP\data\3lib_newComet_params_0.05mzTolerance\041117_DDA_Rep5.mzXML in > msms_run_summary tag ... > processed altogether 76587 results > INFO: Results written to file: /tmp/a07480/params/Rep5_2.pep.xml > command completed in 12 sec > running: "C:/TPP/bin/DatabaseParser > "/tmp/a07480/params/Rep5_2.pep.xml""command completed in 0 sec > running: "C:/TPP/bin/RefreshParser "/tmp/a07480/params/Rep5_2.pep.xml" > "c:/TPP/data/dbase/UniProt_Human_reviewed_05-05-2016_reversed.fasta"" > - Searching the tree... > - Linking duplicate entries... > - Printing results... > > - Building Commentz-Walter keyword tree...command completed in 5 sec > running: "C:/TPP/bin/PeptideProphetParser "/tmp/a07480/params/Rep5_2.pep.xml" > MINPROB=0.05 PPM"using PPM mass difference > (Comet) > init with Comet trypsin > MS Instrument info: Manufacturer: Thermo Scientific, Model: Q Exactive > Orbitrap, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN > > INFO: Processing standard MixtureModel ... > PeptideProphet (TPP v5.0.0 Typhoon, Build 201610111222-7423 > (Windows_NT-x86_64)) AKeller@ISB > read in 0 1+, 50647 2+, 20114 3+, 4175 4+, 597 5+, 159 6+, and 0 7+ spectra. > Initialising statistical models ... > Iterations: .........10.........20.........30.....WARNING: Mixture model > quality test failed for charge (2+).WARNING: Mixture model quality test > failed for charge (3+). > model complete after 36 iterations > command completed in 3 sec > running: "C:/TPP/bin/ProphetModels.pl -i > /tmp/a07480/params/Rep5_2.pep.xml"Analyzing /tmp/a07480/params/Rep5_2.pep.xml > ... > Parsing search results > "c:\TPP\data\3lib_newComet_params_0.05mzTolerance\041117_DDA_Rep5 (Comet)"... > => Found 3406 hits. (0 decoys, 0 excluded) > => Total so far: 3406 hits. (0 decoys, 0 excluded) > command completed in 1 sec > moving tempfile /tmp/a07480/params/rep5_2.pep.xml to rep5_2.pep.xml > running: "C:/TPP/cgi-bin/PepXMLViewer.cgi -I > /tmp/a07480/params/rep5_2.pep.xml.a07480"command completed in 0 sec > running: "C:/TPP/bin/tpp_models.pl "c:/TPP/data/params/Rep5_2.pep.xml""File: > c:/TPP/data/params/Rep5_2.pep.xml > - in ms run: > c:\TPP\data\3lib_newComet_params_0.05mzTolerance\041117_DDA_Rep5... > ------------------------------------------------------------------------------- > TPP DASHBOARD -- started at Fri Jun 16 18:59:42 2017 > ------------------------------------------------------------------------------- > File c:/TPP/data/params/Rep5_2.pep.xml is pepxml > Found fval (+1) model... > Found ntt (+1) model... > Found nmc (+1) model... > Found massd (+1) model... > Found fval (+2) model... > Found ntt (+2) model... > Found nmc (+2) model... > Found massd (+2) model... > Found fval (+3) model... > Found ntt (+3) model... > Found nmc (+3) model... > Found massd (+3) model... > Found fval (+4) model... > Found ntt (+4) model... > Found nmc (+4) model... > Found massd (+4) model... > Found fval (+5) model... > Found ntt (+5) model... > Found nmc (+5) model... > Found massd (+5) model... > Found fval (+6) model... > Found ntt (+6) model... > Found nmc (+6) model... > Found massd (+6) model... > Found fval (+7) model... > Found ntt (+7) model... > Found nmc (+7) model... > Found massd (+7) model... > --> Trying to write file c:/TPP/data/params/Rep5_2.pep-MODELS.html > ------------------------------------------------------------------------------- > Finished at Fri Jun 16 18:59:43 2017 with 0 errors. > ------------------------------------------------------------------------------- > > > command completed in 1 sec > C:/TPP/bin/InteractParser "/tmp/a07480/params/Rep5_2.pep.xml" > "041117_DDA_Rep5.pep.xml" -L"7" 12 sec > C:/TPP/bin/DatabaseParser "/tmp/a07480/params/Rep5_2.pep.xml" > C:/TPP/bin/RefreshParser "/tmp/a07480/params/Rep5_2.pep.xml" > "c:/TPP/data/dbase/UniProt_Human_reviewed_05-05-2016_reversed.fasta" 5 sec > C:/TPP/bin/PeptideProphetParser "/tmp/a07480/params/Rep5_2.pep.xml" > MINPROB=0.05 PPM 3 sec > C:/TPP/bin/ProphetModels.pl -i /tmp/a07480/params/Rep5_2.pep.xml 1 sec > C:/TPP/cgi-bin/PepXMLViewer.cgi -I /tmp/a07480/params/rep5_2.pep.xml.a07480 0 > sec > C:/TPP/bin/tpp_models.pl "c:/TPP/data/params/Rep5_2.pep.xml" 1 sec > job completed in 22 sec > > > El sábado, 17 de junio de 2017, 9:54:15 (UTC-7), David Shteynberg escribió: >> >> Without seeing your search results, my guess is that in your high res >> search the filter is so stringent it excludes many of the correct results >> you see using the low res filter in the other search. >> >> -David >> >> On Fri, Jun 16, 2017 at 7:58 PM, Carolina <[email protected]> >> wrote: >> >>> Hello David, >>> It worked perfectly. Thank you again for your help! >>> Now I am dealing with some weird problem, I believe. I searched the same >>> raw files on Comet with comet params for low resolution, and I did another >>> search with params for high resolution. Both results were run with >>> peptideprophet but in the low res one I did not get any warning, and in the >>> High res I got warning on the mass model for charges +2 and +3, so I got a >>> few proteins comparing with the other one. Would you know what the problem >>> could be? Thank you so much. I am pretty new using TPP, so I am learning. >>> Sorry for bothering. >>> Carolina >>> >>> El jueves, 15 de junio de 2017, 17:05:23 (UTC-7), David Shteynberg >>> escribió: >>>> >>>> Hello Carolina, >>>> >>>> I was able to track down the issue in the analysis. By default, >>>> PeptideProphet multiplies the PSM probability by the LIB probability when >>>> processing SpectraST results. In your particular workflow, the LIB >>>> probability appears to be 0 for all entries, which forces all >>>> PeptideProphet probabilities to 0 also. To avoid this PERFECTLIB flag must >>>> be enabled for PeptideProphet analysis of your data. The option -OB does >>>> this in xinteract (and also the "Analyze Peptide" Petunia page). When >>>> using Petunia GUI "Analyze Peptides" , add "-OB" to the Advanced >>>> Commandline Options. Please report back if it fails to analyze with this >>>> option set. >>>> >>>> Cheers, >>>> -David >>>> >>>> >>>> >>>> On Tue, Jun 13, 2017 at 10:52 AM, David Shteynberg < >>>> [email protected]> wrote: >>>> >>>>> Having multiple decoys in your data is not a problem, in fact it >>>>> gives you the ability to estimate the FDR in different ways. It appears >>>>> that there is significant overlap between the negative and positive >>>>> distribution models so PeptideProphet tries to invalidate the mixture >>>>> model. Force fitting the distribution is usually not advisable. You can >>>>> try different models, for example try turning off "Use >>>>> Non-parametric model". If you are able to forward the dataset as a link >>>>> on >>>>> the cloud I would be able to suggest other analysis paths. >>>>> >>>>> Cheers, >>>>> David >>>>> >>>>> On Tue, Jun 13, 2017 at 9:47 AM, Carolina <[email protected]> >>>>> wrote: >>>>> >>>>>> Hello David, >>>>>> thank you for the answer. I have more than 2858 unique proteins, and >>>>>> 10051 unique peptides. I think that should be enough IDs. The >>>>>> PeptideProphet parameters were: >>>>>> >>>>>> Filter out results below this PeptideProphet probability:0.05 >>>>>> Minimum peptide length considered in the analysis: 6 >>>>>> Force the fitting of the mixture model (bypass automatic mixture >>>>>> model checks) >>>>>> Use decoy hits to pin down the negative distribution. Decoy protein >>>>>> names begin with: DECOY >>>>>> Use Non-parametric model (can only be used with >>>>>> decoy option) >>>>>> Report decoy hits with a computed probability >>>>>> (based on the model learned). >>>>>> >>>>>> I also tried different combinations, with the two decoy options >>>>>> together or not selected, but I alwasy got negative prob. The third >>>>>> parameter I tried without it, but I got an error *Command FAILED* >>>>>> RETURN CODE:65280 >>>>>> >>>>>> Found 33182 Decoys, and 167398 Non-Decoys >>>>>> Iterations: .........10.........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+). >>>>>> model complete after 26 iterations >>>>>> command completed in 114 sec >>>>>> running: "C:/TPP/bin/ProphetModels.pl -i >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml -d >>>>>> "DECOY""Analyzing >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml ... >>>>>> Parsing search results >>>>>> "c:/TPP/data/Results_SpectraST_Search_3lib/041117_DDA_Rep1 >>>>>> (SpectraST)"... >>>>>> => Found 0 hits. (0 decoys, 0 excluded) >>>>>> => Total so far: 0 hits. (0 decoys, 0 excluded) >>>>>> Parsing search results >>>>>> "c:/TPP/data/Results_SpectraST_Search_3lib/041117_DDA_Rep4 >>>>>> (SpectraST)"... >>>>>> => Found 0 hits. (0 decoys, 0 excluded) >>>>>> => Total so far: 0 hits. (0 decoys, 0 excluded) >>>>>> Parsing search results >>>>>> "c:/TPP/data/Results_SpectraST_Search_3lib/041117_DDA_Rep5 >>>>>> (SpectraST)"... >>>>>> => Found 0 hits. (0 decoys, 0 excluded) >>>>>> => Total so far: 0 hits. (0 decoys, 0 excluded) >>>>>> command completed in 0 sec >>>>>> moving tempfile >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml to >>>>>> interact.pep.xml >>>>>> running: "C:/TPP/cgi-bin/PepXMLViewer.cgi -I >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml.a08552" >>>>>> command "C:/TPP/cgi-bin/PepXMLViewer.cgi -I >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml.a08552" >>>>>> failed: Unknown error >>>>>> >>>>>> command "C:/TPP/cgi-bin/PepXMLViewer.cgi -I >>>>>> /tmp/a08552/Results_SpectraST_Search_3lib/interact.pep.xml.a08552" >>>>>> exited with non-zero exit code: 255 >>>>>> QUIT - the job is incomplete >>>>>> >>>>>> >>>>>> So in order to avoid that error, I needed to select the third >>>>>> parameter. >>>>>> Let me know what kind of problem do you think I am having. I just >>>>>> realized, that I had DECOYs on my library, but also reverse in the >>>>>> database >>>>>> fasta file. Would that be an issue? I will try to fix that and see if it >>>>>> gets better. >>>>>> Thanks abain for your time. >>>>>> Carolina >>>>>> >>>>>> El martes, 13 de junio de 2017, 8:07:12 (UTC-7), David Shteynberg >>>>>> escribió: >>>>>>> >>>>>>> Negative probabilities are output as place holders when there aren't >>>>>>> enough IDs to estimate a valid mixture model. When PeptideProphet >>>>>>> cannot >>>>>>> compute a probability but based on other models thinks the ID is >>>>>>> correct it >>>>>>> reports it with a negative probability equal to the absolute charge. It >>>>>>> would be helpful to know your PeptideProphet parameters to help >>>>>>> troubleshoot the failure to report a mixture model probability. >>>>>>> >>>>>>> -David >>>>>>> >>>>>>> On Mon, Jun 12, 2017 at 5:15 PM, Carolina <[email protected] >>>>>>> > wrote: >>>>>>> >>>>>>>> Hello, >>>>>>>> I built a library with decoys with SpectraST, and then, performed >>>>>>>> the search of raw files also with SpectraST against that library. The >>>>>>>> results were analyzed with peptide prophet but the probabilities are >>>>>>>> all >>>>>>>> negative, from -2 to -7. My intention is to get a FDR on my results, >>>>>>>> and >>>>>>>> that probability can not be right. Would you know what it might be >>>>>>>> going >>>>>>>> on? Thanks, Carolina >>>>>>>> >>>>>>>> -- >>>>>>>> You received this message because you are subscribed to the Google >>>>>>>> Groups "spctools-discuss" group. >>>>>>>> To unsubscribe from this group and stop receiving emails from it, >>>>>>>> send an email to [email protected]. >>>>>>>> To post to this group, send email to [email protected]. >>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>> p/spctools-discuss. >>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>> >>>>>>> >>>>>>> -- >>>>>> You received this message because you are subscribed to the Google >>>>>> Groups "spctools-discuss" group. >>>>>> To unsubscribe from this group and stop receiving emails from it, >>>>>> send an email to [email protected]. >>>>>> To post to this group, send email to [email protected]. >>>>>> Visit this group at https://groups.google.com/group/spctools-discuss. >>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>> >>>>> >>>>> >>>> -- >>> You received this message because you are subscribed to the Google >>> Groups "spctools-discuss" group. >>> To unsubscribe from this group and stop receiving emails from it, send >>> an email to [email protected]. >>> To post to this group, send email to [email protected]. >>> Visit this group at https://groups.google.com/group/spctools-discuss. >>> For more options, visit https://groups.google.com/d/optout. >>> >> >> -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected]. > To post to this group, send email to [email protected]. > Visit this group at https://groups.google.com/group/spctools-discuss. > For more options, visit https://groups.google.com/d/optout. > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To post to this group, send email to [email protected]. Visit this group at https://groups.google.com/group/spctools-discuss. For more options, visit https://groups.google.com/d/optout.
