Hi, Ok. I will send to you both. Also, I hope you got my other answer where I told you that the trypsin was the problem... do not know why...
El lunes, 19 de junio de 2017, 11:20:48 (UTC-7), David Shteynberg escribió: > > 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] > <javascript:>> 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/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] <javascript:>. >> To post to this group, send email to [email protected] >> <javascript:>. >> 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. 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