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] > <javascript:>> 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] <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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