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] <javascript:>> 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] >> <javascript:>> 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] <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. 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.
