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 <carolina.fernandez.costa@ > gmail.com> 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.
