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]
> <javascript:>> 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
>>
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