I've uploaded the files (and some notes for interpretation) to Dropbox and hopefully you will have received a link? (I used a contact email from an old paper that is hopefully still valid!).
Please let me know if you're unable to access the files / if you need any further files/info Thanks Pete On Thursday, 21 June 2018 11:11:58 UTC-7, David Shteynberg wrote: > > Hi Pete, > > Yes it would be helpful if I could take a look and you prot.xml and > pep.xml files. Can you put them on a shared drive and email me the link > directly? > > Thanks, > -David > > On Thu, Jun 21, 2018 at 11:05 AM, <[email protected] <javascript:>> > wrote: > >> Hi David, >> >> The 1% FDR I used is decoy-based, which works out as using a protein >> prophet cutoff of ~0.8 (in both cases). >> >> The dataset is large (~ 420,000 total spectra reported in the prot.xml) - >> perhaps this has an effect? >> >> Would it be useful for me to send across my prot.xml files (or pep.xmls?) >> >> Thanks >> Pete >> >> >> On Thursday, 21 June 2018 10:55:11 UTC-7, David Shteynberg wrote: >>> >>> Hi Pete, >>> >>> Thank you for the summary. I had a question about the 1% FDR. Is this >>> decoy-based or model-based? I am wondering what protein counts you will >>> observe when you compare at the 1% decoy-based FDR between running NSP in >>> both iProphet and ProteinProphet, running NSP only in iProphet and running >>> NSP only in ProteinProphet? >>> >>> Thank you, >>> -David >>> >>> >>> On Thu, Jun 21, 2018 at 10:39 AM, <[email protected]> wrote: >>> >>>> Hi David, >>>> >>>> Thanks a lot for your reply. >>>> >>>> Your first answer makes a big difference - I had been combining >>>> iprophet results for a second round which had previously combined multiple >>>> search engines. By combining the results from different search engines >>>> only >>>> in the second iProphet run, my numbers are now more consistent >>>> irrespective >>>> of order of combination. >>>> >>>> Regarding the second point, switching NSP off in protein prophet (after >>>> running NSP in iProphet) makes quite a big difference to my final protein >>>> numbers (2632 entries versus 2336 at 1% FDR). >>>> >>>> Taking a look at the additional matches, they are all single peptide >>>> sequence hits - however, from manual inspection of several of these >>>> though, >>>> there are often multiple matches with high peptide prophet scores across >>>> several different biological replicates, and the spectra look good. Some >>>> of >>>> them do look like single matches though (albeit with good looking spectra). >>>> >>>> Given that both are 1% FDR, it's difficult to choose the most >>>> appropriate method to choose. From what I've said, I think the risk of >>>> false negatives is greater (running both NSP models) than the risk of >>>> false >>>> positives (running iprophet NSP only) - my thoughts are that it will be >>>> better to use NSP 'off' in protein prophet, but to consider protein IDs >>>> from single peptide sequence hits as being less confident. >>>> >>>> Thanks again for your help, >>>> Pete >>>> >>>> >>>> >>>> >>>> On Wednesday, 20 June 2018 13:29:06 UTC-7, David Shteynberg wrote: >>>>> >>>>> Hello Pete, >>>>> >>>>> I think the answer to your first question is it depends on the >>>>> specifics of your analysis. >>>>> >>>>> You can pass iProphet files through iProphet again, since it will just >>>>> use the PeptideProphet probabilities which are not modified (only >>>>> reported) >>>>> by iProphet. If the iProphet is from a single search engine this should >>>>> be >>>>> just fine. However, if the iProphet file contains results from multiple >>>>> search engines then you probably don't want to combine it with iProphet >>>>> again as in this case each spectrum search result comes from the highest >>>>> scoring search engine, so not all the information will be available for >>>>> iProphet in the second analysis. Also, for your large analysis that is >>>>> currently failing in Petunia you might consider running the tool on the >>>>> commandline. >>>>> >>>>> For your second question, the NSP model can be disabled on the >>>>> commandline using the NONSP flag. The ProteinProphet NSP model is >>>>> implemented differently in iProphet and in ProteinProphet. Although, in >>>>> theory applying the models both times could be problematic due to their >>>>> similarity, in practice the models are different enought that during >>>>> testing I have not observed deleterious effects from using both the >>>>> iProphet and the ProteinProphet NSP models. You can try running the tools >>>>> in different ways and comparing the performance. Using both NSP models >>>>> is >>>>> the current default and you would have to explicitly disable the models >>>>> when you run each tool. >>>>> >>>>> I hope this helps. >>>>> >>>>> -David >>>>> >>>>> >>>>> >>>>> >>>>> On Tue, Jun 19, 2018 at 5:29 PM, <[email protected]> wrote: >>>>> >>>>>> Hi All, >>>>>> >>>>>> I have 2 questions I'd be grateful if people could help answer: >>>>>> >>>>>> 1) >>>>>> Is it valid to combine multiple iprophet.pep.xml files by passing >>>>>> through iprophet for a second time? - alternatively, is it valid to >>>>>> combine >>>>>> a single iprophet.pep.xml file with interact.pep.xml files in iprophet? >>>>>> >>>>>> I am trying to combine a lot of different experiments / search >>>>>> engines results etc, and have been combining in iprophet - but I appear >>>>>> to >>>>>> have maxed out the number interact.pep.xml files to pass into iprophet. >>>>>> Beyond a certain number of files (doesn't appear to be file-specific), >>>>>> iProphet fails. As a workaround, I wondered if I could simply run half >>>>>> of >>>>>> the files through iProphet, then combine the resulting file with the >>>>>> remaining files to be run, by running iprophet again prior to running >>>>>> protein prophet. - Would this be valid? >>>>>> >>>>>> 2) >>>>>> I attended a TPP course last year in which the course notes stated >>>>>> that NSP should be switched off in iProphet, if NSP model is to be used >>>>>> in >>>>>> protein prophet. >>>>>> >>>>>> I am using petunia (running protein prophet on the iprophet results), >>>>>> and I cannot see a NSP option in the protein prophet parameters. Does >>>>>> this >>>>>> mean that NSP is not being used when I run protein prophet? ... (i.e. am >>>>>> I >>>>>> ok to leave NSP on in iProphet?) >>>>>> >>>>>> >>>>>> Thanks >>>>>> Pete >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> -- >>>>>> 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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