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] <javascript:>> > 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] <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.
