It appears you forgot to include the _DECOY version of the database. Can you check?
On Tue, Feb 9, 2021 at 3:27 PM Asif Ahmed <kh.asifahme...@gmail.com> wrote: > Hi David, > > In my sample- N15 fed (heavy) female fly mated with N14 (normal) male fly, > after mating, I dissected the female reproductive organs and process the > sample using S-trap kit. > So, "theoretically" in the mated female reproductive tract proteome, there > would be plenty of female proteins (which would be N15) and a tiny amount > of male proteins (N14 proteins). > Our aim is to identify male-originated proteins from the samples and for > now, I just focused on normal search rather than N15 labelling search. > The protocol worked well for PD's SequestHT, Comet and Tandem search > giving ~150 hits, and now trying to add MsGf+ in the analysis.w > For the database, we are using trinity assembly of male reproductive organ > RNAseq, made a 6 frame translation of the assembly and add decoys (with > prefix of DECOY_ ). > > For peptide prophet in petunia, I used the following parameters, as shown > in the tutorial (not any unusual settings at all). > > *Use accurate mass binning, using PPM, * > *Use decoy hits to pin down the negative distribution. Decoy Protein names > begin with: DECOY_, * > *Use Non-parametric model and * > *Report decoy hits with a computed probability* > > Please find the datasets containing: > Thermo RAW data file (201010_P31528_Sample_S1_QE_HFX_HpH_7_5p2.raw), > mzML file (201010_P31528_Sample_S1_QE_HFX_HpH_7_5p2.mzML), > mzid file with conf (asiftest_instrument2_S1.mzid and MSGFPlus_conf.txt) , > Trinity database fasta file (with decoy), and resulted file from peptide > prophet (MsGF_inst1_interact.ipro.pep.xml). > > > https://drive.google.com/file/d/1rULaUtY9j2Ke7N6Z_hIa_a_JyY3admvj/view?usp=sharing > > ASIF > > > On Wednesday, February 10, 2021 at 8:48:19 AM UTC+11 David Shteynberg > wrote: > >> You can compress the directory and post your dataset in the cloud and I >> will pull it down. Perhaps you can start with your search parameters. >> N15 labelling creates mass-shifts on every amino acid, how are you setting >> these? What PeptideProphet options are you using? Any unusual options you >> are setting to get this data to process? >> >> Thanks, >> -David >> >> >> On Tue, Feb 9, 2021 at 1:29 PM Asif Ahmed <kh.asif...@gmail.com> wrote: >> >>> Hi David, >>> >>> Thanks for your reply and appreciate your interpretation. >>> >>> How can i share the dataset with you? I assume you might need the mzXML >>> file (~1.2gb), mzid file (/pepxml file) and the database file? >>> >>> Asif >>> >>> On Wed, 10 Feb 2021 at 5:43 am, 'David Shteynberg' via spctools-discuss < >>> spctools...@googlegroups.com> wrote: >>> >>>> Hello Asif, >>>> >>>> Unfortunately this analysis tells me that the DECOY-estimated FDR >>>> (error rate) is about 50%-60% amongst the highest scoring proteins in this >>>> analysis. I don't believe these are "acceptable" results. The problem is >>>> likely somewhere upstream of the ProteinProphet analysis, I cannot exactly >>>> tell without seeing more of the dataset. >>>> >>>> Best, >>>> -David >>>> >>>> On Tue, Feb 9, 2021, 5:00 AM Asif Ahmed <kh.asif...@gmail.com> wrote: >>>> >>>>> Hi, >>>>> >>>>> I ran a MSgf+ search (with decoy) for my samples (a mixture of N14 and >>>>> N15 proteins, want to detect N14 proteins in the sample) converted >>>>> resulted >>>>> .mzid file to pepXML using IDconvert, changed the paths using "update >>>>> path" >>>>> and run Peptideprophet *(Use accurate mass binning, using PPM , Use >>>>> decoy hits to pin down the negative distribution , Decoy Protein names >>>>> begin with: DECOY_, Use Non-parametric model and report decoy hits)* >>>>> and iprophet and protein prophet combined (as default settings) in >>>>> Petunia. >>>>> >>>>> The run went well without any error, but the models of "Learned NSP >>>>> distribution" as well as others are showing some abnormality. Can you >>>>> advise me, based on the fitted models, can I can accept the result at 0.99 >>>>> to 1 probability? >>>>> >>>>> ASIF >>>>> >>>>> [image: msgf1.PNG] >>>>> >>>>> [image: msgf2.PNG] >>>>> >>>>> -- >>>>> 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 spctools-discu...@googlegroups.com. >>>>> To view this discussion on the web visit >>>>> https://groups.google.com/d/msgid/spctools-discuss/0649dd4c-223f-4928-94f2-a17e3c7f0e76n%40googlegroups.com >>>>> <https://groups.google.com/d/msgid/spctools-discuss/0649dd4c-223f-4928-94f2-a17e3c7f0e76n%40googlegroups.com?utm_medium=email&utm_source=footer> >>>>> . >>>>> >>>> -- >>>> You received this message because you are subscribed to a topic in the >>>> Google Groups "spctools-discuss" group. >>>> To unsubscribe from this topic, visit >>>> https://groups.google.com/d/topic/spctools-discuss/tYk3ZszMyzM/unsubscribe >>>> . >>>> To unsubscribe from this group and all its topics, send an email to >>>> spctools-discu...@googlegroups.com. >>>> To view this discussion on the web visit >>>> https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D8DaS089_k5x6xJtQOk1U19_JMaxdASgDiSs7crdT9_ew%40mail.gmail.com >>>> <https://groups.google.com/d/msgid/spctools-discuss/CAGJJY%3D8DaS089_k5x6xJtQOk1U19_JMaxdASgDiSs7crdT9_ew%40mail.gmail.com?utm_medium=email&utm_source=footer> >>>> . >>>> >>> -- >>> *Khandaker Asif Ahmed* >>> PhD Student| Applied BioSciences, Macquarie University, NSW, Australia >>> Postgraduate Research Student| CSIRO Land and Water Flagship, Black >>> Mountain, ACT, Australia >>> *Address:* Room No: S1.03, Building No: 101, CSIRO Clunies Ross Street, >>> Black Mountain, ACT 2601, Australia >>> *Alternative Email:* khandaker...@csiro.au >>> <http://khandakerasif.ah...@csiro.au/>; khandaker-...@students.mq.edu.au >>> . >>> *Mobile:* (+61)0434018803 <+61%20434%20018%20803>, *Skype ID:* >>> kh.asifratul >>> Linkedin <https://www.linkedin.com/in/khandaker-asif-ahmed-24b461115/> >>> | ResearchGate >>> <https://www.researchgate.net/profile/Khandaker_Asif_Ahmed> | Google >>> Scholar >>> <https://scholar.google.com.au/citations?user=rUJ9DVAAAAAJ&hl=en> >>> >>> -- >>> 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 spctools-discu...@googlegroups.com. >>> >> To view this discussion on the web visit >>> https://groups.google.com/d/msgid/spctools-discuss/CABV_oz83MntSD%2BAsN3-5AYcs2B7JT%2BZAvE8r43Nvt%3DX9Yzf06A%40mail.gmail.com >>> <https://groups.google.com/d/msgid/spctools-discuss/CABV_oz83MntSD%2BAsN3-5AYcs2B7JT%2BZAvE8r43Nvt%3DX9Yzf06A%40mail.gmail.com?utm_medium=email&utm_source=footer> >>> . >>> >> -- > 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 spctools-discuss+unsubscr...@googlegroups.com. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/69a5fe95-dce8-4e3c-8873-f4d607046896n%40googlegroups.com > <https://groups.google.com/d/msgid/spctools-discuss/69a5fe95-dce8-4e3c-8873-f4d607046896n%40googlegroups.com?utm_medium=email&utm_source=footer> > . > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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