Hi Asif, I am not sure what happened but the decoys in your search are tagged XXX_ in the search and DECOY_ in the database.
<search_hit hit_rank="1" peptide="GGGGGGGGGGGWGWVGGWGRGGGGER" peptide_prev_aa="-" peptide_next_aa="K" protein=" XXX_ORF1_TRINITY_DN327608_c2_g1_i1:114:212_UNMAPPED" num_tot_proteins="0" calc_neutral_pep_mass="2241.979927335605" massdiff="0.033447265625" num_tol_term="2" num_missed_cleavages="1" protein_descr="originally identified as XXX_ORF1_TRINITY_DN327608_c2_g1_i1:114:212 in database e:/TPP_DataFolder/dbase/Trinity_TEA_ORFfinder_Proteins_1oct2020_DECOY.fasta"> I think you have to first, carefully check your search parameters, that they are compatible with N15 labeling, and second, verify you are using the correct database in your search, if you plan to use DECOY_ in the TPP analysis the search algorithm should not "know" about them (they should be hidden from the search algorithm because they will be used to validate its performance.) Cheers, -David On Tue, Feb 9, 2021 at 4:26 PM Asif Ahmed <kh.asifahme...@gmail.com> wrote: > Apologies. > > Please find the database with decoy > > https://drive.google.com/file/d/18ijaNWgmIompoJ0m99V0bCD4j54hXRlR/view?usp=sharing > > ASIF > > > On Wednesday, February 10, 2021 at 10:55:02 AM UTC+11 David Shteynberg > wrote: > >> 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.asif...@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-discu...@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. > 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/3e169acf-c2e8-44df-bafa-234e0cb72ca9n%40googlegroups.com > <https://groups.google.com/d/msgid/spctools-discuss/3e169acf-c2e8-44df-bafa-234e0cb72ca9n%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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