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. 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