philosophers on Linux analysis for AP-MS data

[Bin-cc]

Hi,

Thank you for doing such an outstanding work. There are some questions I would like to ask you. I've run into some problems running philosopher on a compute cluster (CentOS 7 Linux). The philosopher.yml and comet.params are referenced from this. And the log is as follows.

time="14:58:00" level=info msg="Executing Pipeline  v4.0.0"
time="14:58:00" level=info msg="Creating workspace"
time="14:58:00" level=warning msg="A meta data folder was found and will not be overwritten. "
time="14:58:00" level=info msg="Initiating the workspace on /bin/A1BG/A1BG_1"
time="14:58:00" level=info msg="Creating workspace"
time="14:58:00" level=warning msg="A meta data folder was found and will not be overwritten. "
time="14:58:00" level=info msg="Initiating the workspace on /bin/A1BG/A1BG_2"
time="14:58:00" level=info msg="Creating workspace"
time="14:58:00" level=warning msg="A meta data folder was found and will not be overwritten. "
time="14:58:00" level=info msg="Annotating the database"
time="14:58:12" level=info msg="Running the Database Search"
time="14:58:12" level=fatal msg="Missing parameter file or data file for analysis. "

The reason seems to be that the system cannot find my comet.params or .mzml file. I'm sure my file path is fine. The same operation I have successfully implemented on Windows, but for some reason I am having problems on Linux. I was a little confused as to why this was the case. Thank you!

Best ragards,

Bin

[Bin-cc]

Additions: I am successfully running philosopher v3.4.13 on Windows but I also encountered the following error with v 4.0.0.

INFO[17:21:15] Protein inference results                     decoy=28 target=2269
INFO[17:21:15] Converged to 0.10 % FDR with 938 Proteins     decoy=0 threshold=0.9843 total=938
FATA[17:21:15] Cannot read file. open .meta\razor.bin: The system cannot find the file specified.

The combine.pep.xml and combine.prot.xml files have been created, but combined_peptide.tsv and combined_protein.tsv do not appear. Is there anything I missed? Thank you.

[Bin-cc]

Question Update,

I seem to have found the answer to my initial question, and it was because the .yml file was written incorrectly. I was able to run to the next step when I used the .yml you provided here instead of my own manual changes.

However, a new problem arises when writing to the interact.prot.xml. The logs are as follows.

time="21:20:16" level=info msg="Executing Pipeline  v4.0.0"
time="21:20:16" level=info msg="Creating workspace"
time="21:20:16" level=warning msg="A meta data folder was found and will not be overwritten. "
time="21:20:16" level=info msg="Initiating the workspace on /Bin/ABCG2/ABCG2_1"
time="21:20:16" level=info msg="Creating workspace"
time="21:20:16" level=warning msg="A meta data folder was found and will not be overwritten. "
time="21:20:16" level=info msg="Initiating the workspace on /Bin/ABCG2/ABCG2_2"
time="21:20:16" level=info msg="Creating workspace"
time="21:20:16" level=warning msg="A meta data folder was found and will not be overwritten. "
time="21:20:16" level=info msg="Annotating the database"
time="21:20:28" level=info msg="Running the Database Search"
time="21:26:08" level=info msg="Running the validation and inference on /Bin/ABCG2/ABCG2_1"
time="21:26:08" level=info msg="Executing PeptideProphet on /Bin/ABCG2/ABCG2_1"
Skipping file /Bin/ABCG2/ABCG2_1/interact.pep.xml, which already contains peptideprophet results ...
INFO: Results written to file: /Bin/ABCG2/ABCG2_1/interact.pep.xml
  - Building Commentz-Walter keyword tree... PeptideProphet  (TPP v5.2.1-dev Flammagenitus, Build 201906251008-exported (Linux-x86_64)) AKeller@ISB
 read in 0 1+, 12823 2+, 7274 3+, 1418 4+, 228 5+, 77 6+, and 0 7+ spectra.
Found 8767 Decoys, and 13053 Non-Decoys
WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (6+).
WARNING: Mixture model quality test failed for charge (7+).
time="21:26:54" level=info msg="Running the validation and inference on /Bin/ABCG2/ABCG2_2"
time="21:26:54" level=info msg="Executing PeptideProphet on /Bin/ABCG2/ABCG2_2"
Skipping file /Bin/ABCG2/ABCG2_2/interact.pep.xml, which already contains peptideprophet results ...
INFO: Results written to file: /Bin/ABCG2/ABCG2_2/interact.pep.xml
  - Building Commentz-Walter keyword tree... PeptideProphet  (TPP v5.2.1-dev Flammagenitus, Build 201906251008-exported (Linux-x86_64)) AKeller@ISB
 read in 0 1+, 10692 2+, 5334 3+, 1295 4+, 262 5+, 74 6+, and 0 7+ spectra.
Found 7348 Decoys, and 10309 Non-Decoys
WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (5+).
WARNING: Mixture model quality test failed for charge (6+).
WARNING: Mixture model quality test failed for charge (7+).
time="21:27:31" level=info msg="Running the validation and inference on /Bin/ABCG2/ABCG2_1"
time="21:27:31" level=info msg="Executing ProteinProphet on /Bin/ABCG2/ABCG2_1"
INFO: mu=3.56854e-06, db_size=299840006
time="21:27:37" level=fatal msg="Cannot write file. open /Bin/ABCG2/ABCG2_1/interact.prot.xml: no such file or directory"

This seems to be a problem with version 4.0.0, how do I fix it? Thank you.

Bin

[prvst]

Please check if the path is correct or if you have permissions to write to that location.

[Bin-cc]

Hi,

Thank you for your reply, My path was probably fine, as the previous steps were able to be completed without problems. The same problem I sometimes encounter on Windows. Yet the same files and operations sometimes work fine on Windows, and I don't quite know why.

[Bin-cc]

Hi,

I seem to have found the reason for the error. Because I copied the files directly from Windows to Linux, I did not initialize the working environment. After the modification I was able to run version 3.4.0 successfully on Linux, but version 4.0.0 still does not work successfully, and the same error on Windows.

time="17:00:49" level=info msg="Protein inference results" decoy=8 target=1128
time="17:00:49" level=info msg="Converged to 0.99 % FDR with 608 Proteins" decoy=6 threshold=0.9119 total=614
time="17:00:49" level=fatal msg="Cannot read file. open .meta/razor.bin: no such file or directory"

The combine.pep.xml and combine.prot.xml have been created, but combined_peptide.tsv and combined_protein.tsv do not appear.Compared with 3.4.0, is there anything to pay attention to when running 4.0.0?

Thank you.
Bin

[prvst]

You need to start from the beginning, if you changed computers, clean or remove your workspace. Also, make sure to use a parameter file from the current version, as they also get updated.

[Bin-cc]

Thank you for the reply. I had taken care to use the current version of the parameter file and initialized the workspace. But it still reported an error that the .meta/razor.bin file cannot be found, as described in my previous question.

[prvst]

@Bin-cc I released a new version last week, please feel free to try it, it should have the fix for your problem.

[Bin-cc]

Hi,

Thank you for telling me about this latest news. However, I tried the latest version and it still reports an error.

INFO[20:42:39] Protein inference results                     decoy=28 target=2269
INFO[20:42:39] Converged to 0.00 % FDR with 1862 Proteins    decoy=0 threshold=0.9931 total=1862
FATA[20:42:39] Cannot read file. open .meta\razor.bin: The system cannot find the file specified.

The parameters of the .yml file are set as follows.

# Philosopher pipeline configuration file
# Version 4.0.1
#
# The pipeline mode automates the processing done by Philosopher and other tools. First, check
# the steps you want to execute in the commands section and change them to
# 'yes'. For each selected command, go to its section and adjust the parameters
# accordingly to your analysis.
#
# If you want to include MSFragger and TMT-Integrator into your analysis, you will
# have to download them separately, and then add their location in their 
# configuration section.
#
# Usage:
# philosopher pipeline --config <this_configuration_file> [list_of_data_set_folders]

analytics: true                                  # reports when a workspace is created for usage statistics
slackToken:                                      # specify the Slack API token (how to generate a token: https://api.slack.com/legacy/custom-integrations/legacy-tokens)
slackChannel:                                    # specify the channel name, or
slackUserID:                                     # specify a user ID for a direct message

Steps:
  Database Search: yes                           # peptide to spectrum matching with Comet or MSFragger
  Peptide Validation: yes                        # peptide assignment validation with PeptideProphet
  PTM Localization: no                           # PTM site localization with PTMProphet
  Protein Inference: yes                         # protein identification validation with ProteinProphet
  Label-Free Quantification: yes                 # precursor label-free quantification inspired by moFF
  Isobaric Quantification: no                    # isobaric labeling-based relative quantification for TMT and iTRAQ
  Bio Cluster Quantification: no                 # protein report based on Uniprot protein clusters
  FDR Filtering: yes                             # statistical filtering, validation and false discovery r ates assessment
  Individual Reports: yes                        # multi-level reporting for both narrow-searches and open-searches
  Integrated Reports: yes                        # combined analysis of LC-MS/MS results inspired by Abacus
  Integrated Isobaric Quantification: no         # integrates channel abundances from multiple isobaric-tagged samples with TMT-Integrator                        

Database Search:                                 # MSFragger-3.4 & Comet v2019011
  protein_database: E:\Uniprot.fasta.fas               # path to the target-decoy protein database
  decoy_tag: rev_                                # prefix tag used added to decoy sequences
  search_engine: comet                           # search engine options include "comet" and "msfragger"
  comet:                                         # Comet v2019011
    noindex: true                                # skip mzML file indexing
    param: E:\comet.params                                     # comet parameter file (default "comet.params.txt")
    raw: mzML                                    # format of the spectra file
  msfragger:                                     # MSFragger v3.0
    path:                                       # path to MSFragger jar
    memory: 8                                    # how much memory in GB to use
    param:                                      # MSFragger parameter file
    raw: mzML                                    # spectra format
    num_threads: 0                               # 0=poll CPU to set num threads; else specify num threads directly (max 64)
    precursor_mass_lower: -50                    # lower bound of the precursor mass window
    precursor_mass_upper: 50                     # upper bound of the precursor mass window
    precursor_mass_units: 1                      # 0=Daltons, 1=ppm
    precursor_true_tolerance: 20                 # true precursor mass tolerance (window is +/- this value)
    precursor_true_units: 1                      # 0=Daltons, 1=ppm
    fragment_mass_tolerance: 20                  # fragment mass tolerance (window is +/- this value)
    fragment_mass_units: 1                       # fragment mass tolerance units (0 for Da, 1 for ppm)
    calibrate_mass: 2                            # 0=Off, 1=On, 2=On and find optimal parameters
    deisotope: 1                                 # activates deisotoping.
    isotope_error: 0/1/2                         # 0=off, 0/1/2 (standard C13 error)
    mass_offsets: 0                              # allow for additional precursor mass window shifts. Multiplexed with isotope_error. mass_offsets = 0/79.966 can be used as a restricted ‘open’ search that looks for unmodified and phosphorylated peptides (on any residue)
    precursor_mass_mode: selected                # selected or isolated
    localize_delta_mass: 0                       # this allows shifted fragment ions - fragment ions with mass increased by the calculated mass difference, to be included in scoring
    delta_mass_exclude_ranges: (-1.5,3.5)        # exclude mass range for shifted ions searching
    fragment_ion_series: b,y                     # ion series used in search
    search_enzyme_name: Trypsin                  # name of enzyme to be written to the pepXML file
    search_enzyme_cutafter: KR                   # residues after which the enzyme cuts
    search_enzyme_butnotafter: P                 # residues that the enzyme will not cut before
    num_enzyme_termini: 2                        # 2 for enzymatic, 1 for semi-enzymatic, 0 for nonspecific digestion
    allowed_missed_cleavage: 1                   # maximum value is 5
    clip_nTerm_M: 1                              # specifies the trimming of a protein N-terminal methionine as a variable modification (0 or 1)
    variable_mod_01: 15.99490 M 3                # variable modification
    variable_mod_02: 42.01060 [^ 1               # variable modification
    variable_mod_03:                             # variable modification
    variable_mod_04:                             # variable modification
    variable_mod_05:                             # variable modification
    variable_mod_06:                             # variable modification
    variable_mod_07:                             # variable modification
    allow_multiple_variable_mods_on_residue: 0   # static mods are not considered
    max_variable_mods_per_peptide: 3             # maximum of 5
    max_variable_mods_combinations: 5000         # maximum of 65534, limits number of modified peptides generated from sequence
    output_file_extension: pepXML                # file extension of output files
    output_format: pepXML                        # file format of output files (pepXML or tsv)
    output_report_topN: 1                        # reports top N PSMs per input spectrum
    output_max_expect: 50                        # suppresses reporting of PSM if top hit has expectation greater than this threshold
    report_alternative_proteins: 0               # 0=no, 1=yes
    precursor_charge: 1 4                        # assume range of potential precursor charge states. Only relevant when override_charge is set to 1
    override_charge: 0                           # 0=no, 1=yes to override existing precursor charge states with precursor_charge parameter
    digest_min_length: 7                         # minimum length of peptides to be generated during in-silico digestion
    digest_max_length: 50                        # maximum length of peptides to be generated during in-silico digestion
    digest_mass_range: 500.0 5000.0              # mass range of peptides to be generated during in-silico digestion in Daltons
    max_fragment_charge: 2                       # maximum charge state for theoretical fragments to match (1-4)
    track_zero_topN: 0                           # in addition to topN results, keep track of top results in zero bin
    zero_bin_accept_expect: 0                    # boost top zero bin entry to top if it has expect under 0.01 - set to 0 to disable
    zero_bin_mult_expect: 1                      # disabled if above passes - multiply expect of zero bin for ordering purposes (does not affect reported expect)
    add_topN_complementary: 0                    # inserts complementary ions corresponding to the top N most intense fragments in each experimental spectra
    minimum_peaks: 15                            # required minimum number of peaks in spectrum to search (default 10)
    use_topN_peaks: 100                          # pre-process experimental spectrum to only use top N peaks
    min_fragments_modelling: 2                   # minimum number of matched peaks in PSM for inclusion in statistical modeling
    min_matched_fragments: 4                     # minimum number of matched peaks for PSM to be reported
    minimum_ratio: 0.01                          # filters out all peaks in experimental spectrum less intense than this multiple of the base peak intensity
    clear_mz_range: 0.0 0.0                      # for iTRAQ/TMT type data; will clear out all peaks in the specified m/z range
    remove_precursor_peak: 0                     # remove precursor peaks from tandem mass spectra. 0=not remove; 1=remove the peak with precursor charge; 2=remove the peaks with all charge states.
    remove_precursor_range: -1.5,1.5             # m/z range in removing precursor peaks. Unit: Da.
    intensity_transform: 0                       # transform peaks intensities with sqrt root. 0=not transform; 1=transform using sqrt root.
    mass_diff_to_variable_mod: 0			           # Put mass diff as a variable modification. 0 for no; 1 for yes and change the original mass diff and the calculated mass accordingly; 2 for yes but do not change the original mass diff.
    labile_search_mode: "off"                    # type of search (nglycan, labile, or off). Off means non-labile/typical search.
    restrict_deltamass_to: all                   # Specify amino acids on which delta masses (mass offsets or search modifications) can occur. Allowed values are single letter codes (e.g. ACD), must
    diagnostic_intensity_filter: 0			         # [nglycan/labile search_mode only]. Minimum relative intensity for SUM of all detected oxonium ions to achieve for spectrum to contain diagnostic fragment evidence. Calculated relative to spectrum base peak. 0 <= value.
    Y_type_masses:                               # [nglycan/labile search_mode only]. Specify fragments of labile mods that are commonly retained on intact peptides (e.g. Y ions for glycans). Only used if 'Y' is included in fragment_ion_series.
    diagnostic_fragments:                        # [nglycan/labile search_mode only]. Specify diagnostic fragments of labile mods that appear in the low m/z region. Only used if diagnostic_intensity_filter > 0.
    add_Cterm_peptide: 0.000000                  # c-term peptide fixed modifications
    add_Cterm_protein: 0.000000                  # c-term protein fixed modifications
    add_Nterm_peptide: 0.000000                  # n-term peptide fixed modifications
    add_Nterm_protein: 0.000000                  # n-term protein fixed modifications
    add_A_alanine: 0.000000                      # alanine fixed modifications 
    add_C_cysteine: 57.021464                    # cysteine fixed modifications 
    add_D_aspartic_acid: 0.000000                # aspartic acid fixed modifications
    add_E_glutamic_acid: 0.000000                # glutamic acid fixed modifications
    add_F_phenylalanine: 0.000000                # phenylalanine fixed modifications
    add_G_glycine: 0.000000                      # glycine fixed modifications
    add_H_histidine: 0.000000                    # histidine fixed modifications
    add_I_isoleucine: 0.000000                   # isoleucine fixed modifications
    add_K_lysine: 0.000000                       # lysine fixed modifications
    add_L_leucine: 0.000000                      # leucine fixed modifications
    add_M_methionine: 0.000000                   # methionine fixed modifications
    add_N_asparagine: 0.000000                   # asparagine fixed modifications
    add_P_proline: 0.000000                      # proline fixed modifications
    add_Q_glutamine: 0.000000                    # glutamine fixed modifications
    add_R_arginine: 0.000000                     # arginine fixed modifications
    add_S_serine: 0.000000                       # serine fixed modifications
    add_T_threonine: 0.000000                    # threonine fixed modifications
    add_V_valine: 0.000000                       # valine fixed modifications
    add_W_tryptophan: 0.000000                   # tryptophan fixed modifications
    add_Y_tyrosine: 0.000000                     # tyrosine fixed modifications

Peptide Validation:                              # PeptideProphet v5.2
  concurrent: false                              # Concurrent execution of multiple instaces
  extension: pep.xml                              # pepXML file extension
  clevel: 0                                      # set Conservative Level in neg_stdev from the neg_mean, low numbers are less conservative, high numbers are more conservative
  accmass: true                                  # use Accurate Mass model binning
  decoyprobs: true                               # compute possible non-zero probabilities for Decoy entries on the last iteration
  enzyme: trypsin                                # enzyme used in sample (optional)
  exclude: false                                 # exclude deltaCn*, Mascot*, and Comet* results from results (default Penalize * results)
  expectscore: true                              # use expectation value as the only contributor to the f-value for modeling
  forcedistr: false                              # bypass quality control checks, report model despite bad modeling
  glyc: false                                    # enable peptide Glyco motif model
  icat: false                                    # apply ICAT model (default Autodetect ICAT)
  instrwarn: false                               # warn and continue if combined data was generated by different instrument models
  leave: false                                   # leave alone deltaCn*, Mascot*, and Comet* results from results (default Penalize * results)
  maldi: false                                   # enable MALDI mode
  masswidth: 5                                   # model mass width (default 5)
  minpeplen: 7                                   # minimum peptide length not rejected (default 7)
  minpintt: 2                                    # minimum number of NTT in a peptide used for positive pI model (default 2)
  minpiprob: 0.9                                 # minimum probability after first pass of a peptide used for positive pI model (default 0.9)
  minprob: 0.05                                  # report results with minimum probability (default 0.05)
  minrtntt: 2                                    # minimum number of NTT in a peptide used for positive RT model (default 2)
  minrtprob: 0.9                                 # minimum probability after first pass of a peptide used for positive RT model (default 0.9)
  neggamma: false                                # use Gamma distribution to model the negative hits
  noicat: false                                  # do no apply ICAT model (default Autodetect ICAT)
  nomass: false                                  # disable mass model
  nonmc: false                                   # disable NMC missed cleavage model
  nonparam: true                                 # use semi-parametric modeling, must be used in conjunction with --decoy option
  nontt: false                                   # disable NTT enzymatic termini model
  optimizefval: false                            # (SpectraST only) optimize f-value function f(dot,delta) using PCA
  phospho: false                                 # enable peptide Phospho motif model
  pi: false                                      # enable peptide pI model
  ppm: true                                      # use PPM mass error instead of Dalton for mass modeling
  zero: false                                    # report results with minimum probability 0

PTM Localization:                                # PTMProphet v6.0
  autodirect: false                              # use direct evidence when the lability is high, use in combination with LABILITY
  cions:                                         # use specified C-term ions, separate multiple ions by commas (default: y for CID, z for ETD)
  direct: false                                  # use only direct evidence for evaluating PTM site probabilities
  em: 2                                          # set EM models to 0 (no EM), 1 (Intensity EM Model Applied) or 2 (Intensity and Matched Peaks EM Models Applied)
  static: false                                  # use static fragppmtol for all PSMs instead of dynamically estimates offsets and tolerances
  fragppmtol: 15                                 # when computing PSM-specific mass_offset and mass_tolerance, use specified default +/- MS2 mz tolerance on fragment ions
  ifrags: false                                  # use internal fragments for localization
  keepold: false                                 # retain old PTMProphet results in the pepXML file
  lability: false                                # compute Lability of PTMs
  massdiffmode: false                            # use the Mass Difference and localize
  excludemassdiffmin: 0                          # Minimum mass difference excluded for MASSDIFFFMODE analysis (default=0)
  excludemassdiffmax: 0                          # Maximun mass difference excluded for MASSDIFFFMODE analysis (default=0)
  massoffset: 0                                  # adjust the massdiff by offset (0 = use default)
  maxfragz: 0                                    # limit maximum fragment charge (default: 0=precursor charge, negative values subtract from precursor charge)
  maxthreads: 4                                  # use specified number of threads for processing
  mino: 0                                        # use specified number of pseudo-counts when computing Oscore (0 = use default)
  minprob: 0                                     # use specified minimum probability to evaluate peptides
  mods:                                          # specify modifications
  nions:                                         # use specified N-term ions, separate multiple ions by commas (default: a,b for CID, c for ETD)
  nominofactor: false                            # disable MINO factor correction when MINO= is set greater than 0 (default: apply MINO factor correction)
  ppmtol: 1                                      # use specified +/- MS1 ppm tolerance on peptides which may have a slight offset depending on search parameters
  verbose: false                                 # produce Warnings to help troubleshoot potential PTM shuffling or mass difference issues

Protein Inference:                               # ProteinProphet v5.2
  accuracy: false                                # equivalent to --minprob 0
  allpeps: false                                 # consider all possible peptides in the database in the confidence model
  confem: false                                  # use the EM to compute probability given the confidence
  delude: false                                  # do NOT use peptide degeneracy information when assessing proteins
  excludezeros: false                            # exclude zero prob entries
  fpkm: false                                    # model protein FPKM values
  glyc: false                                    # highlight peptide N-glycosylation motif
  icat: false                                    # highlight peptide cysteines
  instances: false                               # use Expected Number of Ion Instances to adjust the peptide probabilities prior to NSP adjustment
  iprophet: false                                # input is from iProphet
  logprobs: false                                # use the log of the probabilities in the Confidence calculations
  maxppmdiff: 20                                 # maximum peptide mass difference in PPM (default 20)
  minprob: 0.05                                  # peptideProphet probabilty threshold (default 0.05)
  mufactor: 1                                    # fudge factor to scale MU calculation (default 1)
  nogroupwts: false                              # check peptide's Protein weight against the threshold (default: check peptide's Protein Group weight against threshold)
  nonsp: false                                   # do not use NSP model
  nooccam: false                                 # non-conservative maximum protein list
  noprotlen: false                               # do not report protein length
  normprotlen: false                             # normalize NSP using Protein Length
  protmw: false                                  # get protein mol weights
  softoccam: false                               # peptide weights are apportioned equally among proteins within each Protein Group (less conservative protein count estimate)
  unmapped: false                                # report results for UNMAPPED proteins

Label-Free Quantification:                       # Freequant
  peakTimeWindow: 0.4                            # specify the time windows for the peak (minute) (default 0.4)
  retentionTimeWindow: 3                         # specify the retention time window for xic (minute) (default 3)
  tolerance: 10                                  # m/z tolerance in ppm (default 10)
  raw: false                                     # read raw files instead of converted mzML, or mzXML
  faims: false                                   # use FAIMS information for the quantification

Isobaric Quantification:                         # Labelquant
  bestPSM: false                                 # select the best PSMs for protein quantification
  level: 2                                       # ms level for the quantification
  minProb: 0.7                                   # only use PSMs with a minimum probability score
  plex:                                          # number of channels
  purity: 0.5                                    # ion purity threshold (default 0.5)
  removeLow: 0.0                                 # ignore the lower 3% PSMs based on their summed abundances
  tolerance: 20                                  # m/z tolerance in ppm (default 20)
  uniqueOnly: false                              # report quantification based on only unique peptides
  brand: tmt                                     # isobaric labeling brand (tmt, itraq)
  raw: false                                     # read raw files instead of converted mzML, or mzXML

Bio Cluster Quantification:                      # BioQuant
  organismUniProtID:                             # UniProt proteome ID
  level: 0.9                                     # cluster identity level (default 0.9)

DR Filtering:                                   # Filter
  psmFDR: 0.01                                   # psm FDR level (default 0.01)
  peptideFDR: 0.01                               # peptide FDR level (default 0.01)
  ionFDR: 0.01                                   # peptide ion FDR level (default 0.01)
  proteinFDR: 0.001                               # protein FDR level (default 0.01)
  peptideProbability: 0.7                        # top peptide probability threshold for the FDR filtering (default 0.7)
  proteinProbability: 0.5                        # protein probability threshold for the FDR filtering (not used with the razor algorithm) (default 0.5)
  peptideWeight: 1                               # threshold for defining peptide uniqueness (default 1)
  razor: false                                   # use razor peptides for protein FDR scoring
  picked: false                                  # apply the picked FDR algorithm before the protein scoring
  mapMods: false                                 # map modifications acquired by an open search
  models: false                                  # print model distribution
  sequential: false                              # alternative algorithm that estimates FDR using both filtered PSM and Protein lists

Individual Reports:                              # Report
  msstats: false                                 # create an output compatible to MSstats
  withDecoys: false                              # add decoy observations to reports
  mzID: false                                    # create a mzID output
            
Integrated Reports:                              # Abacus
  protein: true                                  # global level protein report
  peptide: true                                  # global level peptide report
  proteinProbability: 0.9                        # minimum protein probability (default 0.9)
  peptideProbability: 0.5                        # minimum peptide probability (default 0.5)
  uniqueOnly: false                              # report TMT quantification based on only unique peptides
  reprint: true                                 # create abacus reports using the Reprint format

Integrated Isobaric Quantification:              # TMT-Integrator v3.2.0
  path:                                          # path to TMT-Integrator jar
  memory: 6                                      # memory allocation, in Gb
  output:                                        # the location of output files
  channel_num: 10                                # number of channels in the multiplex (e.g. 10, 11)
  ref_tag: Bridge                                # unique tag for identifying the reference channel (Bridge sample added to each multiplex)
  groupby: -1                                    # level of data summarization(0: PSM aggregation to the gene level; 1: protein; 2: peptide sequence; 3: PTM site; -1: generate reports at all levels)
  psm_norm: false                                # perform additional retention time-based normalization at the PSM level
  outlier_removal: true                          # perform outlier removal
  prot_norm: -1                                  # normalization (0: None; 1: MD (median centering); 2: GN (median centering + variance scaling); -1: generate reports with all normalization options)
  min_pep_prob: 0.9                              # minimum PSM probability threshold (in addition to FDR-based filtering by Philosopher)
  min_purity: 0.5                                # ion purity score threshold
  min_percent: 0.05                              # remove low intensity PSMs (e.g. value of 0.05 indicates removal of PSMs with the summed TMT reporter ions intensity in the lowest 5% of all PSMs)
  unique_pep: false                              # allow PSMs with unique peptides only (if true) or unique plus razor peptides (if false), as classified by Philosopher and defined in PSM.tsv files
  unique_gene: 0                                 # additional, gene-level uniqueness filter (0: allow all PSMs; 1: remove PSMs mapping to more than one GENE with evidence of expression in the dataset; 2:remove all PSMs mapping to more than one GENE in the fasta file)
  best_psm: true                                 # keep the best PSM only (highest summed TMT intensity) among all redundant PSMs within the same LC-MS run
  prot_exclude: none                             # exclude proteins with specified tags at the beginning of the accession number (e.g. none: no exclusion; sp|,tr| : exclude protein with sp| or tr|)
  allow_overlabel: true                          # allow PSMs with TMT on S (when overlabeling on S was allowed in the database search)
  allow_unlabeled: true                          # allow PSMs without TMT tag or acetylation on the peptide n-terminus 
  mod_tag: none                                  # PTM info for generation of PTM-specific reports (none: for Global data; S[167],T[181],Y[243]: for Phospho; K[170]: for K-Acetyl)
  min_site_prob: -1                              # site localization confidence threshold (-1: for Global; 0: as determined by the search engine; above 0 (e.g. 0.75): PTMProphet probability, to be used with phosphorylation only)
  ms1_int: true                                  # use MS1 precursor ion intensity (if true) or MS2 summed TMT reporter ion intensity (if false) as part of the reference sample abundance estimation 
  top3_pep: true                                 # use top 3 most intense peptide ions as part of the reference sample abundance estimation
  print_RefInt: false                            # print individual reference sample abundance estimates for each multiplex in the final reports (in addition to the combined reference sample abundance estimate)
  add_Ref: -1                                    # add an artificial reference channel if there is no reference channel (-1: don't add the reference; 0: use summation as the reference; 1: use average as the reference; 2: use median as the reference)
  max_pep_prob_thres: 0                          # the threshold for maximum peptide probability
  min_ntt: 0                                     # minimum allowed number of enzymatic termini
  aggregation_method: 0                          # the aggregation method from the PSM level to the specified level (0: median, 1: weighted-ratio)

[prvst]

Since it's a new version, you need to remove the .meta folders from your workspace and start from scratch. I also advise you to generate a new parameter file because it contains changes.

[Bin-cc]

Hi,

I tried to use the parameter file obtained from v4.1.0 windows.zip extracted and then modified it based on previous experience, but he kept reporting errors at the PeptideProphet stage.

INFO[11:03:18] Running the validation and inference on E:\A1BG\A1BG_1
INFO[11:03:18] Executing PeptideProphet on E:\A1BG\A1BG_1
panic: runtime error: index out of range [0] with length 0

goroutine 1 [running]:
philosopher/lib/ext/peptideprophet.interactParser(0xc01ee341e0, 0x59, 0xc01ee34240, 0x58, 0xc01ee342a0, 0x5f, 0xc01ee341e0, 0x59, 0xc01ee34240, 0x58, ...)
        /workspace/philosopher/lib/ext/peptideprophet/peptideprophet.go:216 +0x22d4
philosopher/lib/ext/peptideprophet.PeptideProphet.Execute(0xc01ee341e0, 0x59, 0xc01ee34240, 0x58, 0xc01ee342a0, 0x5f, 0xc01ee341e0, 0x59, 0xc01ee34240, 0x58, ...)
        /workspace/philosopher/lib/ext/peptideprophet/peptideprophet.go:123 +0x1f0
philosopher/lib/ext/peptideprophet.Run(0xc0000ca090, 0x24, 0xc00001a0d8, 0x12, 0xc000024140, 0x46, 0xc0000ca0c0, 0x21, 0xc00001a0f0, 0x18, ...)
        /workspace/philosopher/lib/ext/peptideprophet/peptideprophet.go:70 +0x508
philosopher/lib/pip.PeptideProphet(0xc000178000, 0x24, 0xc01aaae110, 0x7, 0xc0000c80a0, 0x46, 0xc00025a018, 0x16, 0xc01aaae130, 0xd, ...)
        /workspace/philosopher/lib/pip/pip.go:284 +0x638
philosopher/cmd.glob..func13(0x906cf80, 0xc0000b20c0, 0x2, 0x4)
        /workspace/philosopher/cmd/pipeline.go:85 +0x1025
github.com/spf13/cobra.(*Command).execute(0x906cf80, 0xc0000b2080, 0x4, 0x4, 0x906cf80, 0xc0000b2080)
        /home/prvst/go/pkg/mod/github.com/spf13/[email protected]/command.go:844 +0x2c2
github.com/spf13/cobra.(*Command).ExecuteC(0x906da00, 0xdbbb01, 0x0, 0x0)
        /home/prvst/go/pkg/mod/github.com/spf13/[email protected]/command.go:945 +0x336
github.com/spf13/cobra.(*Command).Execute(...)
        /home/prvst/go/pkg/mod/github.com/spf13/[email protected]/command.go:885
philosopher/cmd.Execute()
        /workspace/philosopher/cmd/root.go:35 +0x34
main.main()
        /workspace/philosopher/main.go:22 +0x75

If I use the previous parameter file I can continue to run it until I finally get the error above.

Could you please provide the latest files of philosopher.yml and comet.params suitable for AP-MS, as here? So that I can determine if the problem is a misconfiguration of my parameters. Thank you.

Best wishes,
Bin

[sarah-haynes]

Hi Bin, we are still looking into this. You can try changing extension: pep.xml in the Peptide Validation section if it was set to pepXML in the last .yml file used above, but you may still run into the .meta/razor.bin issue. Thank you for your patience!

Sarah

[Bin-cc]

Hi Haynes,

Thank you and your team for your hard work, and it really makes me feel more confident about philosopher. I have changed extension: pepXML to pep.xml, but the result is as you predicated, there is still the .meta/razor.bin problem.

It does seem to be a difficult problem to solve. Thank you for your dedication. I will continue to follow the latest developments.

Best regards,
Bin

[Bin-cc]

Hi prvst,

I not sure whether your group have fixed the bug above, but I recently ran philosopher 4.1.1 and also encountered same problem. Are there any methods for me to solve it?

Thanks.

Best,
Bin

[prvst]

Hi Bin-cc, could you set Protein Inference: no, and try again using the latest version?

[Bin-cc]

Hi,

I have followded your suggestion, but unfortunately, it doesn't work. The same problem as follows. What detail have I ignored? Thanks.

Best,
Bin

Computing 4226 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
Calculating sensitivity...and error tables...
Computing MU for 14387 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%
INFO: mu=2.83878e-07, db_size=299836695
INFO[10:12:45] Protein inference results                     decoy=20 target=4206
INFO[10:12:45] Converged to 1.03 % FDR with 1549 Proteins    decoy=16 threshold=0.9197 total=1565
2022/01/04 10:12:46 RemoveAll .: invalid argument

[prvst]

@Bin-cc, I'm creating an issue ticket from this thread, it's easier for us to keep up with the conversation.

[prvst]

Please see #277 for the results from your search.