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vdemichev / DiaNN

DIA-NN - a universal automated software suite for DIA proteomics data analysis.
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How to load .raw files in CLI? #947

Open tannishthadas opened 6 months ago

tannishthadas commented 6 months ago

I am using this in the command line using the debian package of diann to process my samples.

/usr/diann/1.8.1# ./diann-1.8.1 --f "/home/19012024_PE_10min.raw" --f "/home/19012024_PE_30min.raw" --f "/home/19012024_PE_5min.raw" --f "/home/19012024_PE_UT.raw" --lib "" --threads 4 --verbose 3 --out /home/report.tsv --qvalue 0.01 --matrices --out-lib /home/mouse_uniprot_predicted_library.tsv --gen-spec-lib --predictor --fasta /home/uniprotkb_proteome_UP000000589_2024_01_18.fasta --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --cut K,R --missed-cleavages 1 --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --unimod4 --var-mods 2 --var-mod UniMod:35,15.994915,M --var-mod UniMod:21,42.010565,*n --monitor-mod UniMod:21 --reanalyse --smart-profiling --cut --duplicate-proteins

This is the output:

DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) Compiled on Apr 15 2022 08:45:18 Current date and time: Thu Feb 22 09:56:19 2024 Logical CPU cores: 8 Thread number set to 4 Output will be filtered at 0.01 FDR Precursor/protein x samples expression level matrices will be saved along with the main report A spectral library will be generated Deep learning will be used to generate a new in silico spectral library from peptides provided Library-free search enabled Min fragment m/z set to 200 Max fragment m/z set to 1800 N-terminal methionine excision enabled In silico digest will involve cuts at K,R Maximum number of missed cleavages set to 1 Min peptide length set to 7 Max peptide length set to 30 Min precursor m/z set to 300 Max precursor m/z set to 1800 Min precursor charge set to 1 Max precursor charge set to 4 Cysteine carbamidomethylation enabled as a fixed modification Maximum number of variable modifications set to 2 Modification UniMod:35 with mass delta 15.9949 at M will be considered as variable Modification UniMod:21 with mass delta 42.0106 at *n will be considered as variable A spectral library will be created from the DIA runs and used to reanalyse them; .quant files will only be saved to disk during the first step When generating a spectral library, in silico predicted spectra will be retained if deemed more reliable than experimental ones Duplicate proteins in FASTA files will not be skipped WARNING: mass mismatch for the modification UniMod:21: 42.0106 specified, but DIA-NN has 79.9663 recorded. Consider using --clear-mods to make DIA-NN forget all modifications it knows or, alternatively, use a different name for the modification in combination with --original-mods DIA-NN will optimise the mass accuracy automatically using the first run in the experiment. This is useful primarily for quick initial analyses, when it is not yet known which mass accuracy setting works best for a particular acquisition scheme. Exclusion of fragments shared between heavy and light peptides from quantification is not supported in FASTA digest mode - disabled; to enable, generate an in silico predicted spectral library and analyse with this library The following variable modifications will be scored: UniMod:21

4 files will be processed [0:00] Loading FASTA /home/uniprotkb_proteome_UP000000589_2024_01_18.fasta [0:00] Processing FASTA [0:00] Assembling elution groups [0:00] 20782 precursors generated [0:00] Gene names missing for some isoforms [0:00] Library contains 982 proteins, and 872 genes [0:00] Encoding peptides for spectra and RTs prediction WARNING: skipping 8820 precursors, unrecognised modifications? If these precursors are important, consider forcing prediction of spectra/RTs/IMs for these precursors anyway, using the --strip-unknown-mods option [0:01] Predicting spectra and IMs Predictions generated: 6011 [0:17] Predicting RTs Predictions generated: 3403 [0:18] Decoding predicted spectra and IMs [0:18] Decoding RTs [0:18] Saving the library to /home/mouse_uniprot_predicted_library.predicted.speclib [0:18] Initialising library

[0:18] First pass: generating a spectral library from DIA data [0:18] File #1/4 [0:18] Loading run /home/19012024_PE_10min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:18] 0 library precursors are potentially detectable [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Using MS1 mass accuracy: 20 ppm [0:18] Using mass accuracy: 20 ppm [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Removing low confidence identifications [0:18] Searching PTM decoys [0:18] Removing interfering precursors [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Too few confident identifications, neural networks will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Calculating protein q-values [0:18] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:18] Quantification [0:18] Scoring PTMs [0:18] Quantification information saved to /home/19012024_PE_10min.raw.quant.

[0:18] File #2/4 [0:18] Loading run /home/19012024_PE_30min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:18] 0 library precursors are potentially detectable [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Using MS1 mass accuracy: 20 ppm [0:18] Using mass accuracy: 20 ppm [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Removing low confidence identifications [0:18] Searching PTM decoys [0:18] Removing interfering precursors [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Too few confident identifications, neural networks will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Calculating protein q-values [0:18] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:18] Quantification [0:18] Scoring PTMs [0:18] Quantification information saved to /home/19012024_PE_30min.raw.quant.

[0:18] File #3/4 [0:18] Loading run /home/19012024_PE_5min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:18] 0 library precursors are potentially detectable [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Using MS1 mass accuracy: 20 ppm [0:18] Using mass accuracy: 20 ppm [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Removing low confidence identifications [0:18] Searching PTM decoys [0:18] Removing interfering precursors [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Too few confident identifications, neural networks will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Calculating protein q-values [0:18] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:18] Quantification [0:18] Scoring PTMs [0:18] Quantification information saved to /home/19012024_PE_5min.raw.quant.

[0:18] File #4/4 [0:18] Loading run /home/19012024_PE_UT.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:18] 0 library precursors are potentially detectable [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Using MS1 mass accuracy: 20 ppm [0:18] Using mass accuracy: 20 ppm [0:18] Processing [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:18] Precursor search [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Removing low confidence identifications [0:18] Searching PTM decoys [0:18] Removing interfering precursors [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Optimising weights WARNING: too few training precursors, classifier will not be used [0:18] Too few confident identifications, neural networks will not be used [0:18] Calculating q-values [0:18] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:18] Calibrating retention times [0:18] Calculating protein q-values [0:18] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:18] Quantification [0:18] Scoring PTMs [0:18] Quantification information saved to /home/19012024_PE_UT.raw.quant.

ERROR: DIA-NN tried but failed to load the following files: /home/19012024_PE_10min.raw, /home/19012024_PE_30min.raw, /home/19012024_PE_5min.raw, /home/19012024_PE_UT.raw [0:18] Cross-run analysis [0:18] Reading quantification information: 4 files [0:18] Quantifying peptides WARNING: not enough peptides for normalisation [0:18] Assembling protein groups [0:18] Quantifying proteins [0:18] Calculating q-values for protein and gene groups [0:18] Calculating global q-values for protein and gene groups [0:18] Writing report [0:18] Report saved to /home/report-first-pass.tsv. [0:18] Saving precursor levels matrix [0:18] Precursor levels matrix (1% precursor and protein group FDR) saved to /home/report-first-pass.pr_matrix.tsv. [0:18] Saving protein group levels matrix [0:18] Saving gene group levels matrix [0:18] Saving unique genes levels matrix [0:18] Stats report saved to /home/report-first-pass.stats.tsv [0:18] Generating spectral library: [0:18] 0 precursors passing the FDR threshold are to be extracted [0:18] Saving spectral library to /home/mouse_uniprot_predicted_library.tsv [0:18] 0 precursors saved [0:18] Loading the generated library and saving it in the .speclib format [0:18] Loading spectral library /home/mouse_uniprot_predicted_library.tsv [0:18] Spectral library loaded: 0 protein isoforms, 0 protein groups and 0 precursors in 1 elution groups. [0:18] Loading protein annotations from FASTA /home/uniprotkb_proteome_UP000000589_2024_01_18.fasta [0:19] Library contains 0 proteins, and 0 genes [0:19] Saving the library to /home/mouse_uniprot_predicted_library.tsv.speclib

[0:19] Second pass: using the newly created spectral library to reanalyse the data [0:19] File #1/4 [0:19] Loading run /home/19012024_PE_10min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:19] 0 library precursors are potentially detectable [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Using MS1 mass accuracy: 20 ppm [0:19] Using mass accuracy: 20 ppm [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Removing low confidence identifications [0:19] Searching PTM decoys [0:19] Removing interfering precursors [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Too few confident identifications, neural networks will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Calculating protein q-values [0:19] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:19] Quantification [0:19] Scoring PTMs

[0:19] File #2/4 [0:19] Loading run /home/19012024_PE_30min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:19] 0 library precursors are potentially detectable [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Using MS1 mass accuracy: 20 ppm [0:19] Using mass accuracy: 20 ppm [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Removing low confidence identifications [0:19] Searching PTM decoys [0:19] Removing interfering precursors [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Too few confident identifications, neural networks will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Calculating protein q-values [0:19] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:19] Quantification [0:19] Scoring PTMs

[0:19] File #3/4 [0:19] Loading run /home/19012024_PE_5min.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:19] 0 library precursors are potentially detectable [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Using MS1 mass accuracy: 20 ppm [0:19] Using mass accuracy: 20 ppm [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Removing low confidence identifications [0:19] Searching PTM decoys [0:19] Removing interfering precursors [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Too few confident identifications, neural networks will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Calculating protein q-values [0:19] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:19] Quantification [0:19] Scoring PTMs

[0:19] File #4/4 [0:19] Loading run /home/19012024_PE_UT.raw Thermo RAW file format not supported. Thermo RAW file format not supported. Thermo RAW file format not supported. No MS2 spectra: aborting ERROR: cannot load the file, skipping [0:19] 0 library precursors are potentially detectable [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Recalibrating with mass accuracy 0.0001, 3e-05 (MS2, MS1) [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Using MS1 mass accuracy: 20 ppm [0:19] Using mass accuracy: 20 ppm [0:19] Processing [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Restoring classifier and weights to 1 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 0 0 0 0 || 0 0 0 0 0 0 [0:19] Precursor search [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Removing low confidence identifications [0:19] Searching PTM decoys [0:19] Removing interfering precursors [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Optimising weights WARNING: too few training precursors, classifier will not be used [0:19] Too few confident identifications, neural networks will not be used [0:19] Calculating q-values [0:19] Number of IDs at 50%, 5%, 1%, 0.1% FDR: 0, 0, 0, 0 [0:19] Calibrating retention times [0:19] Calculating protein q-values [0:19] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [0:19] Quantification [0:19] Scoring PTMs

ERROR: DIA-NN tried but failed to load the following files: /home/19012024_PE_10min.raw, /home/19012024_PE_30min.raw, /home/19012024_PE_5min.raw, /home/19012024_PE_UT.raw [0:19] Cross-run analysis [0:19] Reading quantification information: 4 files [0:19] Quantifying peptides WARNING: not enough peptides for normalisation [0:19] Quantifying proteins [0:19] Calculating q-values for protein and gene groups [0:19] Calculating global q-values for protein and gene groups [0:19] Writing report [0:19] Report saved to /home/report.tsv. [0:19] Saving precursor levels matrix [0:19] Precursor levels matrix (1% precursor and protein group FDR) saved to /home/report.pr_matrix.tsv. [0:19] Saving protein group levels matrix [0:19] Saving gene group levels matrix [0:19] Saving unique genes levels matrix [0:19] Stats report saved to /home/report.stats.tsv [0:19] Log saved to /home/report.log.txt Finished

Please help me about how to read the .raw files here? Is there an alternative to thermo ms file reader?

vdemichev commented 6 months ago

Thermo RAW files are not supported by the native Linux build. Can either

an-altosian commented 2 months ago

Hello @vdemichev , I tried to use Wine 7.8 to run DIA-NN on .raw files. I downloaded the specific version of MSFileRead and invokedwine MSFileReader_x86_x64.exe. Then I used DIA-NN to process my data. The library construction step worked fine, but once it touched the samples, I got 

Could not read Thermo RAW file. The Thermo .dll likely was not loaded or out of date.

So, how to install MSFileReader so that DIA-NN can find and use it?

an-altosian commented 2 months ago

Seems like MSFileReader has to be installed via their GUI. It did not work from command line.