The maximum variable modification option in diann is not working.

Dear developers, I am unsure why the maximum variable modification option is not working. Do you have any suggestions? I am also not getting any results when running the software. What could be the reasons behind these two issues? diann diann

PS. My software is installed on the Win11 platform. DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) Compiled on Apr 14 2022 15:31:19 Current date and time: Thu Apr 18 09:39:02 2024 CPU: GenuineIntel 13th Gen Intel(R) Core(TM) i5-13600K SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2 Logical CPU cores: 20 diann.exe --f E:\MS\DIA-NN\Hela\3-1.raw --lib --threads 10 --verbose 1 --out E:\MS\DIA-NN\Hela\report.tsv --qvalue 0.01 --matrices --out-lib C:\DIA-NN\1.8.1\report-lib.tsv --gen-spec-lib --predictor --fasta E:\MS\DIA-NN\Hela\hg38.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 --reanalyse --relaxed-prot-inf --smart-profiling --pg-level 1 --peak-center --no-ifs-removal

Thread number set to 10 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 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 Highly heuristic protein grouping will be used, to reduce the number of protein groups obtained; this mode is recommended for benchmarking protein ID numbers; use with caution for anything else When generating a spectral library, in silico predicted spectra will be retained if deemed more reliable than experimental ones Implicit protein grouping: protein names; this determines which peptides are considered 'proteotypic' and thus affects protein FDR calculation Fixed-width center of each elution peak will be used for quantification Interference removal from fragment elution curves disabled 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 WARNING: MBR turned off, two or more raw files are required

1 files will be processed [0:00] Loading FASTA E:\MS\DIA-NN\Hela\hg38.fasta [0:02] Processing FASTA [0:06] Assembling elution groups [0:10] 4297569 precursors generated [0:10] Gene names missing for some isoforms [0:10] Library contains 20570 proteins, and 20319 genes [0:10] [0:15] [17:58] [21:39] [21:51] [21:54] Saving the library to C:\DIA-NN\1.8.1\report-lib.predicted.speclib [22:01] Initialising library

[22:03] File #1/1 [22:03] Loading run E:\MS\DIA-NN\Hela\3-1.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [22:03] 0 library precursors are potentially detectable [22:03] Processing... [22:03] Using MS1 mass accuracy: 20 ppm [22:03] Using mass accuracy: 20 ppm [22:03] Removing low confidence identifications [22:03] Removing interfering precursors [22:03] Too few confident identifications, neural networks will not be used [22:03] Number of IDs at 0.01 FDR: 0 [22:03] Calculating protein q-values [22:03] Number of proteins identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [22:03] Quantification [22:03] Quantification information saved to E:\MS\DIA-NN\Hela\3-1.raw.quant.

ERROR: DIA-NN tried but failed to load the following files: E:\MS\DIA-NN\Hela\3-1.raw [22:03] Cross-run analysis [22:03] Reading quantification information: 1 files [22:03] Quantifying peptides [22:03] Assembling protein groups [22:06] Quantifying proteins [22:06] Calculating q-values for protein and gene groups [22:06] Calculating global q-values for protein and gene groups [22:06] Writing report [22:06] Report saved to E:\MS\DIA-NN\Hela\report.tsv. [22:06] Saving precursor levels matrix [22:06] Precursor levels matrix (1% precursor and protein group FDR) saved to E:\MS\DIA-NN\Hela\report.pr_matrix.tsv. [22:06] Saving protein group levels matrix [22:06] Saving gene group levels matrix [22:06] Saving unique genes levels matrix [22:06] Stats report saved to E:\MS\DIA-NN\Hela\report.stats.tsv [22:06] Generating spectral library: [22:06] 0 precursors passing the FDR threshold are to be extracted [22:06] Saving spectral library to C:\DIA-NN\1.8.1\report-lib.tsv [22:06] 0 precursors saved [22:06] Loading the generated library and saving it in the .speclib format [22:06] Loading spectral library C:\DIA-NN\1.8.1\report-lib.tsv [22:06] Spectral library loaded: 0 protein isoforms, 0 protein groups and 0 precursors in 1 elution groups. [22:06] Loading protein annotations from FASTA E:\MS\DIA-NN\Hela\hg38.fasta [22:07] Library contains 0 proteins, and 0 genes [22:07] Saving the library to C:\DIA-NN\1.8.1\report-lib.tsv.speclib

Finished

vdemichev / DiaNN

The maximum variable modification option in diann is not working. #994