Help! No search data - Githubissues

I am trying to direct-search HeLa raw file in DIA-NN 18. There is no searched data at the end. Below is the report.log which says [25:57] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw No MS2 spectra: aborting This HeLa raw files are searched using Spectronaut 1.8 and PD, so the raw file is good condition. I also re-install DIA-NN and -re-searched it, but no luck. Can anyone help me what's going on in my DIA-NN search? Thanks, Jae jae.choi@thermofisher.com

DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) Compiled on Apr 14 2022 15:31:19 Current date and time: Wed Oct 4 15:54:15 2023 CPU: GenuineIntel Intel(R) Xeon(R) W-2235 CPU @ 3.80GHz SIMD instructions: AVX AVX2 AVX512CD AVX512F FMA SSE4.1 SSE4.2 Logical CPU cores: 12 diann.exe --f D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw --f D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R2.raw --f D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R3.raw --lib --threads 6 --verbose 1 --out D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report.tsv --qvalue 0.01 --matrices --out-lib D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-lib.tsv --gen-spec-lib --predictor --fasta D:\uniprot_human (20426 reviewed)_2023_10_04.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 --peak-center --no-ifs-removal

Thread number set to 6 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 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

3 files will be processed [0:00] Loading FASTA D:\uniprot_human (20426 reviewed)_2023_10_04.fasta [0:03] Processing FASTA [0:10] Assembling elution groups [0:16] 4296496 precursors generated [0:16] Gene names missing for some isoforms [0:16] Library contains 20402 proteins, and 20187 genes [0:16] [0:24] [22:38] [25:31] [25:44] [25:47] Saving the library to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-lib.predicted.speclib [25:54] Initialising library

[25:57] First pass: generating a spectral library from DIA data [25:57] File #1/3 [25:57] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [25:57] 0 library precursors are potentially detectable [25:57] Processing... [25:57] Using MS1 mass accuracy: 20 ppm [25:57] Using mass accuracy: 20 ppm [25:57] Removing low confidence identifications [25:57] Removing interfering precursors [25:57] Too few confident identifications, neural networks will not be used [25:57] Number of IDs at 0.01 FDR: 0 [25:57] Calculating protein q-values [25:57] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [25:57] Quantification [25:57] Quantification information saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw.quant.

[25:57] File #2/3 [25:57] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R2.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [25:57] 0 library precursors are potentially detectable [25:57] Processing... [25:57] Using MS1 mass accuracy: 20 ppm [25:57] Using mass accuracy: 20 ppm [25:57] Removing low confidence identifications [25:57] Removing interfering precursors [25:57] Too few confident identifications, neural networks will not be used [25:57] Number of IDs at 0.01 FDR: 0 [25:57] Calculating protein q-values [25:57] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [25:57] Quantification [25:57] Quantification information saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R2.raw.quant.

[25:57] File #3/3 [25:57] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R3.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [25:57] 0 library precursors are potentially detectable [25:57] Processing... [25:57] Using MS1 mass accuracy: 20 ppm [25:57] Using mass accuracy: 20 ppm [25:57] Removing low confidence identifications [25:57] Removing interfering precursors [25:57] Too few confident identifications, neural networks will not be used [25:57] Number of IDs at 0.01 FDR: 0 [25:57] Calculating protein q-values [25:57] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [25:57] Quantification [25:57] Quantification information saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R3.raw.quant.

ERROR: DIA-NN tried but failed to load the following files: D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw, D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R2.raw, D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R3.raw [25:57] Cross-run analysis [25:57] Reading quantification information: 3 files [25:57] Quantifying peptides WARNING: not enough peptides for normalisation [25:57] Assembling protein groups [25:59] Quantifying proteins [26:00] Calculating q-values for protein and gene groups [26:00] Calculating global q-values for protein and gene groups [26:00] Writing report [26:00] Report saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-first-pass.tsv. [26:00] Saving precursor levels matrix [26:00] Precursor levels matrix (1% precursor and protein group FDR) saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-first-pass.pr_matrix.tsv. [26:00] Saving protein group levels matrix [26:00] Saving gene group levels matrix [26:00] Saving unique genes levels matrix [26:00] Stats report saved to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-first-pass.stats.tsv [26:00] Generating spectral library: [26:00] 0 precursors passing the FDR threshold are to be extracted [26:00] Saving spectral library to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-lib.tsv [26:00] 0 precursors saved [26:00] Loading the generated library and saving it in the .speclib format [26:00] Loading spectral library D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-lib.tsv [26:00] Spectral library loaded: 0 protein isoforms, 0 protein groups and 0 precursors in 1 elution groups. [26:00] Loading protein annotations from FASTA D:\uniprot_human (20426 reviewed)_2023_10_04.fasta [26:00] Library contains 0 proteins, and 0 genes [26:00] Saving the library to D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\DIANN\report-lib.tsv.speclib

[26:03] Second pass: using the newly created spectral library to reanalyse the data [26:03] File #1/3 [26:03] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [26:03] 0 library precursors are potentially detectable [26:03] Processing... [26:03] Using MS1 mass accuracy: 20 ppm [26:03] Using mass accuracy: 20 ppm [26:03] Removing low confidence identifications [26:03] Removing interfering precursors [26:03] Too few confident identifications, neural networks will not be used [26:03] Number of IDs at 0.01 FDR: 0 [26:03] Calculating protein q-values [26:03] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [26:03] Quantification

[26:03] File #2/3 [26:03] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R2.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [26:03] 0 library precursors are potentially detectable [26:03] Processing... [26:03] Using MS1 mass accuracy: 20 ppm [26:03] Using mass accuracy: 20 ppm [26:03] Removing low confidence identifications [26:03] Removing interfering precursors [26:03] Too few confident identifications, neural networks will not be used [26:03] Number of IDs at 0.01 FDR: 0 [26:03] Calculating protein q-values [26:03] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [26:03] Quantification

[26:03] File #3/3 [26:03] Loading run D:\Jae_DIA_practice\1_200 ng HeLa digest (3 files)\200ngHeLa_VelocityDIA_OE480_FAIMS_CV45_R3.raw No MS2 spectra: aborting ERROR: cannot load the file, skipping [26:03] 0 library precursors are potentially detectable [26:03] Processing... [26:03] Using MS1 mass accuracy: 20 ppm [26:03] Using mass accuracy: 20 ppm [26:03] Removing low confidence identifications [26:03] Removing interfering precursors [26:03] Too few confident identifications, neural networks will not be used [26:03] Number of IDs at 0.01 FDR: 0 [26:03] Calculating protein q-values [26:03] Number of genes identified at 1% FDR: 0 (precursor-level), 0 (protein-level) (inference performed using proteotypic peptides only) [26:03] Quantification

Finished

vdemichev / DiaNN

Help! No search data #836