DiaNN exits without searching as it tries to load library

[bobbyg198a]

Dear Vadim, I hope you are well. I have used your software successfully many times for SWATH data. This is my first time using it for diaPASEF data. I initially loaded it up with variable mods in the additional options box and it exited after about an hour at the loading fasta step. "--var-mod UniMod:7,0.984016,NQ” "--var-mod UniMod:35,15.994915,P” "--var-mod UniMod:28,-17.026549,Q” "--var-mod UniMod:27,-18.010565,E”

I re-ran with the additional mods removed, just the options in the GUI. It did the same again, please see below. I have rerun again removing the double pass mode and still no joy. Any help you could provide would be greatly appreciated.

best wishes

Bobby

diann.exe --f "F:\ESKCalc\DIA\240509_ESKCalc_A_70_Min_1.5um_DIA189.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_AA_70_Min_1.5um_DIA217.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_BB_70_Min_1.5um_DIA218.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_C_70_Min_1.5um_DIA190.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_CC_70_Min_1.5um_DIA219.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_D_70_Min_1.5um_DIA191.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_DD_70_Min_1.5um_DIA229.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_E_70_Min_1.5um_DIA192.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_EE_70_Min_1.5um_DIA221.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_F_70_Min_1.5um_DIA193.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_FF_70_Min_1.5um_DIA222.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_G_70_Min_1.5um_DIA194.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_GG_70_Min_1.5um_DIA223.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_H_70_Min_1.5um_DIA195.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_HH_70_Min_1.5um_DIA226.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_I_70_Min_1.5um_DIA196.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_II_70_Min_1.5um_DIA227.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_J_70_Min_1.5um_DIA197.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_JJ_70_Min_1.5um_DIA228.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_K_70_Min_1.5um_DIA198.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_L_70_Min_1.5um_DIA201.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_M_70_Min_1.5um_DIA202.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_N_70_Min_1.5um_DIA203.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_O_70_Min_1.5um_DIA204.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_P_70_Min_1.5um_DIA205.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_Q_70_Min_1.5um_DIA206.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_R_70_Min_1.5um_DIA207.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_S_70_Min_1.5um_DIA208.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_T_70_Min_1.5um_DIA209.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_U_70_Min_1.5um_DIA210.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_V_70_Min_1.5um_DIA213.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_W_70_Min_1.5um_DIA214.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_X_70_Min_1.5um_DIA215.d " --f "F:\ESKCalc\DIA\C240509_ESKCalc_Z_70_Min_1.5um_DIA216.d " --lib "" --threads 16 --verbose 1 --out "F:\ESKCalc\report.tsv" --qvalue 0.01 --matrices --out-lib "F:\ESKCalc\lib.tsv" --gen-spec-lib --predictor --fasta "F:\ESKCalc\ESKCalc_combined_database.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 1 --var-mod UniMod:35,15.994915,M --var-mod UniMod:1,42.010565,n --monitor-mod UniMod:1 --double-search --reanalyse --smart-profiling --peak-center --no-ifs-removal --global-norm DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) Compiled on Apr 14 2022 15:31:19 Current date and time: Tue May 28 16:08:25 2024 CPU: AuthenticAMD AMD Ryzen Threadripper 1950X 16-Core Processor SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2 SSE4a Logical CPU cores: 32 Thread number set to 16 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 1 Modification UniMod:35 with mass delta 15.9949 at M will be considered as variable Modification UniMod:1 with mass delta 42.0106 at n will be considered as variable Neural networks will be used for peak selection 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 Fixed-width center of each elution peak will be used for quantification Interference removal from fragment elution curves disabled Median-based local normalisation 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 The following variable modifications will be scored: UniMod:1 WARNING: double-pass mode is incompatible with PTM scoring, turned off

34 files will be processed [0:00] Loading FASTA F:\ESKCalc\ESKCalc_combined_database.fasta

DIA-NN exited DIA-NN-plotter.exe "F:\ESKCalc\report.stats.tsv" "F:\ESKCalc\report.tsv" "F:\ESKCalc\report.pdf" PDF report will be generated in the background

[vdemichev]

Hi Bobby,

Seems like some incompatibility with that specific fasta file, i.e. error in processing it. What happens if you don't change any default options in the GUI whatsoever? If still exits, I can maybe take a look at the FASTA file?

Best, Vadim

[bobbyg198a]

Hi Vadim, Thanks for getting back. Still exits with defaults. I do suspect it is the FASTA it has 2.6 million entries and is 9GB, for a metaproteomic analysis. Used it with mascot before but that was for DDA data. Would you have any ideas how DiaNN would be able to handle a database that size. thanks

Bobby

[uresch]

Dear Vadim and bobbyg198a-I was wondering if the hardware used: CPU: AuthenticAMD AMD Ryzen Threadripper 1950X 16-Core Processor has anything to do with this?