Spectral Library required for DIA-PASEF data analysis

[Kingswood79]

I have some DIA-PASEF (TimsTOF Pro2) runs I'd like to analyze, using a human microbiome FASTA database (>3 GB). DIA-NN failed because of the big DB size and it was recommended to me to try MDFragger-DIA in Fragpipe.
I'm using the 'DIA_SpecLib_Quant_diaPASEF' workflow and load my DIA-PASEF runs and the FASTA DB, however Fragpipe returns that I need to provide the Spectral Library in DIA-NN. I don't have a SPectral Library, the idea is to have MSFRagger create the in silico Spectral Library from the FASTA file.

[fcyu]

Could you share the log file?

Thanks,

Fengchao

[Kingswood79]

There is no log file, since Fragpipe won't start at all.

[fcyu]

Could you set your .d to DIA data type, and try again?

Thanks,

Fengchao

[Kingswood79]

Yeah I already did just after sending my reply and indeed that works (for now)... I'm sorry.. :-/
log_2025-01-09_15-33-10.txt
Thanks!!

[fcyu]

No worries. Let us know if you have any questions in the future.

Best,

Fengchao

[Kingswood79]

log_2025-01-09_17-23-28.txt
DB is too big,

Splitting doesn't work:

[fcyu]

Since diaTracer has finished and generated the _diatracer.mzML files successfully, you could load those diatracer.mzML as DDA data type, .d folders as DIA-Quant data type, and then enable the database split.

Best,

Fengchao

[Kingswood79]

log_2025-01-10_17-24-18.txt
Getting closer..

[fcyu]

The error was thrown from MSBooster. Kevin @yangkl96 , could you take a look?

Thanks,

Fengchao

[yangkl96]

Hello,

Could you please open the command prompt ("cmd" in Windows search bar) and enter the following: C:\DIA-NN\1.9.2\DiaNN.exe --lib D:\DATABASES\FragPipeTest_IGC-DB\spectraRT.tsv --predict --threads 19 --strip-unknown-mods --predict-n-frag 100. This will give a better understanding of why DIA-NN crashed during predictions.

Thanks,
Kevin

[Kingswood79]

Microsoft Windows [Version 10.0.22631.4602]
(c) Microsoft Corporation. All rights reserved.

I:>C:\DIA-NN\1.9.2\DiaNN.exe --lib D:\DATABASES\FragPipeTest_IGC-DB\spectraRT.tsv --predict --threads 19 --strip-unknown-mods --predict-n-frag 100
DIA-NN 1.9.2 (Data-Independent Acquisition by Neural Networks)
Compiled on Oct 17 2024 21:58:43
Current date and time: Mon Jan 13 16:16:12 2025
CPU: GenuineIntel 12th Gen Intel(R) Core(TM) i7-12700
SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2
Logical CPU cores: 20
Predicted spectra will be saved in a binary format
Thread number set to 19
WARNING: unrecognised option [--strip-unknown-mods]
Deep learning predictor will predict 100 fragments
WARNING: protein inference is enabled but no FASTA provided - is this intended?

0 files will be processed
[0:00] Loading spectral library D:\DATABASES\FragPipeTest_IGC-DB\spectraRT.tsv
cannot read the file
ERROR: D:\diann\src\diann.cpp: 35486: Cannot load spectral library

I:>

[Kingswood79]

I assume my IGC human gut DB (DOI https://doi.org/10.1038/nbt.2942), might be not optimal.

[yangkl96]

This is the same error as #1852

Could you please download an earlier version of DIA-NN such as 1.9.1 and retry?