no results written

[animesh]

I tried running the latest version over some timsTOF-pro data containing phospho-enriched peptides

diann.exe --f "F:\promec\TIMSTOF\LARS\2024\241002_zrimac\zr_IMAC_100ug_zoom20_1dia_25pepsep_S1-A2_1_8449.d
" --f "F:\promec\TIMSTOF\LARS\2024\241002_zrimac\zr_IMAC_200ug_zoom20_1dia_25pepsep_S1-A5_1_8450.d
" --f "F:\promec\TIMSTOF\LARS\2024\241002_zrimac\zr_IMAC_300ug_zoom20_1dia_25pepsep_S1-A8_1_8451.d
" --lib "" --threads 6 --verbose 5 --out "F:\promec\TIMSTOF\LARS\2024\241002_zrimac\DIANN1p9p1\report.tsv" --qvalue 0.01 --matrices  --out-lib "F:\promec\TIMSTOF\LARS\2024\241002_zrimac\DIANN1p9p1\reportlib.parquet" --gen-spec-lib --predictor --fasta camprotR_240512_cRAP_20190401_full_tags.fasta --cont-quant-exclude cRAP- --fasta "F:\promec\FastaDB\uniprotkb_proteome_UP000005640_2024_04_18.fasta" --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --cut K*,R* --missed-cleavages 1 --unimod4 --var-mods 2 --var-mod UniMod:35,15.994915,M --var-mod UniMod:1,42.010565,*n --var-mod UniMod:21,79.966331,STY --mass-acc 20 --mass-acc-ms1 20 --individual-mass-acc --individual-windows --peptidoforms --relaxed-prot-inf --rt-profiling 

which seems like finished processing witho ut errors phos.log.txt but i could not find the results in the folder it says it wrote the results to? Spec lib was generated and written though 🫡

[vdemichev]

Hi Ani,

DIA-NN ran out of RAM. There's a 1.9.2 release soon, will have ~2x lower memory consumption. For now, please see suggestions for phospho here, this should help: https://github.com/vdemichev/DiaNN?tab=readme-ov-file#ptms-and-peptidoforms.

Also, most importantly, please generate an in silico library in a separate pipeline step, i.e. without any raw files specified.

Best, Vadim

[animesh]

Thanks @vdemichev 💯 i can try to run on higher RAM machine but then it would be linux. Is 1.9.1 working well on Linux and any specific things to consider?

[vdemichev]

Hi Ani,

On Linux please don't use --matrices, otherwise all the same. But you can also just reduce RAM usage by using the recommended settings for phospho.

Best, Vadim

[animesh]

Dear Vadim,

i am trying to create library following the guidelines,

diann.exe --threads 32 --verbose 5 --out "F:\promec\FastaDB\uniprot-human-iso-jan24.report.tsv" --qvalue 0.01 --out-lib "F:\peomec\FastaDB\uniprot-human-iso-jan24.report-lib.parquet" --gen-spec-lib --predictor --fasta "F:\promec\FastaDB\uniprot-human-iso-jan24.fasta" --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 2 --max-pr-charge 3 --cut K*,R* --missed-cleavages 1 --unimod4 --var-mods 3 --var-mod UniMod:35,15.994915,M --var-mod UniMod:1,42.010565,*n --var-mod UniMod:21,79.966331,STY --peptidoforms --relaxed-prot-inf --rt-profiling
DIA-NN 1.9.1 (Data-Independent Acquisition by Neural Networks)
Compiled on Jul 15 2024 15:40:36
Current date and time: Sat Oct 12 09:56:58 2024
CPU: GenuineIntel Intel(R) Xeon(R) CPU E5-2650 0 @ 2.00GHz
SIMD instructions: AVX SSE4.1 SSE4.2
Logical CPU cores: 32
Thread number set to 32
Output will be filtered at 0.01 FDR
A spectral library will be generated
Deep learning will be used to generate a new in silico spectral library from peptides provided
DIA-NN will carry out FASTA digest for in silico lib generation
Min fragment m/z set to 200
Max fragment m/z set to 1800
N-terminal methionine excision enabled
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 2
Max precursor charge set to 3
In silico digest will involve cuts at K*,R*
Maximum number of missed cleavages set to 1
Cysteine carbamidomethylation enabled as a fixed modification
Maximum number of variable modifications set to 3
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
Modification UniMod:21 with mass delta 79.9663 at STY will be considered as variable
Peptidoform scoring enabled
Heuristic protein grouping will be used, to reduce the number of protein groups obtained; this mode is recommended for benchmarking protein ID numbers, GO/pathway and system-scale analyses
The spectral library (if generated) will retain the original spectra but will include empirically-aligned RTs
The following variable modifications will be scored: UniMod:35 UniMod:1 UniMod:21

0 files will be processed
[0:00] Loading FASTA F:\promec\FastaDB\uniprot-human-iso-jan24.fasta
[0:47] Processing FASTA
[6:13] Assembling elution groups
[9:07] 47672221 precursors generated
[9:07] Gene names missing for some isoforms
[9:07] Library contains 82078 proteins, and 20536 genes
...

does it sound right or i am missing some switches to get it right 🫡

BTW is there 1.9.2 already out there to be tested 🤓

[vdemichev]

Min precursor m/z set to 300 Max precursor m/z set to 1800

Is this the experiment range?

Modification UniMod:35 with mass delta 15.9949 at M will be considered as variable

Increases search space.