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vdemichev / DiaNN

DIA-NN - a universal automated software suite for DIA proteomics data analysis.
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Fragment vs Precursor quantification #902

Open estelle0530 opened 10 months ago

estelle0530 commented 10 months ago

Hi Vlad,

I ran DIA-NN with the following argument on linux with immunepeptidome mzML input. We aimed to profile individual 9-mer peptide which is generated in the 9mer.fa input fasta file. Since we already performed in silico digestion, cut parameter was disabled.

Since we wanted to preserve the most output possible, we set the q value threshold to be least stringent - I realized that though matrix-qvalue was set to 1, it was automatically adjusted to 0.05 in the log.

When I looked at the output, all 'Protein.Group', 'Protein.Ids', 'Protein.Names', 'Genes', 'PG.Quantity', 'PG.Normalised', 'PG.MaxLFQ', 'Genes.Quantity', 'Genes.Normalised', 'Genes.MaxLFQ', 'Genes.MaxLFQ.Unique'Precursor.Quantity and Precursor.Normalised were 0 across samples and peptides. However, Fragment.Quant.Corrected and Fragment.Quant.Raw were non-zero.

I suspected that this could have been a result of no precursor was identified below 0.05 FDR threshold, but we were able to detect significant peptides matches using Spectronaut.

After multiple tries that gave us this result, we would like to know

  1. How do we recover precursor levels from fragment quantification ?
  2. Can this inconsistency be solved by adjusting the commands ?
  3. What may be potential errors on our part that can be changed to enable significant PSM?

diann --f test.mzML --fasta 9mer.fa --gen-spec-lib --matrices --met-excision --fasta-search --cut
-out-lib library.tsv --out output.tsv --var-mods 1 ---lib --reannotate --qvalue 1 --matrix-ch-qvalue 1 --matrix-qvalue 1 --matrix-tr-qvalue 1 --matrix-spec-q 1 --mass-acc 10 --mass-acc-ms1 10 --smart-profiling --verbose 3 --report-lib-info --matrices --var-mod UniMod:1,42.010565,*n --monitor-mod UniMod:1 --relaxed-prot-inf --smart-profiling --peak-center --no-ifs-removal --min-pep-len 7 --max-pep-len 25 --min-pr-charge 1 --max-pr-charge 4

Thank you for your time in advance!

Best, Estelle

vdemichev commented 10 months ago

Hi Estelle,

The problem is probably too few peptides being searched. A single one? Also, please use the main report, not the matrices.

I would suggest to add FASTAs with anything potentially detectable.

If you need to apply different digests to different FASTAs, this is also possible, please let me know if this is the case & I will detail how.

Best, Vadim