Clarification regarding DIA-NN 2.6.1 warning during predicted library search

[d22016]

Hi Demichev,
Warnings_DIANN.docx

I am analyzing my DIA proteomics data using DIA-NN 2.6.1 and would like to clarify an issue regarding the predicted spectral library workflow. I first performed a FASTA-only run using the human UniProt FASTA database and generated the predicted spectral library:

report-lib.predicted.speclib

For the subsequent analysis, I selected this .speclib file and loaded SCIEX ZenoTOF 7600+ .wiff DIA files, without specifying the FASTA file.

However, DIA-NN gives the following warnings:

WARNING: it is recommended to specify a FASTA database and

WARNING: incorrect settings, the in silico-predicted library must be generated in a separate pipeline step and then used to process the raw data

The command generated by DIA-NN for the second run contains:

--lib ...\report-lib.predicted.speclib

but also contains:

--gen-spec-lib --predictor

Therefore, DIA-NN appears to be attempting to generate an in-silico library again despite the previously generated .speclib being supplied.

Could you please advise which GUI settings should be selected in DIA-NN 2.6.1?

[vdemichev]

Hi,

WARNING: it is recommended to specify a FASTA database and

Correct, FASTA needs to be present.

WARNING: incorrect settings, the in silico-predicted library must be generated in a separate pipeline step and then used to process the raw data

Incorrect 'Mode' setting.

Best,
Vadim

[d22016]

Thanks.
I selected the mode: Prediction from FASTA and supplied the previously generated report-lib.predicted.speclib.

However, I am getting the following warning:

WARNING: it is recommended to specify a FASTA database

Could you please clarify which setting is causing the FASTA warning?

[vdemichev]

That's incorrect Mode setting for the second step. See https://github.com/vdemichev/DiaNN#getting-started-with-dia-nn

WARNING: it is recommended to specify a FASTA database

Not sure, how is this warning not clear? It means FASTA database needs to be specified in the respective field.

[d22016]

Thanks Vadim. I was able to resolve the issue.
I have a doubt regarding the parameter - scan window, what does the scan window actually mean? and what type of value it holds?

[vdemichev]

Internal parameter of DIA-NN which should be set to ~average number of dp/peak (whole peak, not FWHM).