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vdemichev / DiaNN

DIA-NN - a universal automated software suite for DIA proteomics data analysis.
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Seeking beginner help regarding DIA-NN library free analysis #1146

Open bluemooninvestor opened 1 month ago

bluemooninvestor commented 1 month ago

First of all, my apologies for asking this simple thing again. However, I am little confused despite reading the manual and talking to Prof Vadim once. It is mentioned in the readme, that it is preferable to do the library free search in two steps. However, the steps are a little unclear to me. Here is how I am doing it. Please let me know if this is correct or any changes are needed.

DIANN1

In the first step, I am selecting only the FASTA file and nothing else (not even RAW files). What should I click to generate the library in this step.

1) Do I click the "FASTA digest for library free search" button or the "Generate Spectra library" button. 2) Do I need to untick the quantities matrices button?

Further, if I have made the library in the first step using just the FASTA

DIANN2

How do I proceed further? What I am doing is that I am selecting the following:

1) RAW file 2) Library generated in step 1 3) FASTA file (do I need to select this at this step?? or is it not necessary since it has been already used for the predicted library?)

What should I click after selecting these 3 files?

DIANN3

1) Should I click the "Generate Spectral Library" again? 2) Should I leave out (untick) two options under "Precursor ion generation" ie "FASTA digest..." and "Deep Learning..." 3) What else do I need to click, if any?

I am sorry if this is silly, but for a beginner all this is quite confusing because the library is generated and regenerated in so many steps, and the readme does not explictly mention the steps for Two-step analysis.

Thank you

vdemichev commented 1 month ago

Hi,

  1. Both get clicked automatically.
  2. Irrelevant.

1 - 3: yes, correct, also select FASTA.

  1. It will get clicked automatically when you select MBR.
  2. Yes, these must not be ticked.

Best, Vadim

bluemooninvestor commented 1 month ago

Thank you very much! Regarding my first query, "Do I click the "FASTA digest for library free search" button or the "Generate Spectra library" button.", neither of these actually get clicked automatically when I select the FASTA (as you can see in the first image). But I will manually select them as you instructed.

Thanks