Can't find my report.tsv file

[Catball79]

Following youtube toturial, I firstly created an new project and moved my "report.tsv" file in it. Running DIAgui, shiny tool appeared. clicking "Select your report file", there is no any file in selection window.

[mgerault]

Hello,

Okay, that's unexpected. On what OS are you running R ? Did you remove the variable WD (which store the path of your working directory) from your R environment ? It seems the shinyFile command cannot detect the path of your working directory.

[Catball79]

Hello,

Okay, that's unexpected. On what OS are you running R ? Did you remove the variable WD (which store the path of your working directory) from your R environment ? It seems the shinyFile command cannot detect the path of your working directory.

Hi, My OS is Windows and I didn't remove WD from my R environment. Thanks for your reply. I have finished the iBAQ quantification with R code, based on the help information of your package. Thank you! I got a new questween. I don't konw whether can I use the iBAQ value to compute the FC between experimental group and control group? Some studies do that.

[mgerault]

Okay, did you try again in another folder or maybe is your working directory protected by a password ? If the problem persist, try to uninstall DIAgui, restart your R session and reinstall it. Right now I don't see a clear cause of the problem.

Yes you can use the iBAQ for the FC calculation as long as you remember you used absolute quantification (iBAQ in that case) for this calculcation.

[Catball79]

Okay, did you try again in another folder or maybe is your working directory protected by a password ? If the problem persist, try to uninstall DIAgui, restart your R session and reinstall it. Right now I don't see a clear cause of the problem.

Yes you can use the iBAQ for the FC calculation as long as you remember you used absolute quantification (iBAQ in that case) for this calculcation.

Thank you for your reply. Yes I have tried to change another disk and got a same question. And there is no file encryption system enabled. Thank you very much.

There is another question based on the iBAQ. In theory, iBAQ is usually used to check the protein abundace in a same sample, LFQ to calculate the difference between several groups. You said I can use the iBAQ value to calculate FC, whether it means the iBAQ value from DIAgui is based on the normalized intensity value (such as RT-dependent). If not, should I take some normalization before FC calculation ?

[mgerault]

What is your R version and shinyFiles package version ? Try to update both and try again; most likely it is just a version problem.

iBAQ (as Top3) in DIAgui, are based on the raw inetnsity, not the normalized one. In theory, absolute quantification is already a form of normalization but you can perform another normalization after, before your FC computation. What I advise is to check your data after the iBAQ calculation with quality control plots like PCA, density, etc. If you see any batch effect or else, apply a normalization.

[Catball79]

What is your R version and shinyFiles package version ? Try to update both and try again; most likely it is just a version problem.

iBAQ (as Top3) in DIAgui, are based on the raw inetnsity, not the normalized one. In theory, absolute quantification is already a form of normalization but you can perform another normalization after, before your FC computation. What I advise is to check your data after the iBAQ calculation with quality control plots like PCA, density, etc. If you see any batch effect or else, apply a normalization.

R version is 4.3.2 and shinyFiles package version is 1.4.3. I'll try later. Thank you.

[mgerault]

Okay so something a bit weird is that the last version of shinyFiles package available on CRAN is 0.9.3 https://cran.r-project.org/web/packages/shinyFiles/index.html Are you sure you downloaded the right package from the right repository ?

[Catball79]

Sorry for the mistake i made. Yes, the shinyFiles is latest.

[mgerault]

And still it's not working after uninstalling and reinstalling everything ?

[Catball79]

I'll send the resolution method once I slove it. Thanks for your guidance.

[Catball79]

Hi, there is a new question when I back to check the warning message. I defined the method "sum", but it still used the max method when I calculate the iBAQ.

[mgerault]

Yes I checked and I forgot to update the warning message but it did use the sum and not the maximum. You can update the package but your data will not change but just the warning message.

[Catball79]

Ok, thank you for your reply🤓

---- Replied Message ---- | From | @.> | | Date | 09/25/2024 17:55 | | To | @.> | | Cc | @.>@.> | | Subject | Re: [mgerault/DIAgui] Can't find my report.tsv file (Issue #5) |

Yes I checked and I forgot to update the warning message but it did use the sum and not the maximum. You can update the package but your data will not change but just the warning message.

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[Catball79]

Hi, new question The raw intensity result from iBAQ calculation proceduer raised my interest. Because the first step is to sum the Precursor.Quantity of the same Protein.Group. First Icalculated the raw intensity result with code as followed: raw <- diann_matrix(report, id.header = "Protein.Group",

• quantity.header = "Precursor.Quantity",
• method = "sum") Then I checked the result by comapring the raw intensity result to the sum value of Precursor.Quantity of a specific P.G ID from the report.tsv. And question occurred.

AOA1W2PQ72 in raw is 96455.7, however in report file, are 67157.90, 29297.80 and 48097.10 separately. Obvious raw value is not the sum value from different precursor, but the sum of 67157.90 and 29297.80.

[mgerault]

Did you check the Q.value and the PG.Q.value of the precursor with intensity 48097.10 ? Remember that the first point of using DIAgui is to refilter the precursors from the report and recompute the quantification. If that precursor didn't fulfill the filtering criteria it was then removed from the sum.

[Catball79]

Did you check the Q.value and the PG.Q.value of the precursor with intensity 48097.10 ? Remember that the first point of using DIAgui is to refilter the precursors from the report and recompute the quantification. If that precursor didn't fulfill the filtering criteria it was then removed from the sum.

I didn't filiter any precursors with Q.value and the PG.Q.value thresthold in today's calculation with any code. And 48097.10 won't filted even apply filter thresthold.

[mgerault]

The diann_matrix function apply by default filter, except if you set the parameters q and pg.q to 1. But indeed that wouldn't apply to this precursor since the default cutoff are 0.01. I have to say this leaves me perplex, I don't have an explaination for now; do you think you could send me the full code you used and your data ?

[Catball79]

code.txt test_report.csv

[mgerault]

Ok so this is indeed because a of a filtration cutoff, this one based on the column Quantity.Quality which states about the measurement quality. The default cutoff is 0.8, the Quantity.Quality of the removed precursor is 0.74 so it wasn't taken in the sum, hence the obtained intensity of 96455.7 This criteria has been added in the last version of DIAgui based on a user suggestion. Change this cutoff if you want to include it in your calculation.

[Catball79]

Ok so this is indeed because a of a filtration cutoff, this one based on the column Quantity.Quality which states about the measurement quality. The default cutoff is 0.8, the Quantity.Quality of the removed precursor is 0.74 so it wasn't taken in the sum, hence the obtained intensity of 96455.7 This criteria has been added in the last version of DIAgui based on a user suggestion. Change this cutoff if you want to include it in your calculation.

Thank you for your rigorous approach to study !

[mgerault]

Also, I think I know why shinyFiles doesn’t find any path when you use the app. In the code you sent me you have a line where you change your working directory with setwd. When you load DIAgui it automatically find your working directory and save its path under the variable WD. The app will take this path to load your reports. But since you change your working directory and didn't change the variable WD the app can't find anything. When you load the package, a warning message state about this.

This is my guess for now. So to make it work either don't change your working directory or update as well the WD variable. Let me know if that worked.

[Catball79]

Also, I think I know why shinyFiles doesn’t find any path when you use the app. In the code you sent me you have a line where you change your working directory with setwd. When you load DIAgui it automatically find your working directory and save its path under the variable WD. The app will take this path to load your reports. But since you change your working directory and didn't change the variable WD the app can't find anything. When you load the package, a warning message state about this.

This is my guess for now. So to make it work either don't change your working directory or update as well the WD variable. Let me know if that worked.

Sorry, it seems doesn't work. And at the first time I using shinyFiles, I didn't set a wd. However, other file inportion function is normal.

[mgerault]

OK then back to square one...