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EncyclopeDIA error sscott13  2025-04-11 09:54
 

Hello,

This is my first go through using Skyline/EncyclopeDIA to look at my samples vs. GPF, so there might be something I'm setting up incorrectly, but I keep hitting a GC overload error. There are a ton of entries in the file, and I'll try parsing it to see if that fits, but I wanted to check to make sure it wasn't something in my setup. I had 6 GPF pools, and 15 samples. The blib is created, but the error comes in when the elib is supposed to be created.

  1. Is there a way to get around this in Skyline? I'm happy to send over whatever files you need.

Thank you,

Shay
 
 
Nick Shulman responded:  2025-04-11 10:13
The amount of memory required to convert a .blib file to a .elib seems to depend on the total number of unique peptide sequences in the FASTA file.
You could reduce the amount of memory needed by using a smaller fasta file and/or by setting Max Missed Cleavages to a smaller number.

There is a little bit more information about this error here:
https://skyline.ms/announcements/home/support/thread.view?rowId=66269

If your computer has less than 24GB of RAM in it, then this might succeed if you did it on a computer with more RAM.
Skyline tells EncyclopeDIA to use the smaller of either 12GB or half the amount of memory in the computer, so increasing the RAM beyond 24GB is unlikely to fix the problem.

How much RAM does your computer have? I could probably send you a special version of Skyline which tells EncyclopeDIA to use more RAM so you can get past this problem if it's not feasible for you to use a smaller FASTA file and/or decrease the number of missed cleavages to get around this problem.
-- Nick
 
sscott13 responded:  2025-04-11 10:35
Hi Nick,

Thanks so much for the prompt response. I have 16.0 GB of RAM. I'm not sure if this is it, but I had a really hard time getting MSConvert to work on my overlapping files (kept getting the minWindowValue too low error) - I attempted to raise it, but it didn't fix it, so i droped the .raw files into the Skyline EncyclopeDIA tool - it's able to read them in, generate the koina blib, then it crashes shortly thereafter. My fasta file is large - I'll try chopping it into bits. Is the reason it's crashing because I'm converting them in the program?

This might be a dumb question (sorry if it is!), but if I have the blib, is there any way to start at that point in Skyline, where I could load the blib and start from the point of elib generation?

Thanks again,

Shay
 
sscott13 responded:  2025-04-11 11:00
Hi sorry to pop this on here again,

For this error- I think the reason why it's failing is because of how MSConvert reads the DIA scheme from my runs -

Is there any way to export the mzml files that are generated from EncyclopeDIA in skyline?

Thank you,,
Shay
 
Nick Shulman responded:  2025-04-11 11:17
If you are getting the error about the number of deconvolution windows changes, that is probably caused by them overlapping in a weird way.
You can find some information about getting past that error here:
https://sourceforge.net/p/proteowizard/mailman/message/37747772/

I am not sure about the answers to your other questions. Someone else might know.
-- Nick