| Nick Shulman responded: |
2024-07-14 21:32 |
The error messages that you seem to have are:
[16:31:53] ERROR: Reading tab file, too few features present.
[16:31:53] No-terminate flag set: ignoring error and using a pseudo-feature of zeroes.
[16:31:53] Exception caught: ERROR: Reading tab file, error reading PSM on line 2. Could not read PSMid.
[16:31:53]
[16:31:53] Fatal Error: Percolator exited with non-zero status: 1
[16:31:53] Sorry, not feeling well today! Try again tomorrow!
[16:31:53] Encountered Fatal Error!
I do not know what those error messages mean, but if you send us your data files we might be able to reproduce the problem and figure out what is going wrong.
I am not sure exactly which files we would need from you, but I imagine we would need your FASTA file as well as the mzML or .raw files that you are trying to search.
You can package your files in a .zip and upload them here:
https://skyline.ms/files.url
-- Nick
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| michal zawadzki33223 responded: |
2024-07-15 00:45 |
Hi Nick,
Thank you for looking into this issue. I have uploaded my files (mzML) and fasta as requested. You can find them in the folder called "Data for Skyline troubleshooting" with a description "EnclyclopeDIA Percolator error"
Many thanks,
Michal
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| Nick Shulman responded: |
2024-07-15 09:24 |
Thank you for sending those files.
It would also be helpful if you could send me the following file:
C:\Users\zawadzki\OneDrive - Vertex Pharmaceuticals\Documents\HeLa QC\DIA\elib_chrom\Inst-Exploris480_Date-2024JUL09_Evts-A2_Meth-90min-400_500mz_DIA_Cond-50cm_01.mzML.features.txt
That seems to be the file which Percolator was reading when it said:
ERROR: Reading tab file, too few features present.
-- Nick
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| michal zawadzki33223 responded: |
2024-07-17 06:08 |
Hi Nick,
I have attempted to run the analysis on a different workstation. This time it managed to run EncyclopeDIA search successfully (see the attached log "Skyline EncyclopeDIA log.txt"). However, when I follow the "Import Peptide Search" wizard afterwards I am getting a message that "Importing the FASTA did not create any target proteins". I am using the same fasta file as I used for the EncyclopeDIA search.
Kind regards,
Michal
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| Nick Shulman responded: |
2024-07-17 07:47 |
The error "Importing the FASTA did not create any target proteins" usually means that no peptides were found during the peptide search.
However, in your output it definitely looks like plenty of peptides were found, so I am not sure what might be causing this message.
I would recommend trying this using Skyline-daily instead of Skyline 23.1.
We are going to release Skyline 24.1 in a week or so, and Skyline 24.1 is going to be very similar to the thing that is currently Skyline-daily.
You can install Skyline-daily by clicking the "Skyline-daily (beta)" button on the main Skyline page:
https://skyline.ms/project/home/software/Skyline/begin.view
We have made a lot of improvements to Import EncyclopeDIA Search in Skyline-daily. (Also, the menu item has moved to "File > Search > EncyclopeDIA Search").
It is likely that whatever problems you are running into with EncyclopeDIA search in Skyline 23.1 have already been fixed in Skyline-daily.
If you encounter any issues with Skyline-daily please let us know.
-- Nick |
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| michal zawadzki33223 responded: |
2024-07-22 00:51 |
Hi Nick,
I have upgraded Skyline to the recently released version 24.1 and tried to repeat the EncyclopeDIA search. I am still getting the same fasta import error as before. Is there a workaround I could try e.g. process the data with EncyclopeDIA wizard and then import results separately using "Import Peptide Search" wizard?
Kind regards,
Michal |
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| Nick Shulman responded: |
2024-07-22 09:41 |
Yes, you can run EncyclopeDIA yourself outside of Skyline and then add the .elib file to Skyline.
After you have used EncyclopeDIA to create your .elib file you would use the Skyline menu item:
File > Import > Peptide Search
on the first page of the Import Peptide Search wizard you should click the "Use existing" radio button and then point Skyline at the .elib file that you have and then go through the rest of the steps of the wizard which will involve importing a FASTA file and extracting chromatograms from raw files.
If you have more than one .elib file then, instead of using the Import Peptide Search wizard, you should go to:
Settings > Peptide Settings
and on the "Library" tab click "Edit List" and add all of your .elib files.
When you are adding these .elib files to that list, there is a checkbox "Use explicit peak bounds" which controls whether to use the peak boundaries from the .elib file (checked) or whether Skyline should do its own peak detection (unchecked). The wizard would have left that checkbox checked.
After you have added your multiple .elib files to the Skyline document, you can either use the "File > Import > FASTA" menu item to add peptides to your document, or you could go to "View > Spectral Libraries" and use the "Add All" button to add the peptides to your document.
After the peptides have been added to the document you can tell Skyline to extract chromatograms using the "File > Import > Results" menu item.
-- Nick |
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Percolator error.txt
Import Peptide Search troubleshooting.docx
UP000005640_9606.fasta