Todd,

Can you send us your Skyline document?
In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

In a .blib spectral library, retention time values can be found in the "RefSpectra" table as well as the "RetentionTimes" table. I would hope that the values in the "RetentionTimes" table would be actual retention time values corresponding to the files that they are supposed to be from. The retention time values in the "RefSpectra" table are not used by Skyline and could be in an arbitrary scale and would not cause any problems for Skyline.
-- Nick

Hi Nick,
Thank you for the quick reply. Please see attached Skyline document.
Todd

Todd,

I see that you used "File > Import > Assay Library" to import a TSV file into Skyline.
The usual way to get DIA-NN results into Skyline would be to use the "File > Import > Peptide Search" menu item and then point Skyline at a .speclib file.
When you import DIA-NN search results that way, Skyline uses the peak boundaries that DIA-NN chose.

I am not sure exactly what you get when you do "File > Import > Assay Library" (I'm not as familiar with that feature in Skyline).
-- Nick

Hi Nick,

Thanks for catching that, I forgot I had to use that function. The reason I used this function was because I got an error when trying to build the Blib library using the speclib file. See below. I'm not sure which of the ERRORs is the root cause, but I can confirm that the peptide AAAGELQEDSGLC(UniMod:4)VLAR2 is present in the parquet that is being referenced. I'm not sure if it matters, but the first ERROR mentions"precursorId", but the column in my parquet is "Precursor.Id".

If you have any thoughts on these errors, that would be helpful.

Todd
---------------------------
Skyline-daily
---------------------------
ERROR: could not find precursorId 'AAAGELQEDSGLC(UniMod:4)VLAR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file report_HumanExp_jck-lib.parquet.skyline.speclib
ERROR: boost::filesystem::remove: The process cannot access the file because it is being used by another process [system:32]: "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"

Command-line: C:\Users\enoni\AppData\Local\Apps\2.0\R8KJE96M.0AL\5P814CJB.DXZ\skyl..tion_9286511f3362df93_0019.0000_b3f34f3a54bf06ec\BlibBuild -s -A -H -o -c 0.95 -i hCSF_DIANN21_DNARHfiltered_wizard -S "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant202505231141.stdin.txt" "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"
Working directory: E:\Data\_CHDI\20250418_hCSF_diapasef
Exit code: 1
---------------------------
OK More Info
---------------------------
Skyline-daily (64-bit) 25.0.9.131 (23f06989d4)

System.IO.IOException: ERROR: could not find precursorId 'AAAGELQEDSGLC(UniMod:4)VLAR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file report_HumanExp_jck-lib.parquet.skyline.speclib
ERROR: boost::filesystem::remove: The process cannot access the file because it is being used by another process [system:32]: "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"

Command-line: C:\Users\enoni\AppData\Local\Apps\2.0\R8KJE96M.0AL\5P814CJB.DXZ\skyl..tion_9286511f3362df93_0019.0000_b3f34f3a54bf06ec\BlibBuild -s -A -H -o -c 0.95 -i hCSF_DIANN21_DNARHfiltered_wizard -S "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant202505231141.stdin.txt" "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"
Working directory: E:\Data\_CHDI\20250418_hCSF_diapasef
Exit code: 1 ---> System.IO.IOException: ERROR: could not find precursorId 'AAAGELQEDSGLC(UniMod:4)VLAR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file report_HumanExp_jck-lib.parquet.skyline.speclib
ERROR: boost::filesystem::remove: The process cannot access the file because it is being used by another process [system:32]: "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"

Command-line: C:\Users\enoni\AppData\Local\Apps\2.0\R8KJE96M.0AL\5P814CJB.DXZ\skyl..tion_9286511f3362df93_0019.0000_b3f34f3a54bf06ec\BlibBuild -s -A -H -o -c 0.95 -i hCSF_DIANN21_DNARHfiltered_wizard -S "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant202505231141.stdin.txt" "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"
Working directory: E:\Data\_CHDI\20250418_hCSF_diapasef
Exit code: 1

Output:
Reading results from report_HumanExp_jck-lib.parquet.skyline.speclib.
Read 6167 entries from speclib.
Reading report headers.
Reading 6171 rows from report.
ERROR: could not find precursorId 'AAAGELQEDSGLC(UniMod:4)VLAR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file report_HumanExp_jck-lib.parquet.skyline.speclib
100%
ERROR: boost::filesystem::remove: The process cannot access the file because it is being used by another process [system:32]: "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"

 ---> System.IO.IOException: ERROR: could not find precursorId 'AAAGELQEDSGLC(UniMod:4)VLAR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file report_HumanExp_jck-lib.parquet.skyline.speclib
ERROR: boost::filesystem::remove: The process cannot access the file because it is being used by another process [system:32]: "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"

Command-line: C:\Users\enoni\AppData\Local\Apps\2.0\R8KJE96M.0AL\5P814CJB.DXZ\skyl..tion_9286511f3362df93_0019.0000_b3f34f3a54bf06ec\BlibBuild -s -A -H -o -c 0.95 -i hCSF_DIANN21_DNARHfiltered_wizard -S "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant202505231141.stdin.txt" "E:\Data\_CHDI\20250418_hCSF_diapasef\hCSF_DIANN21_DNARHfiltered_wizard.redundant.blib"
Working directory: E:\Data\_CHDI\20250418_hCSF_diapasef
Exit code: 1
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_25_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 206
   --- End of inner exception stack trace ---
   --- End of inner exception stack trace ---
   at pwiz.Common.SystemUtil.ProcessRunner.ThrowExceptionWithOutput(Exception exception, String output) in C:\proj\skyline_25_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 266
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_25_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 248
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_25_1\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 493
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_25_1\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 152
---------------------------

That error "is 'report_HumanExp_jck.parquet' the correct report TSV file?" is usually caused by BiblioSpec finding the wrong .tsv (or I guess BiblioSpec supports .parquet now too) file to go with the .speclib that you chose.
DIA-NN makes it very easy to put the results from multiple peptide searches into the same folder.
When BiblioSpec builds a spectral library from a .speclib file, BiblioSpec gets information about m/z's and intensities from the .speclib file, but BiblioSpec needs to find information about scores and retention times from a report TSV file.
BiblioSpec has heuristics for finding the correct TSV file to go with the .speclib file, but, if BiblioSpec choose the wrong TSV file, that TSV file might be missing information for some of the peptides in the .speclib and you will get the error you are seeing.

You might be able to get this to work by deleting the file "report_HumanExp_jck.parquet", in which case BiblioSpec would have to use a different file which might be the correct one.
If that does not work you might need to run your DIA-NN search again and make sure the output folder does not contain results from any other searches.
-- Nick

Hi Nick,

Thanks for the suggestions, but still no luck.

I moved all files from the analysis into one directory and that generated same error message.
I deleted the parquet files, and that reported a different error that no associated report file could be found for the speclib.
I also re-ran the analysis. This is when I remembered that since I was analyzing a single file, DIANN does not automatically generate the "..skyline.speclib" file, only the parquet library. I generated the speclib file in a separate step by loading the report-lib.parquet into DIANN w/o any data and it generated the speclib. Perhaps this is at the core of the issue? Do you have any thoughts, or should I inquire on the DIA-NN support forum?

Todd

Can you zip up all the files you have and upload them here:
https://skyline.ms/files.url

This is the correct forum to be asking questions about importing DIA-NN results into Skyline.
-- Nick

I have uploaded a file containing the skyline share zip and all DIA-NN outputs, hCSF_DIANN21_DNARHfiltered_wizard.zip
Todd

DIA-NN will only generate the Skyline.speclib file when you have multiple files and "MBR" checked.

Mike

One issue is iit really does seem like a bug that DIANN doesn't generate a .speclib file when it's given a single input file.

Read 6167 entries from speclib.
Reading report headers.
Reading 6171 rows from report.

The row count difference is suspicious. The speclib should always have more rows than the report, I think. The error is saying it can't find that precursorId in the speclib, not that it can't find it in the parquet.

But when I run the files you uploaded I get a different error and row counts:

Reading results from c:\test\issues\diann2-parquet\report_HumanExp_jck-lib.parquet.skyline.speclib.
Read 6213 entries from speclib.
Reading report headers.
Reading 6171 rows from report.
ERROR: could not find precursorId 'AIALDPR2' in speclib; is 'report_HumanExp_jck.parquet' the correct report TSV file?
ERROR: reading file c:\test\issues\diann2-parquet\report_HumanExp_jck-lib.parquet.skyline.speclib

And indeed, that precursor is not in the speclib, strongly suggesting that the parquet did not come from that speclib. Can you try analyzing 2 files so you get a speclib by default and see if the issue goes away?

I believe it is intentional. Vadim mentioned that you can only make quantitative comparisons if there are multiple runs. If you have multiple runs then we should be using the two step search in DIANN to increase the detection of peptides between runs. In DIANN this requires turning on "MBR" ... which essentially reduces the library size and leaks information from the search of one replicate to another. So DIANN only outputs the skyline.speclib file if there are multiple runs and "--reanalyse" used from the command line or "MBR" checked in the GUI.

To Matt's comment:
Yes, I can confirm that when I generate the skyline.speclib in a workflow with 2+ files (MBR enabled), then it can be used to create a Skyline library with no errors.

To Mike's comment:
This makes sense to me too for quantitative comparisons.

In this rare case of having one file that we want quick evaluation, I read on the DIANN forums about converting the parquet lib to a skyline.speclib by loading the parquet library into DIANN, but not queuing the raw data, and enabling generate spectral library. This does create a skyline.speclib file, but from our testing here, there is something not quite right about it, as it is missing precursors that are in the main report. Vadim is working on improved handling in DIA-NN v2.2, so I can mention this to him and see if there are plans to support this rare use-case.

Thanks for everyone's troubleshooting and comments.

Todd