Issue opening skyline

[bsphinney]

I got skyline to be recognized thanks to another thread here (thank you!!) but I am getting this error when it tries to build the library. Interestingly I also get this error if I try and build the library myself within skyline. I verified the files it's looking for are indeed in the directory and start with the same leading characters.

Thanks!!

F:\Brett\DIANN19_test>"C:\Program Files\Skyline-daily\SkylineCmd.exe" --timestamp --dir=F:\Brett\DIANN19_test --overwrite --new=report_skyline.sky --import-search-cutoff-score=0.01 --import-search-add-mods --import-search-file="F:\Brett\DIANN19_test\experiment_specific.tsv.skyline.speclib" --pep-max-missed-cleavages=1 --pep-min-length=6 --pep-max-length=50 --pep-exclude-nterminal-aas=0 --tran-precursor-ion-charges="2,3,4,5,6" --tran-product-ion-charges="1,2" --tran-product-ion-types="y,b,p" --tran-product-start-ion="ion 3" --tran-product-end-ion="last ion" --tran-product-clear-special-ions --library-match-tolerance=10ppm --library-product-ions=6 --library-min-product-ions=6 --library-pick-product-ions=filter --full-scan-precursor-analyzer=centroided --full-scan-precursor-isotopes=Count --full-scan-precursor-threshold=3 --full-scan-precursor-res=15 --full-scan-product-analyzer=centroided --full-scan-product-res=15 --ims-library-res=20 --full-scan-rt-filter=ms2_ids --full-scan-rt-filter-tolerance=2 --instrument-min-mz=50 --instrument-max-mz=2000 --full-scan-precursor-isotopes=Count --full-scan-acquisition-method=DIA --full-scan-isolation-scheme="Results only" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-A1_1_5579.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-B1_1_5580.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-C1_1_5581.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-D1_1_5582.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-F1_1_5584.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-G1_1_5585.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_S6-H1_1_5586.d" --import-file="F:\Brett\DIANN19_test\Mouse_liver_250ng_OT_sand_Fail_S6-E1_1_5583.d" --import-search-exclude-library-sources --import-fasta="F:\Brett\DIANN19_test\uniprotkb_proteome_UP000000589_AND_revi_2024_06_12.fasta\uniprotkb_proteome_UP000000589_AND_revi_2024_06_12.fasta" --associate-proteins-shared-peptides=DuplicatedBetweenProteins [2024/06/13 13:27:44] File report_skyline.sky opened. Transition Settings -- Filter changed Settings > Transition Settings -- Filter > Peptide Precursor charges changed from "2" to "2, 3, 4, 5, 6" Settings > Transition Settings -- Filter > Peptide Ion charges changed from "1" to "1, 2" Settings > Transition Settings -- Filter > Ion types changed from "y" to "y, b, p" Settings > Transition Settings -- Filter > Product ion selection start changed from "m/z > precursor" to "ion 3" Settings > Transition Settings -- Filter > Product ion selection end changed from "3 ions" to "last ion" Settings > Transition Settings -- Filter > Special ions: removed all Transition Settings -- Instrument > Max m/z changed to "2000" Settings > Transition Settings -- Instrument > Max m/z changed from "1500" to "2000" Transition Settings -- Library changed Settings > Transition Settings -- Library > Ion match tolerance changed from "0.5" m/z to "10" ppm Settings > Transition Settings -- Library > Minimum product ions changed from "0" to "6" Settings > Transition Settings -- Library > Product ions changed from "3" to "6" Settings > Transition Settings -- Library > Pick changed from "From filtered ion charges and types" to "From filtered product ions" Transition Settings -- Full-Scan changed Settings > Transition Settings -- Full-Scan > Acquisition method changed from "None" to "DIA" Settings > Transition Settings -- Full-Scan > Isolation scheme changed from Missing to "Results only" Settings > Transition Settings -- Full-Scan > Isolation scheme > Isolation width is "Results" Settings > Transition Settings -- Full-Scan > Isolation scheme > Prespecified isolation windows is Empty Settings > Transition Settings -- Full-Scan > Product mass analyzer changed from "None" to "Centroided" Settings > Transition Settings -- Full-Scan > Mass accuracy changed from Missing to "15" Settings > Transition Settings -- Full-Scan > Isotope peaks included changed from "None" to "Count" Settings > Transition Settings -- Full-Scan > Peaks changed from Missing to "3" Settings > Transition Settings -- Full-Scan > Precursor mass analyzer changed from "None" to "Centroided" Settings > Transition Settings -- Full-Scan > Mass accuracy changed from Missing to "15" Settings > Transition Settings -- Full-Scan > Isotope labeling enrichment changed from Missing to "Default" Settings > Transition Settings -- Full-Scan > Retention time filter length changed from "5" to "2" Peptide Settings -- Digestion > Max missed cleavages changed to "1" Settings > Peptide Settings -- Digestion > Max missed cleavages changed from "0" to "1" Peptide Settings -- Filter changed Settings > Peptide Settings -- Filter > Exclude N-terminal AAs changed from "25" to "0" Settings > Peptide Settings -- Filter > Min length changed from "8" to "6" Settings > Peptide Settings -- Filter > Max length changed from "25" to "50" [2024/06/13 13:27:45] Enabling extraction based on spectral library ion mobility values. [2024/06/13 13:27:45] Changing ion mobility spectral library resolving power to 20. [2024/06/13 13:27:45] Creating spectral library from files: [2024/06/13 13:27:45] experiment_specific.tsv.skyline.speclib [2024/06/13 13:27:45] Error: ERROR: unable to determine DIA-NN report filename for 'experiment_specific.tsv.skyline.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)

Command-line: F:\Brett\DIANN19_test\BlibBuild -s -A -H -v warn -o -c 0.01 -i report_skyline -S "F:\Brett\DIANN19_test\report_skyline.redundant202406130127.stdin.txt" "F:\Brett\DIANN19_test\report_skyline.redundant.blib" Working directory: F:\Brett\DIANN19_test

F:\Brett\DIANN19_test>if 2 == 0 "C:\Program Files\Skyline-daily\Skyline-daily.exe" --opendoc F:\Brett\DIANN19_test\report_skyline.sky

[szabzola]

Hi! What was the name of your report tsv file, and library? I had the same error if I just left it at default for testing (report.tsv). But is looks for "somedata-tsv.speclib and somedata-report.tsv", so if I added something (same) before "report" and tsv-speclib in filename it worked. The same issue exists for a while if you create library within Skyline from DIA-NN. But it may be different in your case. Best regards,

[vdemichev]

Hi Brett,

Great to hear from you! I think this whole thing was inspired by the tutorials you made :) I guess what happens here, the library file needs to be named 'similarly' to the main report. That is if the main report is report.tsv, and library is report-lib.tsv (default name generated by DIA-NN GUI; the library generated in the first pass of MBR will aslo be saved to report-lib.tsv.skyline.speclib, which gets passed to Skyline), then Skyline will figure this out and open report.tsv. Will also work if the library is report-exp.tsv or sth like that. But if the names are very different, e.g. report.tsv and new_lib.tsv, then Skyline will not be able to find the main report.

Best, Vadim

[edunivers]

Dear Vadim,

When analyzing the DIA with DIANN with the following parameters and default output names, we get an error when trying to launch Skyline. The error says:

Errors: C:\DIA-NN\1.9\report-lib.tsv.skyline.speclib not found

The file is indeed not generated and we only have the "report-lib.tsv" file as well as the "report-lib.predicted.speclib" file, but not the required one.

Is there any parameter that we are missing?

Command: diann.exe --f "H:\2024MK027\2024MK027_001_01_2ug.raw" --f "H:\2024MK027\2024MK027_002_01_2ug.raw" --lib "" --threads 4 --verbose 1 --out "C:\DIA-NN\1.9\report.tsv" --qvalue 0.01 --matrices --out-lib "C:\DIA-NN\1.9\report-lib.tsv" --gen-spec-lib --predictor --xic --fasta "H:\2024MK027\UP000008227_9823.fasta" --fasta-search --min-fr-mz 350 --max-fr-mz 1850 --met-excision --min-pep-len 7 --max-pep-len 30 --min-pr-mz 500 --max-pr-mz 900 --min-pr-charge 1 --max-pr-charge 4 --cut K,R,!*P --missed-cleavages 1 --unimod4 --var-mods 1 --var-mod UniMod:35,15.994915,M --reanalyse --relaxed-prot-inf --smart-profiling --pg-level 1

[vdemichev]

Hi, Most likely it's because this is 'on the fly' FASTA search. Should work if you generated a predicted library in a separate pipeline step.

Best, Vadim