How Can I use peak visualization function with Skyline

[k12-sasaki]

Hope it finds you well.
I'm using DIA-NN 1.9 and Skyline (64 bit) 24.1.

I'm trying to use following skyline visualiation function.

Skyline. To visualise chromatograms/spectra in Skyline, analyse your experiment with MBR and a FASTA database specified and then click the 'Skyline' button. DIA-NN will automatically launch Skyline (make sure you have Skyline/Skyline daily version 23.1.1.459 or later installed as 'Administrator install'). Currently this workflow does not support multiplexing and will not work with modifications in any format other than UniMod.

I have tried to perform the analysis with the following settings and pressed the skyline button on the DIA-NN after completion, but I got error message like "report-lib.tsv.skyline.speclib not found."

Skyline found: Skyline (64 bit) 24.1.0.199
MSFileReader found: MSFileReader Core 31

diann.exe --f "D:\XXXX\cell_DIA_DMSO-1.raw
" --f "D:\XXXX\cell_DIA_DMSO-2.raw
" --lib "" --threads 32 --verbose 1 --out "D:\XXXX\DIA-NN\report.tsv" --qvalue 0.01 --matrices --out-lib "D:\XXXX\DIA-NN\report-lib.tsv" --gen-spec-lib --predictor --fasta "D:\XXXX\FASTA\human all\uniprot-organism_9606+reviewed_yes.fasta" --fasta-search --min-fr-mz 200 --max-fr-mz 1800 --met-excision --min-pep-len 7 --max-pep-len 30 --min-pr-mz 300 --max-pr-mz 1800 --min-pr-charge 1 --max-pr-charge 4 --cut K*,R* --missed-cleavages 1 --unimod4 --reanalyse --relaxed-prot-inf --rt-profiling
DIA-NN 1.9 (Data-Independent Acquisition by Neural Networks)
Compiled on Jun 8 2024 20:00:31
Current date and time: Thu Nov 28 16:09:33 2024
CPU: GenuineIntel Intel(R) Xeon(R) Gold 6226R CPU @ 2.90GHz
SIMD instructions: AVX AVX2 AVX512CD AVX512F FMA SSE4.1 SSE4.2
Logical CPU cores: 64
Thread number set to 32
Output will be filtered at 0.01 FDR
Precursor/protein x samples expression level matrices will be saved along with the main report
A spectral library will be generated
Deep learning will be used to generate a new in silico spectral library from peptides provided
Library-free search enabled
Min fragment m/z set to 200
Max fragment m/z set to 1800
N-terminal methionine excision enabled
Min peptide length set to 7
Max peptide length set to 30
Min precursor m/z set to 300
Max precursor m/z set to 1800
Min precursor charge set to 1
Max precursor charge set to 4
In silico digest will involve cuts at K*,R*
Maximum number of missed cleavages set to 1
Cysteine carbamidomethylation enabled as a fixed modification
A spectral library will be created from the DIA runs and used to reanalyse them; .quant files will only be saved to disk during the first step
Heuristic protein grouping will be used, to reduce the number of protein groups obtained; this mode is recommended for benchmarking protein ID numbers, GO/pathway and system-scale analyses
The spectral library (if generated) will retain the original spectra but will include empirically-aligned RTs
DIA-NN will optimise the mass accuracy automatically using the first run in the experiment. This is useful primarily for quick initial analyses, when it is not yet known which mass accuracy setting works best for a particular acquisition scheme.
Exclusion of fragments shared between heavy and light peptides from quantification is not supported in FASTA digest mode - disabled; to enable, generate an in silico predicted spectral library and analyse with this library
WARNING: it is strongly recommended to first generate an in silico-predicted library in a separate pipeline step and then use it to process the raw data, now without activating FASTA digest

Are there any points of usage that are incorrect?
I have checked similar problem #1048, but still not resolved.

I would appreciate your kind help.
Thank you so much!

[vdemichev]

Hi,

--fasta-search

Must never be used in combination with raw files analysis, in this case also please see the prerequesites for visualisation with Skyline https://github.com/vdemichev/DiaNN?tab=readme-ov-file#visualisation - I guess here I confusingly did not mention that what's meant is a spectral library speicifed + FASTA is just there for annotation :) I will correct the docs later on. I also recommend switching to 1.9.2.

Best,
Vadim

[k12-sasaki]

Hi, that has been resolved!
Thank you so much for the quick response!

Just one more point, if I run all 5 analyses (Analysis1-5) in pipeline mode and they are all complete, how do I use the skyline visualization feature in Analysis 1?
Thank you very much!