Reading profile mode mzmls not supported

[rodvrees]

Hi, I want to run FlashLFQ on a set of mzML files using the docker container (see command below)

docker run -it -v ./bio-rescoring/PXD041846/mzmls_msconvert:/raws -v ./bio-rescoring:/data smithchemwisc/flashlfq:1.0.3 --idt data/ms2rescore-output/results.ms2rescore.mokapot.flashlfq.txt --rep raws/ --out data/rescore_flashlfq_no_mbr --ppm 5

However, FlashLFQ fails to correctly read in the mzml.

Opening PSM file data/ms2rescore-output/results.ms2rescore.mokapot.flashlfq.txt
Done reading PSMs; found 116554
Setup is OK; read in 116554 identifications; starting FlashLFQ engine
Reading spectra file
Problem opening .mzML file raws/XM001_ctrl_SCX_4.mzML; One or more errors occurred. (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported) (Reading profile mode mzmls not supported)

At first I thought it was because I had incorrectly configured MSConvert, but to me the settings look correct as per the user guide for converting raw files for FlashLFQ:

docker run -v ./bio-rescoring/PXD041846/:/data \
-v ./bio-rescoring/PXD041846/raws/:/raws
chambm/pwiz-skyline-i-agree-to-the-vendor-licenses:latest
wine msconvert '/raws/*.raw' -o /data/mzmls_msconvert/ --filter "peakPicking vendor msLevel=1"

Any idea what I am doing wrong?
Thank you in advance!

[Alexander-Sol]

It seems like the files were not converted correctly. Can you share one of the files and the identification file you're using so I can investigate this issue further?

[rodvrees]

https://drive.google.com/drive/folders/1zKILc84LzGi75seMHuSaOpP52V_-9xhb?usp=sharing

.7z file contains input ID file and one of the mzML files.
I also included a raw file, because I have now also tried using FlashLFQ directly with the raw files (Windows)

This obviously circumvents the above problem, but now I have a new one: all quants are 0.

This also happened when I ran FlashLFQ on mzML's created using ThermoRawFileParser.
At first I thought the mzML conversion was the cause of the failed quants, so I switched to MSConvert to make sure I was converting using the recommended approach (which then led to the above issue), but turns out the problem probably lies elsewhere because it's also happening with the raw files.
Any idea why this would happen?

Any help would be very much appreciated!

[Alexander-Sol]

Thank you for uploading the data. I'm looking into it now, hopefully will have answers for you sometime in the next few days

[rodvrees]

@Alexander-Sol Hi, have you had a moment to look into this?

[Alexander-Sol]

@rodvrees thanks for the reminder. I just ran the data you provided (using the .raw file) and also saw all zero values for the quant results.

I believe the retention time column of the results file is in the incorrect format. FlashLFQ needs the scan retention time in minutes, however, all of the scan retention times are less than 3. If you can convert the retention times to minutes and ensure they're correct, that should fix your issue.