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QUASAR error carmen.gonzaleztejedo  2017-10-25 03:37
 
Dear all,

I am trying to use QUASAR in order to calculate LOD and LLOQs, but it is giving me the next error:



"C:\Program Files\R\R-3.0.1\bin\R.exe" -f "C:\Users\gonzal01\AppData\Local\Apps\2.0\VOVKTNKO.4H1\YJMXWZG1.41A\skyl..tion_e4141a2a22107248_0003.0007_e5f1fd0a6674c91e\Tools\QuaSAR-1_33\QuaSAR-Skyline.R" --slave --no-save --args "C:\Users\gonzal01\AppData\Local\Apps\2.0\VOVKTNKO.4H1\YJMXWZG1.41A\skyl..tion_e4141a2a22107248_0003.0007_e5f1fd0a6674c91e\Tools\QuaSAR-1_33\QuaSAR.R" "C:\Users\gonzal01\AppData\Local\Apps\2.0\VOVKTNKO.4H1\YJMXWZG1.41A\skyl..tion_e4141a2a22107248_0003.0007_e5f1fd0a6674c91e\Tools\QuaSAR-1_33\common.R" "C:\Users\gonzal01\AppData\Local\Temp\QuaSAR_QuaSAR_Input.csv" NULL 231017 "heavy Area" 1 "light Area" fmol/ug 1 1 1 1 1 1 1 1000 1000 0 NULL 0 NULL 231017 0
[1] $Id: QuaSAR-Skyline.R 149 2014-10-23 13:56:13Z manidr $
[1] $Id: QuaSAR.R 149 2014-10-23 13:56:13Z manidr $

Attaching package: 'boot'

The following object is masked from 'package:lattice':

    melanoma

The following object is masked from 'package:gtools':

    inv.logit, logit

KernSmooth 2.23 loaded
Copyright M. P. Wand 1997-2009

Attaching package: 'gplots'

The following object is masked from 'package:stats':

    lowess

[1] Processing arguments ...
[1] >>> Processing part 1 of 1
[1] Initializing ...
Error in calculate(data.subset, concentrationFile, output.prefix = outputPrefix, :
  Duplicate transitions found -- try including neutral loss
Calls: tryCatch -> tryCatchList -> parse.cmdline -> calculate
Execution halted
Finished!





I think I started since the last Skyline Daily update.

Could you please help me with this issue?

Many thanks
 
 
Nick Shulman responded:  2017-10-25 04:02
In February 2015, someone else was running into this same error "Duplicate transitions found..." and this is what was said to do about it:
-----------------
That error occurs when there are multiple transitions with the same (PrecursorCharge, FragmentIon, ProductCharge) for a given peptide and sample. Possible causes could be:

1. You are monitoring the same fragment with different neutral losses -- in this case (as the error message suggests), include the NeutralLoss column to disambiguate the transitions.

2. You have modified peptides, but are not including the modifications in the ModifiedPeptideSequence column of the Skyline report. In such a case, two different modified peptides could have the same sequence, and hence could result in identical transitions.

3. Your sample specification was incorrect. If you have multiple concentrations, each concentration must be a different "sample". Please see the tutorial on how to specify the sample.
-----------------

Is any of that information helpful?

It is conceivable that this is a new issue that was caused by a recent Skyline-Daily update. I did make a change to the way that the Modified Sequence works, although that was not supposed to have any effect on how that column looks in a Report. If the steps listed above do not fix your problem, then you should send us your Skyline document.

In Skyline, you can use the menu item:
File > Share > (Complete)
to create a .zip file containing your Skyline document and supporting files.

You can upload that .zip file here:
https://skyline.ms/files.url
 
carmen.gonzaleztejedo responded:  2017-10-25 07:24
Hi Nick,

Thanks for your quick reply. You are right, I have modified peptides (phospho and non-phospho), but the modification ([+80}) is shown in the Peptide Modified Sequence column in the QUASAR input document. I have tried the different solutions you suggested, but it still giving the error.

I have shared my skyline document in the provided link; its called "20171021_WB_CAG_Response_Curve_HF_Results.sky"

Many thanks for your help.

Carmen
 
Nick Shulman responded:  2017-10-25 14:57
Thanks for sending me your file.

Yes, this is caused by the changes I made to "Modified Sequence". I unfortunately made it so that "Pivot Isotope Label" in a report did not work correctly if the Modified Sequence was one of the columns, so twice as many rows were being sent to the Quasar external tool (the light and heavy ended up on separate rows of the report, instead of side by side like they are supposed to).

I will fix this in the next update of Skyline-Daily.

I believe you can revert to the previous version of Skyline-Daily by going into "Programs and Features" in Control Panel, doing "Uninstall/Change" on Skyline-Daily and choosing "Restore the application to its previous state".

Thank you for reporting this bug!
 
carmen.gonzaleztejedo responded:  2017-10-26 04:05
Hi Nick,

I have restored the application to its previous state but still it is not working, because the report is the same due to the "Pivote Isotope label" option. I checked the version and it's v3.7.1.11446. Is it possible to get again the previous version Skyline-daily 3.7.1.11357? (I have analysed a similar experiment with that version and QUASAR was working at that moment).

Many thanks!
 
Nick Shulman responded:  2017-10-26 14:33
I guess you would actually have to go all the way back to Skyline-Daily 3.7.1.11271 in order to not have this problem.

Another way to work around this problem is to replace your report "Quasar Input".
That is, go to:
File > Export > Report > Edit List
then change "Group" to "External Tools"
and then press "Import" and import the report from the attached file "QuasarInputFixed.skyr".

That will replace the report that the Quasar external tool uses with one that does not have this problem with the PeptideModifiedSequence.
 
carmen.gonzaleztejedo responded:  2017-11-03 03:50
Many thanks for that. I have replaced the report Quasar input and now it is working.