Issue with Skyline command runner

Issue with Skyline command runner shridevi patil  2019-08-12

Hello Skyline Team,

We are using Skyline command runner in our application, we need to arrive at optimized method for data acquisition using Waters Xevo Tq instrument..
We internally use Skyline Daily runner command to execute the Step1: RT,Step2:CEFixed,Step3:CEOptimized and Step4:FinalMethod.

We are facing problem with execution of these command on runner.
--dir="D:\Bit_Dev\WorkFlowManager\TestData\SkyLine" --in="c:\" --import-all-files="C:\Users\Test\AppData\Local\Temp\zft5bmr3" --exp-method-instrument="Waters Xevo TQ" --exp-template="c:\template.exp" --exp-run-length=15 --exp-file="C:\Users\Test\AppData\Local\Temp\sjty44c0\CE.exp" --exp-method-type=scheduled --refine-min-peak-found-ratio=0.1 --tran-product-ion-types="p,y,b" --out="C:\Users\Test\AppData\Local\Temp\ofe4atzp.ftq\" --exp-strategy=Buckets --exp-max-trans=100

is there any mistake in this command. Because return type from execution of Skyline command runner is failure.

Please guide/ correct out understanding.
Thank you

Brendan MacLean responded:  2019-08-12

Hi Shridevi,
Can you post the output from running this command? It is awfully hard to guess why it may have failed without at least the output. For instance, it may have failed simply because one of the specified paths does not exist. I am guessing that is not the case, but the output from the process will likely give us some helpful information.

Thanks for using SkylineRunner to automate your Skyline data processing.


shridevi patil responded:  2019-08-12

Thank you Brendan for the response.
Here is the output which is the result on command runner prompt:

C:\Users\Test\AppData\Roaming\Microsoft\Windows\Start Menu\Programs\Skyline-d
aily>SkylineDailyRunner.exe --dir="D:\TestData\SkyLine"
--in=" C:\Users\Test\Desktop\test\" --import-all-files=" C:\Users
\Test\Desktop\testRawdoc\Raw" --exp-method-instrument="Waters Xevo TQ" --exp-
template="D:\TestData\HV1\docs\template.exp" --exp-run-l
ength=15 --exp-file="C:\Users\Test\AppData\Local\Temp\ieni3bk1\CE.exp" --exp-
method-type=scheduled --refine-min-peak-found-ratio=0.1 --tran-product-ion-types
="p,y,b" --out="C:\Users\Test\AppData\Local\Temp\obecro0a.dz0\" --exp-s
trategy=Buckets --exp-max-trans=100
Opening file...
File opened.
Transition Settings -- Filter > Ion types changed to "p, y, b"
Settings > Transition Settings -- Filter > Ion types changed from "y, b" to "p,
y, b"

Adding results...

  1. C:\Users\Test\Desktop\testRawdoc\Raw\Unscheduled_REP01_0001.RAW
    Adding results...
  2. C:\Users\Test\Desktop\testRawdoc\Raw\Unscheduled_REP01_0002.RAW

[1] 61%
[1] 75%
[1] 89%
[2] 50%
[2] 64%
[2] 80%
[2] 97%

Joining file C:\Users\Test\Desktop\test\Unscheduled_REP01_0002_724963149.RAW.
Refining document...
Document unchanged
Saving file...
File saved.
Warning: The vendor Waters Xevo TQ does not match the vendor in either the CE or
DP prediction setting. Continuing exporting a transition list anyway...
Error: to export a scheduled method, you must first choose a retention time pred
ictor in Peptide Settings / Prediction, or import results for all peptides in th
e document.
No method will be exported.

We are still unable to generate CE.exp file for next Step.
Let us know, if we are missing anything bit.
Thank you.

Brendan MacLean responded:  2019-08-13

Hi Shridevi,
I feel like I must be missing something because the error seems obvious to me. Whenever SkylineRunner fails, you should first look at your output for a line that begins with "Error:" near the end of the output.

In this case, that line reads:

Error: to export a scheduled method, you must first choose a retention time predictor in Peptide Settings / Prediction, or import results for all peptides in the document.

Is this error message enough for you to understand what the problem is? Were you expecting that the imported data would cover all of your peptides?

To better understand this outcome, I would simply remove all of your --exp arguments and let Skyline succeed importing the data. Then open the resulting .sky file in Skyline. There you can try File > Export > Method to manually reproduce this error message, which I would expect you to get again from the UI when you try to export a scheduled method.

Then you can use the UI to figure out what peptide(s) in your document are not getting enough results information to have a retention time for scheduling. You should be able to do this pretty easily with View > Retention Times > Regression > Score to Run. Anything without a measured retention time will show up as a point on the x-axis, and you can just click on it to have Skyline select the peptide in the targets view. This is covered in the Targeted Method Refinement tutorial (page 10 - Missing Data):

Hope this helps. Thanks for posting the output.


Brendan MacLean responded:  2019-08-13

One thing I could imagine wanting to add would be --import-replicate-name=Unscheduled_REP01

This would cause your two files


To get imported into a single replicate expected to cover all of your document peptides once. Otherwise, I would expect that when you perform the command as I suggested that you will see two replicates in your saved file:


And each replicate will cover only half of the targets in your document.

Again, the Targeted Method Refinement tutorial does a pretty good job of covering the different ways you can import mass spec files into a Skyline document. The SkylineRunner command-line you have is equivalent to "Add single injection replicates in files" in the UI.

shridevi patil responded:  2019-08-13

Thank you Brendan, I will do as above guidelines. Will update you with the results.
Thank you so much.