differences in the volcano plot data generated from skyline

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differences in the volcano plot data generated from skyline Linwei Li  2021-05-02
 

Hello! I used two ways to generate a volcano plot from skyline, and both of them produced data that are somehow different.
The first way I used is to us the group analysis in MSstats, which produced a viable result. However, despite my target of interest that is down-regulated, the rest of the data points look very flat.
The second way I used is try to extract the data of volcano plot out, however, they produced the same adjusted p value, and all my data points look flat including my target of interest.
I have attached the pdf using the first method (group analysis). The standard I used is "equalize to all medians."
Also, When I set my standard to "relative to global standard," group analysis from MSstats failed to generate one. I wonder what I can do to improve.
Thanks so much!

Best,
Linwei

 
 
Nick Shulman responded:  2021-05-02
Can you send us your Skyline document?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

-- Nick
 
Linwei Li responded:  2021-05-03
Thanks for replying! I have attached the files below.
 
Nick Shulman responded:  2021-05-03
Linwei,

I see that you uploaded your .mzXML files and your .sky file.

What I need you to send me instead is a .sky.zip file.
In your case, the .sky.zip file would contain the Skyline document (.sky), extracted chromatograms (.skyd), and spectral library (.blib).

The way to create a .sky.zip file is by using the menu item in Skyline:
File > Share

Thanks,
-- Nick
 
Linwei Li responded:  2021-05-06
Sorry for the delay. I have uploaded the files mentioned in your response. Let me know if you need more information and thank you so much!

Best,
Linwei

edit:
I think the zip file is larger than the minimum size. What should I do about this? Thanks so much!
 
Linwei Li responded:  2021-05-06
Attached each file in a separate folder. (2) refers to the .blib file and (3) refers to the .skyd file.

Best,
Linwei

Edit: I think the .skyd file is too large to be uploaded.
 
Nick Shulman responded:  2021-05-06
Linwei,

It sounds like you are saying that you uploaded some new files somewhere, but I cannot find any new files. Where did you upload them?

You can upload files here:
https://skyline.ms/files.url

I will also send you an email directly in case it's easier for you to send us files in an email message.
-- Nick
 
Nick Shulman responded:  2021-05-06
Linwei,

Thank you for sending me the rest of those files.
The reason that your volcano plots look the way that they do is that the p-values of the group comparisons really are very close to 1.
With a group comparison, a low p-value such as 0.05 would mean that there was a significant difference between the two groups, whereas a p-value close to 1 means that there is no observable difference between the two groups.
The p-value comes from a Student's T-test, and it measures the variance within the two groups is much less than the variance between the two groups.

When I look at the Peak Area Replicate Comparison graph, I see that this is huge variation within the two groups that you have defined ("DMSO" and "JC19"), and it is much larger than the variation between the two groups, and therefore you get a p-value which is close to 1.

The Group Comparison tutorial is a great way to learn about this feature in Skyline:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_grouped

-- Nick