Email Response:

Hi Molly,

I think that as long as we have the plots displaying the temperature dependent DE genes from the final timepoints (75min) for each genotype, without gene IDs on the plot, that would be great! Please let me know if you have any more questions!

Thank you for all of your help!
Anthony

Code Changes:

• Had to relabel the plot objects so they are all named differently and can be pulled individually for later use.
• Added code that creates the PDF output file,
• Changed the volcano_plot() function in StatFunctions.R script to remove the lab = d$gene to lab = NA so that the genes names are removed from the plot.

Branch: https://bitbucket.org/mdavis4290/molly3-flavonoid-rnaseq/branch/IGBF-3482

Note: I sent the new pdf to Anthony to confirm accuracy but wanted to go ahead and save my code in a branch just in case.

Anthony Response:

Hi Molly,

These look really good! I will begin integrating them into the document.

Thanks again for your help!

Reviewer:

• Please pull the branch and knit markdown
• Does the new VolcanoPlots_NoLabels-SL4-DESeq2-75min.pdf accurately respond to Muday lab request for the volcano plots (see first comment in ticket)

Please describe the code changes made in the branch.

The changes are in a comment above [~aloraine]

I am going to make sure that StatFunctions.R still works in the other markdowns with the new changes.

Testing StatFunctions() changes in all markdowns:

• FindControlVsStressDEGenes-SL4-DESeq2.Rmd = Pass
• FindControlVsStressDEGenes-SL4-edgeR.Rmd = Pass
• FindControlVsStressDEGenes-SL5-DESeq2.Rmd = Pass
• FindControlVsStressDEGenes-SL5-edgeR.Rmd = Pass

Please indicate what changed in the code.
What file changed? What needs to be re-run now that source code has changed?

I looked at the code.
How is the knitted PDF now different?
Please explain in English what you did and how the knitted markdown changed.

It would be a lot cleaner and a lot better if you would create a NEW Markdown called "MakeVolcanoPlotsForPublication.Rmd" that simply reads in the existing results and then uses the volcano plot code to create new SVG files for Muday lab.

That Markdown can explain in plain English what the created plots actually are. It's particularly important to explain the input data used to create the plots.

Adding a whole new purpose to existing code that is already very long and complex is not a good approach, in my opinion.

Updates:

• I created a new markdown for making custom volcano plots = Custom_Volcano_Plots.Rmd
• I don't want to change StatFunctions() so I added my own volcano_plot() function and made edits in my markdown to include labels or no labels under certain statements
• The output pdf file is called VolcanoPlots_NoLabels&Labels-SL4-DESeq2-75min.pdf
• I am currently writing to finalize the markdown and then I will commit the new markdown, knitted pdf of the markdown, and volcano plots output pdf

Commit: https://bitbucket.org/mdavis4290/molly4-flavonoid-rnaseq/commits/d69f08a9bde161908328591f91567f03682de87a
Branch: https://bitbucket.org/mdavis4290/molly4-flavonoid-rnaseq/branch/IGBF-3482

Files to review:

• Custom_Volcano_Plots.pdf
• Custom_Volcano_Plots.Rmd
• VolcanoPlots_NoLabels&Labels-SL4-DESeq2-75min.pdf

Reviewer:

• Pull down the markdown and make sure it knits correctly
• review the knitted pdf and make sure writing and visuals are correct
• review the VolcanoPlots_NoLabels&Labels-SL4-DESeq2-75min.pdf file and make sure it provides the custom volcano plots that muday lab requested (see previous comments)

Change requests:

• Please change the name of the Markdown and its knitted version to MakeCustomVolcanoPlots.Rmd and MakeCustomVolcanoPlots.pdf. This is so that the new Markdown's name will follow the same convention as all the others where we use a verb in imperative voice in the name, e.g., MakeMeASandwich.Rmd versus Sandwich.Rmd
• Please change the name of the PDF file that is produced to "CustomVolcanoPlots-SL4-DeSeq2.pdf" so that it will be more obvious which Markdown created it. I don't think you need to include "NoLabels&Labels" in the name.

The Markdown knits and the PDF output file is created. The code compiles and looks fine.

Questions:

• Is the output PDF file exactly the same as what was sent to Anthony last week?
• I thought the desired image file format was "SVG" - support vector graphics. Instead, a PDF file was made. I guess this change is fine, but I just wanted to confirm that PDF instead of SVG is OK.

Commit: https://bitbucket.org/mdavis4290/molly4-flavonoid-rnaseq/commits/0de18b92acbd220a9a90e40db4c61b682f1c9279
Branch: https://bitbucket.org/mdavis4290/molly4-flavonoid-rnaseq/branch/IGBF-3482

Commit includes: Changing the names of the files.

Answer to questions:

• The output file is the same as the plots I sent last week to Anthony. The only difference is that I combined the labels and no labels onto the same pdf instead of two separate PDF's.
• What was sent to Anthony last week were PDF files and he said they looked great and was going to integrate them into the document (see comments above). So SVG file was not made due to no request for that file to be made. But if you would like me to create this file I can. I also emailed Anthony and am double checking to see if he prefers the PDF or SVG.

Anthony Email Response:

Hi Molly, 

Thank you for checking in! The PDFs worked well so we will move forward with those.

Best,
Anthony

Thanks a lot [~molly]!
Please proceed with PR when ready.

PR: https://bitbucket.org/hotpollen/flavonoid-rnaseq/pull-requests/33

Testing:

Markdown = MakeCustomVolcanoPlots.Rmd

• Muday lab requested plots displaying the temperature dependent DE genes from the final timepoints (75min) for each genotype, without gene IDs on the plot. To do this we made a new markdown that creates custom volcano plots with a custom function. We did this because we did not want to make changes to original functions and markdowns. So this new markdown allows us to take results made from (FindControlVsStressDEGenes-SL4-DESeq2.Rmd -> CvT-DESeq2-SL4.txt) and create custom volcano plots with and without labels at 75min for each genotype.
• Please pull down the new markdown (https://bitbucket.org/hotpollen/flavonoid-rnaseq/src/main/72_F3H_PollenTube/MakeCustomVolcanoPlots.Rmd) and knit the markdown.
• If there are no errors with knitting, next check the MakeCustomVolcanoPlots.pdf and CustomVolcanoPlots-SL4-DeSeq2.pdf output files.
• Do these files answer to their requests?

I pulled code based off of this:

"Please pull down the new markdown (https://bitbucket.org/hotpollen/flavonoid-rnaseq/src/main/72_F3H_PollenTube/MakeCustomVolcanoPlots.Rmd) and knit the markdown."

I knitted the code with success.
A Pdf was produced.
Many Volcano plots exist with that PDF!

Clearly, the plots are from the 75 min time points.
ANd I clearly see plots with the time points and a subsequent plot without the time plot.

This all looks good to me!

Rob

Thanks [~RobertReid]

Moving ticket to done!