Confirming the Analysis with bar plots of gene expression:

• Changed log-fold change threshold to 0.5 and only got 1 DE gene at just 30 min. Changed to 0.1 and got 5 DE genes at just 30 min. Changed to 0.05 and still only got the exact same 5 DE genes at 30min from before. Change to 0 fold change threshold and got 6 DE genes at 30 min. These genes are:

1. Solyc09g092690.3
   
2. Solyc04g011440.4
   
3. Solyc06g076560.2
   
4. Solyc01g102960.3
   
5. Solyc03g117630.1
   
6. Solyc03g120090.1
   

• Reviewed the 6 DE genes at 30 min in the expression barplot Dr. Loraine made to confirm analysis (https://ann-loraine-gmail.shinyapps.io/72_F3H_PollenTube/) seen above. These genes meet the statistical design of being DE across genotype VF36 plus ARE and the temperatures 28C and 34C. In other words, we are answering the question: is the condition effect different across genotypes?
• Wrote in more detail of what was used and why in the markdown.

Branch: https://bitbucket.org/mdavis4290/molly3-flavonoid-rnaseq/branch/IGBF-3472

Reviewer:

• Please pull changes from FindTreatmentEffectAcrossGenotypes-DESeq2.Rmd markdown branch and make sure everything runs properly with knit
• Review writing and comprehension of the analysis
• Is the interaction term and log2foldchange explained properly
• Are the output files present in results folder (MvW-temp-[assembly].xlsx) and structurally make sense
• Do the barplots in the previous comment of the 6 DE genes support the analysis? If so then they can also be added to the Flavonoid repo. Another ticket could be made for this.

I have pulled down the code.

Running this on console because it asked me to.
tinytex::install_tinytex()

Ran the code in RStudio.

Got the 2 xlsx files (SL4 and SL5).
They look to have 4 tabs, one for each time point.

And look as one would expect.

As expected, those p-values leave us with nothing!
That does seem to not coincide with the bar plots that we see in the comments above. So confusing.

There should be 6 DE genes when the threshold for log2foldchange is 0. The code from the branch in this ticket should have a threshold of 0 and the 6 significant genes are only present at 30 minutes. The code is at the very top of the markdown that indicates the threshold. Do you see this occurrence? It is also stated in the knitted pdf how many DE genes are present at each time chunk. The 6 genes I found to be significant are the 6 genes in the barplots in the above comment. Thanks!
[~RobertReid]

Confirmed !!

I see the 6 genes below the P-adjusted cutoff at 30 minutes (SL4) once the LFC threshold is set to 0.

Are there any other pieces here that need review?

That is all that needs to be reviewed. Thank you!

Creating a PR!

Pull Request: https://bitbucket.org/hotpollen/flavonoid-rnaseq/pull-requests/29

Testing:

• Had no issues knitting the markdown.
• Results files are saved and organized properly.
• Comprehension & grammar are fine. Might need to improve further later on.

Moving to done!