RNA Seq Fescue Project

[DarrianTalamantes]

Update May 28th

Today, I was able to complete the pipeline up till deseq2 or possibly featurecounts. The Star script is finished, and now, within the Star rule, I am using samtools to separate the mapped reads form the unmapped reads and then separate the large bam file into many small bam files. This will allow me to use feature counts in the next step.

https://github.com/DarrianTalamantes/RNA_seq_fescue/blob/c3a3945ef43f2ef106164fd7afc550bade32c13f/rules/star.smk#L53C1-L78C12

[DarrianTalamantes]

Update 2

Pipeline update

The snakemake pipeline was completed around June 10th. It goes all the way up to feature counts. I decided to make DeSeq2 a standalone R analysis.

DeSeq2 update

I read about and think I created valid methods to visualize the differences between different treatment groups. May need to learn more to add endophyte as third factor.

https://github.com/DarrianTalamantes/RNA_seq_fescue/blob/7c62d4bdc4261827dbdbe6f6c96109474a83993f/Non_Pipeline/DeSeq2_Analysis.R#L113C1-L135C2

[DarrianTalamantes]

Corrected Volcano Plots

Here I redid the volcano plots and made the design more in line with how I think the variation will affect the experiment. https://github.com/DarrianTalamantes/RNA_seq_fescue/blob/c9de2f437184c90c0d3f7664a14efd0e9f371c82/Non_Pipeline/DeSeq2_Analysis.R#L36C1-L39C99

I then also made the filter more stringent on what "genes" I can count for the analysis. https://github.com/DarrianTalamantes/RNA_seq_fescue/blob/c9de2f437184c90c0d3f7664a14efd0e9f371c82/Non_Pipeline/DeSeq2_Analysis.R#L45C1-L47C19

[DarrianTalamantes]

Created First Heatmap.

I am having trouble finding out how to parse the data to make meaningful comparisons with the heatmaps and the dds object. It seems like I'll have to recreate the object every time I wanna look at different comparisons. I say this because the contrast function that I used to make the various heatmaps is only available when running the results function. However the heatmap tutorial does not use this result object. I suppose the next steps are to figure out if you can use the log2foldchange of the results function to make a heatmap.

https://github.com/DarrianTalamantes/RNA_seq_fescue/blob/eaa9078d81f9be5f175154a652fbbd0b06071400/Non_Pipeline/DeSeq2_Analysis.R#L155C1-L179C15

[DarrianTalamantes]

Pairwise Comparisons

I added in epichloe as a factor in my comparisons. Now I can compare two factors at once. Here I do comparisons of the same treatments but epichloe negative vs positive. I notice that the epichloe positive has less DEGs.

[DarrianTalamantes]

Removing CTE25 and CTE31

In these graphs I removed the clones above because they did not have data for certain months. (I will add them in later when I get the data and run it all through the pipeline again)

This looks to see if this changes PC1, PC2, and PC3 to anything related to the treatments. It does not.

seeing that PC1 is still Clone

Seeing if endophyte PC2 and PC3, it is not.

PC2 and PC3 is month and year. Time is a combination of these two.