RNA seq Analysis

I took a look at some RNA seq data that was previously acquired by a post-doc in our lab and I've been doing various things to just practice analyzing large datasets. Here's some interesting things I've done:

(note: K = KW20, U = HI_0659-,  T = HI_0660-,  S = sxy-)

Coverage of sxy mRNA

Nucleotide-resolution of the sxy mRNA reads. There appears to be less expression in the HI_0659 knockout and possibly more in the HI_0660 knockout. The two KW20 replicates appear to differ but I think it is a timing issue since 10 min and 30 min lines in the B replicate appear to be averaged versions of the 10 min and 30 min lines in the A replicate.

 

 Number of fragments that start/end at a particular base (KW20)

The common drop around base #255 (especially at 30 min) interested me for quite a bit as a possible cleavage site but it could also be due to specific secondary structural elements that encourage strand breakage during fragmentation. I plan on creating graphs like these for the rest of the genome so I'll see if this is a common thing.

Timecourse for all genes

I wrote an R script that creates a timecourse-type graph for all the genes under every condition. Here's what they look like:

The competence genes actually stand out because the strains really differ at the 30 min mark:

I also tried normalizing the score a couple different ways. First I normalized by the sum of the fragment counts for the whole genome for each condition, second I used the value at the 75% quartile for each condition. Here's a comparison (chosen at random):

 

1. Normalized by sum

2. Normalized by quartile

I think I prefer the second method.

Finally, one interesting thing I noticed was that the trp operon appeared to have similar characteristics to the competence proteins:

 One trpR regulated operon

  Another trpR regulated operon

trpR

I'm not sure if there is a known connection between the trp operon and competence regulation? There appears to be according to these graphs (and others). Perhaps it's something to pursue.