edgeR practice: crab 2019 data

Haven’t posted in a long long time… so in today’s post I’ll detail what I’ve been up to with the crab project: paper progress and trying out edgeR for differential gene expression analyses.

Testing out edgeR

Non-crab test

We used edgeR in the Ecology of Infectious Marine Disease course at FHL in summer 2019 to find differentially expressed genes in eelgrass with and without exposure to a pathogen, Labyrinthula zostera. Very convenient timing that I took that class becuase it was before any analyses were being performed for the crab project.

I went back thorugh the github repo we had from the class and tested out edgeR by reproducing this script: https://github.com/eimd-2019/project-EWD-transcriptomics/blob/master/analyses/EdgeR/GRASS_START_GENE_MEE_Contrasts_CAB_7_10_nonZostera_at.R

Things went smoothly and I was able to reproduce everything after I got the ‘targets.txt’ file from Colleen.

Trying with crab data: 2019 dataset

The 2019 RNAseq data was a combination of temperature treatments, but there were four main groupings:

  • Day 26 Uninfected
  • Day 26 Infected
  • Day 12 Uninfected
  • Day 12 Infected

Steven and Sam helped with getting a file of gene counts for each sample (link: here)

And I made a ‘targets’ file that lists out the samples and their treatments (link: targets_2019.txt). I originally had the sample treatments listed as “26_Uninf”, etc., but there’s a part in the script where contrasts between different groupings need to be made, and having numbers in the treatment descriptions was messing things up, so I changed it to:

  • “E” = day 12 (stands for ‘earlier’)
  • “L” = day 26 (stands for ‘later’)
  • “U” = uninfected
  • “I” = infected
    After that was fixed, the code worked well.

Link to the Rmd file of testing out edgeR with the crab 2019 dataset:

  • markdown with figures: here
  • script: here

It worked, but I need to go through more slowly and with the manual at hand to make sure all the steps make sense for the crab data. (Manual for edgeR: here

Crab project updates

Paper draft: here

methods:

  • have written methods for RNA extraction for each data set
  • have written what I can for methods of crab collection and experimental set-up

Results:

  • starting to get some differential gene expression progress in that I’m testing out edgeR and working on comparisons
  • will work on making a mortality figure

Other: We got the data back from NWGC that I sent out a while back (Sam’s working with it) - notebook post detailing what the samples are: here

General timeline:
This week (4/13) - focusing on crab results
Next week (4/20) - working on discussion
week after (4/27) - working on intro

Goal to have draft of full thesis done by somewhere around May 4th to send to committee.

Written on April 15, 2020