Hello, I am working on getting differential gene expression between an infected and uninfected population using direct RNA-seq from a MinION. When loading my values into DESeq and creating a heatmap and MAplot things look strange and I'm not entirely clear on how to interpret it.
My MAplot: Graph Obviously doesn't look right but I can't tell why/what this might indicate.
Heat Map: Graph2 Likewise in this heatmap, the DEGs charted all have next to no variance in expression against themselves despite these being the most differentially expressed genes.
For context I have been loosely following this RNA-seq script.
I can't tell if I'm failing to normalize things properly or have some greater misunderstanding of my own process/output. Feel free to ask for more info, I'm not quite sure what is useful for me to supply.
Edit: Also, I am aware of the possible pitfalls of using long-read sequencing for expression data but circumstance requires I use nanopore unfortunately.
Please give some details on the number of replicates and the samples (cell lines, primary).
I'm working with two replicates of an uninfected control, and 8h, 24h, 48h infected samples. Already this is disqualifying in terms of experimental design but due to unfortunate circumstance this is all the data I have to work with for the time being.
Have you seen this repo from Jared Simpson for transcript quantification?