Questions about Reference Sequence in DESeq2

Question

[williamsbrian5064]

I am trying to use Geneious to do some expression analysis using Dseq2. I was going through the tutorial and the reference sequence they used was super complete (NC_006841). It was annotated and it had the name of the proteins, min, max, length, and direction. I'm just wondering where they even got that reference sequence from? The best I can get is just a fasta file with the genome. I'm working with Drosophila so I was guessing there is a reference sequencing out similar to the one they used in their tutorial. (https://www.geneious.com/tutorials/expression-analysis-deseq2/)

Would it be under "Assembly" on NCBI nucleotide?

[h.mon]

Which Drosophila? Did you look at Flybase?

[williamsbrian5064]

Drosophila Melanogaster. I've looked at flybase but I'm not entirely sure what kind of file I'm looking for? They didn't explain much in the tutorial.

[GenoMax]

Contact Geneious tech support since you have a license for the software. They can tell you where the annotations came from.

[williamsbrian5064]

I just don't think mapping the RNA-seq reads to a Drosohpilia reference will tell me much about expression. I feel like I'm missing something here.

Answer 1

[h.mon]

I can't help you with Geneious, as I don't use it - so far, I haven't even been able to pronounce its name, so I pretend it don't exist. There are several detailed workflows and tutorials out there (one example, among many, here), but generally speaking, you have to:

1) map your sequencing reads to a reference genome or transcriptome

2) summarize read counts using an annotation

3) perform differential expression analysis

Flybase has a very complete D. melanogaster genome and annotation, but it should be the same or very close to those from Ensembl or NCBI.