I thinking of using a single reference genome, however I was thinking of concatenating the two reference genomes together to create this. I'm not sure if this is an overly simplistic approach, or if it would be appropriate in this case. I've seen an approach like this in this paper for example: https://pubmed.ncbi.nlm.nih.gov/32442239/
Currently working on a bulk RNA-seq project with two non-model species in the same genus. Each species has its own genome and annotation, and i've using nf-core/rnaseq with Salmon for quantification. I’m running DESeq2 for differential analysis. Not sure the best way to do the cross-species part. Should I be mapping orthologs after DE and comparing those? What do people usually do with species-specific genes that don’t have clear orthologs? And what’s the usual approach for functional analysis like GO or pathways when working across species?
I also have F1 hybrids and F2 backcrosses that I’d like to include somehow, but I’m not sure what the best approach is.
If anyone knows of bulk RNA-seq studies that compare species like this, that'd also be really helpful
2 answers
Hard to generalise without knowing exactly what you want out of this project, but there are some general pointers based on my experience.
Should I be mapping orthologs after DE and comparing those?
This is probably the simplest and most accurate approach, but also the most limiting. You may need to limit analyses to 1-to-1 orthologs as 1-to-many and many-to-many orthologs can make interpretation considerably more tricky.
Another alternative is just map all the reads to a single reference genome rather than both. Makes interpretation easier, but definitely skews data towards whichever samples are evolutionarily closer to the reference genome you are using. Even within the same species, population level differences affect mapping efficiency.
What do people usually do with species-specific genes that don’t have clear orthologs?
If the whole goal of the project is to compare expression patterns across different species, these genes are not the most useful. More of a footnote where you can describe general patterns where it looks like the species respond differently to their environment. Maybe there is more here, but nothing comes to mind at the moment.
And what’s the usual approach for functional analysis like GO or pathways when working across species?
You can annotate GO terms yourself without too much hassle if you have the compute. The alternative is inherit GO terms or other functional annotations from orthologs if one assembly is annotated and the other isn't. Again, this can get messy and is probably more hassle than just annotating yourself.
I don't think that is a good idea. By concatenating your two references you don't solve the unique genes or orthologs issues. If anything, you are probably just splitting reads more making it even more difficult to understand results.
I faced a similar issue previously and outlined my approach here.
- Investigating differential expression within species using DESeq2
- Inferring orthogroups with Orthofinder, then mapping differentially expressed genes to these orthogroups
- Testing for overlaps in differentially expressed orthogroups between species (and assessing significance)
I know publication isn't always a guarantee of "correctness" per se, but we didn't get any pushback from reviewers for our paper.
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