What information can we extract from this experiment: RNA-seq of a High-salt tolerant cultivar and a wildtype, both were cultivated in a NORMAL salt condition?

Question

[GiantSilverSoy]

Hi, recently I was assigned of analyzing the 2 RNA-seq data of:

• A high-salt tolerant cultivar induced from ionizing radiation exposure
• And its parental wildtype

The difficulty is that they were both cultivated in a normal salt condition. No RNA-seq data in the high-salt condition are available to compare. I think Differential expression analysis is meaningless in this case. SNP analysis might be better but still the SNPs on transcripts that only expressed in high-salt condition cannot be found.

Has anyone ever encountered this (bad) experiment design? and what do you think?

Thanks

[ATpoint]

Your question is lacking details. What is the aim of the study, what is the exact design, how many replicates per group, and what is actually the question to be answered.

[GiantSilverSoy]

There are 3 replicates per group. The initial aim was to investigate which genes were responsible for the high-salt tolerance. However, they failed to carry out a high salt experiment. In the end there were only 2 groups, mutant and WT, both in normal condition. And that’s why I said it was a bad experiment design.

My question is: what types of analysis (DE, Variant calling or anything else) that I should do to extract something useful out of these data?

[ATpoint]

First of all, do not give yourself a hard time figuring out what you should analyze. Ask the provider of the data what they want, and tell them about the limitations of the data towards the experimental design. You are not responsible for poor experimental design, so do not accept that they blame you for it.

That having said, what you can always do is to check which genes are differentially expressed. n=3 is small, but maybe you get something with edgeR, DESeq2 or whatever you prefer. Variant calling is generally difficult with RNA-seq data, and for this scenario, I do not see what you could get out of it. This phenotype is most likely a lot more complex that a few SNPs. It is difficult to advice anything, because I of course do not know about the details. The question would also be how this tolerant cultivar was generated. Was this traditional breeding or is this a transgenic organism? Therefore, talk to the wet-lab guys and openly communicate the issues and ask exactly what they want. It is not your job to figure out for them what they "might maybe probably potentially" want to do with the data towards the biological question. This has to come from them.

Answer 1

[benformatics]

With your data you can find out which genes are differentially expressed in the salt-tolerant cultivar compared to the wild-type under wild-type conditions (assuming WT means low/no salt).

If high-tolerance to salt is due to an acute transcriptional mechanism

If response/resistance to high salt concentrations is an acute transcriptional/transcriptomic change then you may not discover much (but a negative result is still an informative result). In this situation you would expect exposure to salt (or specific salt %) to induce transcriptional changes and thus because this did not occur during in your experiment, you would see minimal/no changes.

If high-tolerance to salt is due to a chronic transcriptional mechanism

In contrast, if the resistance to high salt concentrations is a chronic/global transcriptomic change then you may see salt-tolerance related genes (if such things exist) or other related pathways to be differentially expressed between the two samples.