RNA seq for differential gene expression analysis with only 2 biological replicates

Question

[WMA]

Hello Everyone,

So, unfortunately, I sent my RNA samples to a genome sequencing company; 3 treated and 3 controls, and only 2 treated and 1 control have passed the quality check at their facility.

Also unfortunately, : | , I am going to proceed with the sequencing since this is for my master degree experiment and money and time is a problem. . Any advice? or pep talk haha

Thank youuu

[ATpoint]

Not much to comment here. Results are very unreliable, try to focus on genes with larger fold changes and small FDRs, not something like FC=1.24, FDR=0.049. Develop a hypothesis and confirm it with further experiments to be sure that you're not chasing ghosts. It is a rather "poor" experiment to build a thesis on unfortunately to be honest.

[WMA]

Yes, I know :( Thank you.

[ATpoint]

With confirm I mean try to do experiments that tell you whether the biology the RNA-seq suggests is true. Not validating individual genes, rather pathways or anything you can measure on the cellular level.

[noahhelton98]

I think the only thing to do is on the wet lab side of things - ie if you have genes of interest do RT-qPCR to confirm their differential expression. Unfortunately without multiple replicates the statistics of the RNA-seq experiment won't be super useful, but I think with a little bit of validations experimentally you can still have a great story!!

[WMA]

Thank you all.

Answer 1

[Istvan Albert]

a correction to your title you have only 1 biological replicate for control (aka no replicate)

most modern tools won't quite run with this setup, but you can go back into the past, deseq1, or tophat those will produce something out of it.

my empirical observation is is that when multiple samples fail there are interconnected reasons and those same reasons probably affect the rest of the data, even those that "pass". Having one replicate by design is usually far more reliable than ending up with one sample because the rest failed.

so make sure you are not spending too much time/effort attempting to salvage something that is not salvageable

[ATpoint]

2 vs 1 can be run on all standard tools. The dispersion from the replicated group is then used for the entire analysis.

[Istvan Albert]

Ah interesting, good to know, 1 vs1 did not work once and I assumed the two replicates to be treated independently.

[WMA]

Thank you.