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Let's say we have two samples, sample 1 and sample 2, and we're running an RNA seq experiment. Consider genes A and B for both. For simplicity, let's say that when we run the RNA seq experiment, we get the following results:

gene A sample 1 count: 2

gene B sample 1 count: 4

gene A sample 2 count: 2000

gene B sample 2 count: 4000

Based on my understanding, RNA seq gives relative results. So I guess in this guess, how can you tell whether either one of the following are true:

1- You sequenced sample 2 a thousand fold more compared to sample 1

2- Sample 2 really has a thousand fold more expression in both genes A and B

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  1. We cannot be sure about this. For all we know, they could have been sequenced to the same depth and there is a biological difference that drives the 1000x (ridiculously large) fold change.
  2. See above

The only thing you can say for sure is that gene B is expressed twice as much as gene A in samples 1 and 2. That's what "relative" means - relative expression of genes within a sample.

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  • $\begingroup$ thanks this was helpful. just want to see what others have to say as well before I accept the answer $\endgroup$ Jun 13 '20 at 14:41
  • $\begingroup$ Oh of course. I’m no expert at this, but this is what I’ve encountered in our experiments. $\endgroup$
    – Ram RS
    Jun 14 '20 at 6:08
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You obviously have to normalize for differences in depth followed by a proper statistical test to get fold changes and significances. Sequencing depth is a technical confounder that has to be removed, and that is what normalization does. You have been posting a lot of very basic RNA-seq questions, I suggest you read the RNA-seq Hitchhikers guide: https://peerj.com/preprints/27283/ before posting new questions first.

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The process of RNA-seq is a sampling process of all the RNA molecules. We cannot make reliable comparisons before we know how deep we sample. And since we know it is a sample from a population, the absolute value does not make sense.

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  • $\begingroup$ So can we know how deep we sample? And knowing that, what would that allow us to do exactly? And how does this tie to sequencing "depth"? $\endgroup$ Jun 13 '20 at 14:38
  • $\begingroup$ This is usually the sequencing depth when I think about this problem. $\endgroup$
    – Phoenix Mu
    Jun 14 '20 at 2:01

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