Hi, I have a theoretical question. I know that it's important to do biological reps when doing differential expression RNA-seq experiments (3 seems to be a minimum). I very much see the point when it's from 3 different frogs/trees/chickens/humans. However I'm encountering situations where people are doing 3 biological reps which are derived from pooled samples (e.g. a pool of lots of individuals, where for practical reasons you can't get enough RNA from one individual).
In this case, what would be the advantage of doing biological reps (for example, doing 3 biological reps, with 3 different library preps/multiplex tags) and what is the difference between this situation and the situation where you just do 3x as much sequencing on one sample, and later randomly split the reads (bioinformatically) into 3 pools?
To me it's a similar situation:
Situation 1: you have a pool of samples, split it into 3, extract RNA and do some sequencing, as opposed to..
Situation 2: you have a pool of samples, extract RNA and do some sequencing, and split it into 3.
What's the difference biologically/statistically? Does it really make sense to have 3x the library-prep/multiplexing costs for situation 1?
The advantage for the latter situation would be a (considerable) saving in library prep / multiplexing costs (I'm talking about Illumina PE sequencing).
Thoughts anyone?
In this case, what would be the advantage of doing biological reps (for example, doing 3 biological reps, with 3 different library preps/multiplex tags) and what is the difference between this situation and the situation where you just do 3x as much sequencing on one sample, and later randomly split the reads (bioinformatically) into 3 pools?
To me it's a similar situation:
Situation 1: you have a pool of samples, split it into 3, extract RNA and do some sequencing, as opposed to..
Situation 2: you have a pool of samples, extract RNA and do some sequencing, and split it into 3.
What's the difference biologically/statistically? Does it really make sense to have 3x the library-prep/multiplexing costs for situation 1?
The advantage for the latter situation would be a (considerable) saving in library prep / multiplexing costs (I'm talking about Illumina PE sequencing).
Thoughts anyone?
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