Hi all,
I want to make following comparison:
Sample-time1 + Sample-time2 + Sample-time3 + Sample-time4 (sequencing of pooled RNA)
vs
Sample-time1 + Sample-time2 + Sample-time3 + Sample-time4 (samples sequenced individually and in-silico pooled)
Each approach has 3 replications.
The aim is to check if there are differences between the two approaches.
And I have a question, at which level should I do in-silico pooling? Joining fastq files, counts ...
Pooling at the count-level is easier, but if it is correct should I calculate sum or average of values?
I know pooling is a frequently disscussed topic and there are many resources about it, but it concerns mainly physical pooling. I haven't found any information about in-silico pooling.
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boczniak767
I suppose it depends on what part of the process you are looking to evaluate. I would be inclined to generate a separate counts matrix for each sample, which will allow you to run some QC you wouldn't otherwise be able to do if you concatenate FASTQ files. I would them sum counts across samples within each replicate.
Thanks for input, I'd like to check if both methods give comparable results. The "pooled" data are already analyzed (QC result is fine) and I'm working on joint exploratory data analysis of data from both methods.
I agree, there is no reason to concatenate FASTQ files, because one would lose some information that way.
Each replicate should be measured as it was collected and then the results should be combined at the end.
In the end, what you want is to compare variability across the two approaches, and for that you would need to know how much it varies within each approach.
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