Question: Uneven number of technical replicates for biological replicates in RNA-seq
gravatar for microbiotaiota
8 weeks ago by
microbiotaiota20 wrote:

Hi everyone, I am new to RNA-seq analysis and I'm trying to understand what to do with my data.

I have 60 biological samples (30 control and 30 diseased, paired samples). Each biological sample has 1 or two technical replicates. I will concatenate the .fastqc files for technical replicates to yield the biological replicate file. My issue is that biological replicates with only 1 technical replicate will have proportionally less reads than biological samples with 2 technical replicates. Is this correct? Is there anyway to overcome this? Is this an acceptable method?

Alternatively, I can work with the technical replicates and then average the reads for biological samples that have 2 technical replicates, so that proportionally biological replicates with 1 technical replicate are proportional to biological replicates with 2 technical replicates.

Either method (concatenating or averaging) when it comes to differential expression will compare relative expression values.

Reading the below Biostars discussion was useful: Technical replicates in RNAseq


ADD COMMENTlink modified 8 weeks ago by Ido Tamir5.1k • written 8 weeks ago by microbiotaiota20

I think maybe concatenate technical replicates and take this action as one batch group? For example with replicates is 1 without replicate is 0.

ADD REPLYlink written 8 weeks ago by MatthewP730

Thank you. this was confusing me

ADD REPLYlink written 8 weeks ago by microbiotaiota20

if DEG is the goal of this analysis, than one of the first steps in this process is correction for library size, so whether one sample has two or no or more technical repeats is not an issue. Much more critical is that you have biological replicates for each sample!

ADD REPLYlink modified 8 weeks ago • written 8 weeks ago by lieven.sterck8.5k

Thank you, I'll go ahead and concatenate the files.

ADD REPLYlink written 8 weeks ago by microbiotaiota20
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