RNA-seq different reads numbers of two samples is trouble?

Question

[szp770]

Hi, for RNA-seq analysis, I did experiments for two samples, each with two repeats, sample1(both repeat1 and repeat2) have 70M raw reads, but sample2(both repeat1 and repeat2) only have 30M raw reads, so for the differential analysis, is it a trouble? Thanks!

[Nitin Narwade]

I think there are multiple factors you need to consider.

1. The treatment/Biological conditions (What if the treatment is impacting on global gene expression program)
2. RNA quantity/quality
3. prior to sequencing Number of cells (Maybe)
4. Sequencing depth
5. Alignment rate
6. Alignment to the specific genomic regions (What if the majority of reads are aligning to the other genomic regions and not on the exons)

etc. etc.

I would just give it try and check the results.

Answer 1

[MatthewP]

Different read depth(coverage) is not a problem for differential analysis, for example you can use DESeq2. But I would recommend to have at least 3 repeats each sample, not 2 repeats. Here I mean biology repeats.