I'd like your opinion on the best way to compare two technical replicates.
First, I give you some preliminary information on the experiment. I isolated total RNA from the same sample (mammalian cell line) by usining the same kit (Qiagen) but two different protocols for DNA elimination (using the Qiagen gDNA eliminator column or the Qiagen DNase). Looking at the RNA at the TapeStation, the first protocol led to the complete loss of the RNA 28s subunit. This was weird...so I decided to go deeper.
As the final goal is the gene expression analysis (DE), RNA-seq libraries for these two samples were constructed and sequenced. I obtained a different coverage for the two libraries: 28 M reads and 44 M reads. Most probably due to the flowcell balancing that was not optimal (as declared by the service provider).
Now I'd like to know if the two libraries are similar (theorically they should be identical, but we know that this is impossible!), and which is the best one. I checked the number of expressed genes counting the genes with a minimum of x raw reads, but this is cearly impacted by the coverage. So the 44M llibrary showed a higher number of genes.
Do you have any suggestion to evaluate the similarity between the two samples and to determine which is the best one?
Thank you all Best Marianna