Dear Bioconductor community, I am confronted to a puzzling question. I can't find specific answers on biostars or other websites, and can't figure it out. I have been working on RNA from olfactory epithelium. I had to extract the full sample provided as olfactory receptors can be locally distributed (I had to be sure I have everyone). Some tissues were too "big" so I extracted them in two batches. I prepared separate libraries for each of them. In total I have 27 libraries, and for three samples I have two replicates. I am not sure if there are real technical replicates. I saw one post where it was not recommended to use duplicatecorrelation if I just have a few replicates. If I average the expression, I might lose power on some gene expression because I may capture some genes in the second extraction that were not in the first. The overall library size is quite similar though. Hopefully it should not make a lot of difference except for some genes but I wonder if I should sum the count post normalization (to account for library size difference between replicates) or if I should average them. I hope my question makes sense, any suggestions would be appreciated! Best regards Alice

If you just took some tissue, cut it into two chunks and then processed in parallel, that (IMO) is the definition of a technical replicate. When aligning, I would just tell your aligner that you have two FASTQ files (or four, if PE) for those samples and have all the alignments dumped into a single BAM file. Or you could align separately and just sum up the counts. Your choice.