although (or exactly because of that) I read a lot of manuals and papers dealing with RNA-Seq for the analysis of differential expressed genes, I´m unfortunately don´t really sure about the pipeline/parameter I should use. So, I really hope that some of you can give me any advice.
My experimental setup in short: 2 WT samples and 2 patient samples (same family, same unknown mutation), 150 bp paired-end. I´m searching for genes that are differentially expressed. Especially, I´m searching for a gene which is not expressed in the patients, but in the WT (perhaps because of a deletion of an exon or suchlike). I merged the WT and patient samples and analyze the files with the help of TopHat2 and Cuffdiff2. As I learned in some forum discussions, I can´t use any statistics for filtering, because I don´t have biological replicates. So I´ve just calculated the TPMs and ranked them regarding their log2fold changes. But I have different problems: many of the genes have in one condition FPKM/TPM = 0 and I don´t know, how to proceed with them. Should I add a pseudocount? Or should I eliminate them? But regarding my questioning I ´don´t want to cut them off actually. Can anyone help me with this topic?
Thanks a lot in advance!!