Hi Lisa,

Which package do you use to count reads per gene? In my experience, I use featureCounts, which count the reads that align to the exons. Thus, DESeq2 differential expression analysis will be specific for those reads. Also, you can inspect your bam file to visualize the coverage of your reads and check if they align to the exons or introns.

Regards,

Andrés

Thank you Andres-I will take a look at featureCounts. I got the data from someone else, but can ask those questions.

The gene I am investigating is not annotated completely in Gencode, and a deleted exon (which is annotated) is not impacting the DGE analysis. (Even though by ddPCR it is the highest exon expressed at that age.) I've done deep sequencing on it and still confirmed high expression for this exon so I think their is something strange about the DGE analysis. Do you think there could be anything else that might account for the difference?