Why would there be a spike in coverage within a gene in RNA seq data? [closed]

Question

I'm looking at RNA seq data in Geneious for Salmonella typhimurium and I occasionally see a spike in coverage for a sequence of RNA within a gene. Why is there a section of the gene that seems to be transcribed much more often than any other part of it? The name of the gene in question is ftsI which is an essential cell division protein that may link upstream cytoplasmic cell division proteins with downstream periplasmic cell division proteins.

Answer 1

Without knowing how your libraries have been prepared and sequenced it is hard to tell if that is due to a technical or biological source.

However, there are a few bias sources in RNA-seq (which aren't an issue in DNA-seq)

One of them is that if you used a "random-hexamer" protocol, they're known to not be so random after all. See: Hansen KD, Brenner SE, Dudoit S. Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Res. 2010;38: e131.

You might also have a contamination from other species that would present a sequence identity for that part more specifically.

Answer 2

Some regions might be more present than others in the sequencing data because some exons might be more often included in the mature transcript (alternative splicing). Check whether the peaks matches with exons. Edit: This will likely not be the case since you are dealing with a bacteria: as far as I know, splicing is very uncommon in bacteria

Another likely explanation could be that there are biases during library preparation or sequencing.

In both scenarios, the biases would be post-transcriptional.

Yet another possibility would be that other transcription units are present within your gene of interest. This sometimes happen, especially when dealing with compact genomes.