3' Single Cell RNA Seq Reads mapping to promoter region of gene

Question

[rekharichardson]

Hi! I am trying to visualize my reads from a 3' Single cell RNA seq run in IGV. I am using the bam file from the cell ranger output. For some reason, I am encountering a lot of genes where the reads appear to be piling up at the promoter. This doesn't make a whole lot of sense to me, because I was expecting the read pileups to be at the 3' end of the gene. I am not sure how I am getting these reads.

For example, one of the genes I am looking at is nppb. It looks like there is a read pileup at the promoter region there. When I try to make a feature plot for this gene, it is absent from my data (making me think that these reads are not being associated with the nppb gene). I have two questions about this. 1) Do you think that these reads are supposed to be associated with the gene nppb? If so, why are they piling up here and not at the 3' end? Do you think this is a sequencing issue or a mapping issue? 2) If these reads are not supposed to be associated with the nppb gene, where are they coming from?

Thank you so much in advance.

Answer 1

[benformatics]

Nppb is a ridiculously tiny gene (less than 1.5 kb in its full length - let's not even mention splicing!). If you look up how the 10x protocol works, then it is fully expected to have fragments of this size present (i.e of the entire gene length). So yes if they are exonic they are likely reads from Nppb and there is probably no issue. In terms of your feature plot... no idea.

Also how are you even defining the "promoter"? The promoter wouldn't be expected to produce reads present in an RNA-seq experiment. So attributing reads upstream of the 5'-most exon to the gene would go against the experimental expectations.

There is a MIRc inside the first intron but unlikely that will affect the promoter.