Reason(s) for low "Fraction Reads in Cell"
"Fraction Reads in Cell" values in the summary metrics came out around 30% from in first samples of snRNA sequencing. By definition, it sounds like a simple term to understand, but I couldn't figure out the reason why the value is so low and what I can do it to fix it in the future.
I would greatly appreciate any suggestions!
Thanks a lot in advance!
Re: Reason(s) for low "Fraction Reads in Cell"
Low fraction of reads in cell can primarily be caused due to:
1) The lower fraction of reads in cells can be caused by ambient RNA (background) in your sample. This ambient RNA comes from lysed/dead cells in your sample. So Cell Ranger is able to confidently align the reads from ambient RNA to the transcriptome/genome but they are not associated with a valid cell containing GEM.
2) Low value of this metric may also indicate that the cell-calling heuristic did not work well. For example, there may be higher variation in RNA content than expected (more cells with lower RNA content), say in tumor cells.
Cell Ranger's algorithm for partitioning barcodes as cells vs barcode is based on the idea that barcodes for cells should have more transcript counts associated with them than the background barcodes. This can be visualized by the UMI vs barcode plot in the web_summary. More details on the cell filtering algorithm can be found here and here. If you suspect that Cell Ranger's cell calling algorithm did not work well for your sample, please re-run cellranger count again with --force-cells option to call the expected number of cells.
Please feel free to email at firstname.lastname@example.org in case you have further questions.