Fraction reads in cells CELLRANGER

Question

[GiuliaAC]

Hi,

I’ve just analyzed single cell sequencing human samples (sequenced by nextseq550) with CellRanger 3.0.2 (mkfastq and count).

Looking at the web_summary, I got a really low percentage in Fraction Reads in Cells (about 50%). Is it due to sequencing problems or computational analysis?

Thank you

[nlehmann]

It's hard to say without seeing your data. Could you share your report or a screen shot ?

Answer 1

[swbarnes2]

That's a library prep thing.

Ideally, you want a small number of cell barcodes having a ton of reads, and if a bunch more cell barcodes have just a few reads each, you figure that's ambient cruft that didn't come from cells, and you ignore it.

Low % of reads in cells means that lots of your reads are from barcodes that don't have a whole lot of reads, which means it's all ambient cruft, and you shouldn't be counting it.

I suppose if you messed around with the white list of barcodes,or used a too-low "force-cells" argument, you could make the software draw that conclusion, but if you ran everything on default, and you got that message, it's not you, it's the library prep.

However, Cellranger 3.1.0 has a smarter algorithm for deciding what is or isn't a good cell, so you might get a better result if you upgraded.

[FantasticAI]

I got the same alert by using cell ranger V5.0, everything is on default. Do you think it is library prep thing too?