I was wondering what others thought about the multiplet calculations in the various 10x user guides.
I have received some very helpful feedback from 10x, but I think something about the numbers being exactly the same still doesn't seem quite right to me. For example, you can compare the following tables:
3` GEX V2 (single-barcode):
That table comes from page 18 in the V2 user manual.
3` GEX V3 (single-barcode):
That table comes from page 17 in the V3 user manual.
3` GEX V3.1 (single-barcode):
That table comes from page 18 in the V3.1 single-barcode user manual.
3` GEX V3.1 (dual-barcode):
That table comes from page 18 in the V3.1 dual-barcode user manual.
I would usually expect dual-barcode libraries to have less index-hopping than single-barcode libraries (which can be a different cause of multiplets). However, I think this is meant to emphasize multiplets that specifically come from the Chromium Controller. So, in terms of the ballpark measurements, I think think that I agree with that (although I would not conclude that the multiplet rate was robustly slightly higher with the dual-barcode libraries).
My understanding is that those multiplet rates are supposed to be coming from an experiment with human and mouse cells like Figure 2 in Zheng et al. 2017:
Likewise, I think Figure S1a emphasizes how the multiplet rates shouldn't be exactly the same (since you can see the difference from the trendline):
When I noted that the multiplet rates were the same but the loaded cells were slightly different between the user manuals (I assumed from slightly different estimates), I was provided the following explanation: "We load Beads: super-Poissonian and Cells: Poissionian to control for the multiple rate. That is why you see the [multiplet] rate uniform across chemistries though the # of cells recovered vary slightly."
I was also provided a link to the Zhang et al. 2019 review (I think for the term "super-Poissonian"), and the human-vs-mouse experiment described above was mentioned at a later part of the conversation.
What you all of you think?
Thank you very much,