Question: Why is the number of cells called in Cell Ranger equal to the Targeted Cell Recovery for HT?
Answer: For the standard kits, the number of barcodes called by Cell Ranger will be less than the Targeted Cell Recovery, given that some cells will be partitioned together into the same GEMs (cell multiplets).
As shown in the example below, 6 cells were partitioned into GEMs. However, given that two cells were incorporated into the same GEM, there are only 5 GEMs that are associated with cells. Therefore, Cell Ranger would only call 5 cell barcodes.
Because cell loading for the standard assay does not consider Poisson distribution, barcodes detected are lower than target cell recovery.
For the high throughput assay (HT), chip loading considers Poisson distribution, and additional cells are loaded such that our target cell recovery should be the same as barcodes detected.
This graph shows the expected Targeted Cell Recovery / Cell Barcodes Detected called in Cell Ranger (red).
Using the 3' CellPlex Kit for Cell Multiplexing, we support a Targeted Cell Recovery of up to 60,000 cells with HT.
Here is a tabular representation of the singlets and multiplets obtained across of range of Targeted Cell Recoveries.
We can see the number of singlets and also that the multiplet rate is approximately half that of the standard assay at the same Targeted Cell Recovery. If we consider the Targeted Cell Recovery of 30,000 cells for HT:
- ~55,400 cells will be loaded onto the chip
- ~32,000 GEMS will contain one or more cells
- Of these ~32,000 GEMs, we expect ~27,900 to contain one cell (singlet) and ~4,100 to contain more than one cell (multiplet)
- Note that this is a multiplet rate of ~12.8%
- 4,100 multiplets / 32,000 cell-associated GEMs = ~12.8% multiplets
For reference, for the standard assay at Targeted Cell Recovery of 30K, the number of cells loaded would be ~49,500 or approximately 6K fewer. The multiplet rate is ~24%. Assuming proper mixing of the cell suspension and accurate cell counting, this multiplet rate combined with the higher cell loading makes the number of barcodes called in Cell Ranger equal to the Targeted Cell Recovery.
Please also see:
What affects the cell multiplet rate and cell multiplet filtering?
Why is the HT multiplet rate lower than our standard assay?
Products: Single Cell Gene Expression, Targeted Gene Expression, CellPlex