Question: Why is the multiplet rate different for the Next GEM Single Cell 3’ LT v3.1 assay when compared to other single-cell applications?
Answer: The multiplet rate is the fraction of cell-associated barcodes estimated to be associated with more than one cell. In standard Next GEM Single Cell assays, this rate is driven by the fraction of cell-containing GEM's that contain more than one cell.
The Next GEM Single Cell 3’ LT v3.1 assay was designed with a barcode diversity of ~9.2K total barcodes. The multiplet rate for the 3’ LT v3.1 assay is driven by the increased rate of barcode collisions for GEMs containing cells in addition to the fraction of cell-containing GEM's that contain more than one cell.
In the above example, each gel bead color and letter represents a unique 10x Barcode. There are gel beads with the same color and letter due to the lower barcode diversity in the emulsion. The GEM containing the Grey Gel Bead (E) has two cells and the Purple Gel Beads (B) result in a barcode collision for GEMs containing only one cell. The GEMs containing both Grey and Purple Gel Beads result in a barcode multiplet for the 3’ LT assay
The Next GEM Single Cell 3’ LT v3.1 assay has a similar range of estimated multiplet rates within the supported cell recovery as compared to standard Next GEM single-cell assays:
- Next GEM Single Cell 3’ LT v3.1 assay
- Supported cell recovery: 100-1,000 cells
- Estimated Multiplet rates within supported cell recovery: ~0.8 - ~8%
- Next GEM Single Cell 3’ v3.1 assay
- Supported cell recovery: 500-10,000
- Estimated Multiplet rates within supported cell recovery: ~0.4 - ~8%
When using the 3' LT v3.1 assay at 10-fold fewer cell capture relative to our standard 3' v3.1 assay, the comparable multiplet rate enables customers to use the LT kit at 10-fold fewer cells for pilot experiments and then scale up with minimal batch effects between kits.
Products: Single Cell Gene Expression