Question: Is index hopping observed for Single Cell 3' libraries sequenced on the HiSeq 4000?
Answer: We have not observed evidence of index hopping when sequencing Single Cell 3' libraries. This doesn't preclude the possibility that it occurs but the design of the 10x Single Cell assay scheme is such that the probability of switching events is extremely low.
For example, because the Single Cell 3' v2 assay contains a 10x Barcode, and because a typical sample only uses 1,000-10,000 out of 737,000 possible 10x Barcodes, the chance of a molecule switching to a new sample index and having a 10x Barcode from the destination sample is reduced by a factor of 100-1,000. For example, if the sample index switching rate is 1% and a sample has 5,000 cells, then there is an effective switching rate of roughly 0.01%. This corresponds to 1 misassigned UMI count per cell (out of typically tens of thousands). In traditional Smart-Seq libraries, the origin cell is specified solely by the i5 and i7 indexes, which gives you an increased risk (factor of 100-1,000) for a sample index to switch from one cell to another.
For Single Cell 3' v3 libraries, the probability is even lower due to the increased 10x Barcode diversity (3,500,000 compared to 737,000 unique 10x Barcodes).
In addition, the Sample Index PCR step as well as the stringent size selection with SPRIselect will help remove excess primer that can lead to index hopping during the 10x Single Cell v2 workflow.
Products: Single Cell 3'