Reads lacking an insert sequence between gene-specific primers or mapped to off-target loci are discarded. We use amplicon read completeness in each barcode to call cells from barcodes and require barcodes to have at least 80 % data completeness for the working amplicons. This method has been effective at improving the data completeness in called cells and reducing the noise from bad barcodes.
The barcodes are identified as cells using a three-step process:
- We select the barcodes that pass a total reads cutoff, which is defined by the number of amplicons in the panel times 10 reads.
- We calculate the panel performance as 0.2 * the mean of all amplicon reads for all qualified barcodes. If this value is more than 10, then we use 10 as the threshold. Otherwise, the threshold is 0.2 • the mean value. We identify good-performing amplicons as those that pass the panel performance threshold.
- We identify the barcodes that have at least 80 % of these good-performing amplicons. These barcodes are the called cells.