The Singular Genomics Demultiplex software will check for barcode collisions in the input sample sheet relative to the allowed-mismatches and the min-delta parameters. If the minimum hamming distance (the number of different bases) between any two sample barcodes is less than the allowed-mismatches or less than or equal to the min-delta parameter, the Singular Genomics Demultiplex software will report an error indicating the failing comparison. This error is to prevent the generation of nonsensical demultiplexing results.
For example, if the sample sheet contains Sample1 with a barcode sequence of AAAAAAAA and Sample2 with a barcode sequence of AAAAAATT. The hamming distance between Sample1 and Sample2 is 2. Therefore, the maximum allowed-mismatch parameter is 2 while the maximum min-delta is 1. If these two parameters are set higher, then barcode collision would occur. For example, a barcode with the sequence AAAAAAAA is a perfect match (0 mismatches) for Sample1 and has 2 mismatches compared to Sample2. If the min-delta parameter is set at 2 or higher, this perfect sequence matches to Sample1 would be discarded into Undetermined because the best match (to Sample1) is not 2 hamming distances better than the second best match (to Sample2). With the min-delta parameter set to 2 or higher, all perfectly matching reads to Sample1 or Sample2 would be discarded. Since this would be undesirable and nonsensical behavior for essentially all use cases, the distances are checked before demultiplexing and an error is reported instead.