Notes about the --barcode-separation parameter:

• Larger values (close to the minimum distance of the code) require more strict matching of the predicted signal for a read to be assigned to a barcode.

• Smaller values (for example, 0.2 and below) allow barcode assignment with an expanded tolerance for errors. For example in the extreme case of separation=0, the measured signal may be right in between two predicted barcode signals.

• If --barcode-separation is set at or above the minimum distance of the barcodes in flow space, no reads at all are assigned to a barcode.

• If --barcode-separation is set close to the minimum distance of the barcodes in flow space, very few reads are assigned to a barcode.

• If --barcode-separation is too small, the risk of cross contamination increases. More ambiguous reads are forced into a barcodeassignment (with a higher rate of error in these assignments).

A rule of thumb for a good --barcode-separation setting is one half of the minimum distance of the barcode set in flow space: