Fig. 1

The workflow of TDFPS-Designer. a Barcode design strategy (initial). Given a k-mer space or a user-defined sequence set, the hash value (Eq. (2)) of each read is calculated, and the sequences are sorted according to the hash value in ascending order. Next, a subset of sequences is selected evenly from the sorted sequence items. The hash values of the selected sequences should have significant distinctness. These selected sequences are then converted into simulated nanopore current signals. Algorithm 1 is used to select the exact signals as barcode signals, ensuring that the DTW distances between these signals are relatively large and greater than a given threshold r. b Demultiplexing strategy. To demultiplex nanopore signals, the barcode regions of reads are identified and compared to the current signals translated from the standard barcode references (items of the barcode sets). Afterwards, the DTW distance matrices between the sequenced barcode signals and the standard barcode current signals are calculated, based on which a top-k selection is carried out to determine the demultiplexing results. c Barcode design strategy (final). Based on the initially designed barcodes, the demultiplexing process is simulated. Barcodes with poor demultiplexing performance are filtered out, resulting in the final kit of barcodes