TDFPS-Designer: an efficient toolkit for barcode design and selection in nanopore sequencing

Abstract Oxford Nanopore Technologies (ONT) offers ultrahigh-throughput multi-sample sequencing but only provides barcode kits that enable up to 96-sample multiplexing. We present TDFPS-Designer, a new toolkit for nanopore sequencing barcode design, which creates significantly more barcodes: 137 with a length of 20 base pairs, 410 at 24 bp, and 1779 at 30 bp, far surpassing ONT’s offerings. It includes GPU-based acceleration for ultra-fast demultiplexing and designs robust barcodes suitable for high-error ONT data. TDFPS-Designer outperforms current methods, improving the demultiplexing recall rate by 20% relative to Guppy, without a reduction in precision.