Generating barcodes for nanopore sequencing data with PRO.

DNA barcodes, short and unique DNA sequences, play a crucial role in sample identification when processing many samples simultaneously, which helps reduce experimental costs. Nevertheless, the low quality of long-read sequencing makes it difficult to identify barcodes accurately, which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run. Here, we present a comprehensive study of the generation of barcodes and develop a tool, PRO, that can be used for selecting optimal barcode sets and demultiplexing. We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete. For practical applications, we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy. Specifically, the maximum size of the barcode kits designed by PRO is 2,292, which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies (ONT). We validated the performance of PRO on a simulated nanopore dataset with high error rates. The demultiplexing accuracy of PRO reached 98.29% for a barcode kit of size 2,922, 4.31% higher than that of Guppy, the official demultiplexing tool. When the size of the barcode kit generated by PRO is the same as the official size provided by ONT, both tools show superior and comparable demultiplexing accuracy.