Barcode Crosstalk in ONT Multiplex Sequencing: Quantification and Mitigation Strategies

Abstract Multiplexing in long-read DNA sequencing using Oxford Nanopore Technologies (ONT) increases throughput and reduces costs by ligating specific native barcodes to individual samples. However, this approach carries the risk of barcode misassignment (barcode crosstalk). Indeed, barcode crosstalk was observed after library preparation following the ONT standard protocol, particularly in samples with low DNA input. It is conceivable that misligation of residual native barcodes can occur during the subsequent sequencing adapter ligation step. To systematically investigate barcode crosstalk, genomic DNA of bacterial type strains with varying input concentrations was processed using three library preparation protocols: the former ONT standard protocol (protocol A, valid until July 2, 2025), an updated ONT protocol (protocol B), and an in-house protocol (protocol C) in which samples were pooled only after sequencing adapter ligation. Sequencing was performed with a Nanopore PromethIon. Protocol A showed substantial barcode crosstalk, especially in low-input samples, with up to 2.4% misassigned reads. Protocol B significantly reduced crosstalk to below 0.01%, likely due to improved washing conditions. Protocol C virtually eliminated crosstalk. These findings indicate that older ONT barcoding protocols should be critically reassessed. While the updated protocol improves accuracy, the in-house approach is recommended for low DNA input and high-precision sequencing.