Minor Library Preparation Modification Significantly Reduces Barcode Crosstalk in ONT Multiplexed Sequencing

ABSTRACT Barcode crosstalk is a potential source of error in indexed multiplex sequencing that can mimic cross-contamination and generate false-positive signals, particularly in experiments with low-biomass samples and imbalanced designs. Here we identify and quantify barcode crosstalk in multiplexed Oxford Nanopore sequencing and introduce post-ligation pooling (PLP). PLP is a drop-in library-preparation modification that prevents barcode crosstalk, rather than simply mitigating its effects. These results highlight the potential for barcode crosstalk to have detrimental effects in multiplexed long-read sequencing and establish PLP as an immediately adoptable mitigation, especially where negative controls and low-abundance signals inform biological interpretation.