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bioRxiv · 10.1101/2025.11.19.689316

A simple library preparation modification significantly reduces barcode crosstalk in ONT multiplexed sequencing

Dai, Q.·Gunsch, C. K.·Granek, J. A.
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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.

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Dai, Q., Gunsch, C. K., Granek, J. A.. 2025-11-19. A simple library preparation modification significantly reduces barcode crosstalk in ONT multiplexed sequencing. https://doi.org/10.1101/2025.11.19.689316

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