Identification, quantification, and elimination of barcode crosstalk in multiplexed Oxford Nanopore sequencing

2025/11/19 by Qing Dai, Claudia K. Gunsch, Joshua A. Granek · 1 voice
Biochemistry, Genetics and Molecular Biology · Decision Sciences · #Genomics and Phylogenetic Studies #Scientific Computing and Data Management #Genomics and Rare Diseases

paper · pdf · doi:10.1101/2025.11.19.689316

openalex publication_date 2025/11/19 · openalex created_date 2025/11/20 · openalex updated_date 2026/07/15

Abstract

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.

Citations

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Discussions

• A simple library preparation modification significantly reduces barcode crosstalk in ONT multiplexed sequencing doi.org/10.1101/2025... Cookbook for Plant Genome Sequences www.preprints.org/manuscrip [bsky, 1 points, 0 comments]

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