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

Barcode based multiplexing enables scalable sequencing on ONT platforms but carries risk of barcode crosstalk. While analyzing environmental samples using the standard ligation-based barcoding kit SQK-NBD114, we detected false-positive reads in negative controls, prompting a quantitative investigation. Using defined microbial DNA and synthetic blanks, we benchmarked several workflows and identified the pre-ligation pooling step as a key contributor to crosstalk. We introduce a post-ligation pooling protocol that reduced misassigned reads from 0.36 percent to 0.023 percent, with further improvement to 0.017 percent when combined with ONT's Short Fragment Buffer wash. These refinements eliminated spurious reads in blanks and improved F1 scores to greater than 99.98 percent across samples, without compromising yield. The PLP plus SFB workflow is simple, compatible with existing kits, and enhances demultiplexing accuracy in barcoded ONT sequencing. It enables confident multiplexing for sensitive applications, including metagenomics, clinical assays, and low input samples, where even rare cross-sample contamination can lead to misleading results.