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

Dai, Q.; Gunsch, C. K.; Granek, J. A.

2025-12-16 genomics
10.1101/2025.11.19.689316 bioRxiv
Show abstract

Barcode crosstalk is a potential source of error for multiplexed high-throughput sequencing. It results in reads misassigned to the wrong sample, mimicking cross-contamination. We identify, quantify, and present a solution to barcode crosstalk in the ONT library preparation. Post-Ligation Pooling improves read misassignment more than ten-fold with a minor modification. Our results highlight the problem of misassignment and a simple solution to improve data quality.

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