A simple and rapid library preparation for Oxford nanopore amplicon sequencing using reverse complement PCR

Oxford Nanopore sequencing is a powerful platform for rapid and scalable amplicon sequencing; however, library preparation for multiplexed samples is laborious using conventional protocols. In this study, we developed a simplified and rapid library preparation method by combining reverse complement PCR (RC-PCR) with Rapid Barcode Primers from the Rapid PCR Barcoding Kit. Multiplexing barcodes and a tag for ligase-free adapter attachment were incorporated during amplification, which reduce post-PCR processing to a single solid-phase reversible immobilization clean-up step, followed by a 5-min adapter attachment step. We evaluated the performance of this method using two DNA barcode loci as test cases and found that successful amplification was achieved at the optimized concentration of reverse-complement probes. Compared with standard Native and Rapid Barcoding Kit protocols, our method substantially reduced hands-on time, while preserving full-length amplicons. These results indicate that our protocol provides a rapid and efficient alternative for nanopore amplicon sequencing and is particularly suited to time-sensitive applications.