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

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Title: A simple and rapid library preparation for Oxford nanopore amplicon sequencing using reverse complement PCR.
Authors: Sonoda S; National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa City, Chiba, 277-0882, Japan. Electronic address: sonoda@nrips.go.jp., Watanabe D; National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa City, Chiba, 277-0882, Japan.
Source: Analytical biochemistry [Anal Biochem] 2026 Sep; Vol. 716, pp. 116163. Date of Electronic Publication: 2026 May 27.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Elsevier Country of Publication: United States NLM ID: 0370535 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0309 (Electronic) Linking ISSN: 00032697 NLM ISO Abbreviation: Anal Biochem Subsets: MEDLINE
Imprint Name(s): Publication: <2000- > : San Diego, CA : Elsevier
Original Publication: Orlando Fl : Academic Press
MeSH Terms: Polymerase Chain Reaction*/methods , Nanopore Sequencing*/methods , Sequence Analysis, DNA*/methods , Gene Library* , Nanopores*, DNA Primers
Abstract: 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.
(Copyright © 2026 Elsevier Inc. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Amplicon sequencing; Oxford nanopore sequencing; Rapid library preparation; Reverse complement PCR (RC-PCR)
Substance Nomenclature: 0 (DNA Primers)
Entry Date(s): Date Created: 20260528 Date Completed: 20260611 Latest Revision: 20260611
Update Code: 20260611
DOI: 10.1016/j.ab.2026.116163
PMID: 42208723
Database: MEDLINE
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  Data: 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.&lt;br /&gt; (Copyright &#169; 2026 Elsevier Inc. All rights reserved.)
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