High-performance protocol for ultra-short DNA sequencing using Oxford Nanopore Technology (ONT)
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
In recent years, Oxford Nanopore Technologies (ONT) has gained substantial attention across various domains of nucleic acid research, owing to its unique advantages over other sequencing platforms. Originally developed for long-read sequencing, ONT technology has evolved, with recent advancements enhancing its applicability beyond long reads to include short, synthetic DNA-based applications. However, sequencing short DNA fragments with nanopore technology often results in lower data quality, likely due to the absence of protocols optimised for these fragment sizes. To address this challenge, we refined the standard ONT library preparation protocol to improve its performance for ultra-short DNA targets. By utilising the same core reagents required for conventional ONT workflows, we introduced targeted alterations to enhance compatibility with shorter fragment lengths. We then benchmarked these adjustments against libraries prepared using the standard ONT protocol. Here, we present a comprehensive, step-by-step protocol that is accessible to researchers of various technical expertise, facilitating high-quality sequencing of ultra-short DNA fragments. This protocol represents a significant improvement in sequencing quality for short DNA sequences using ONT technology, broadening the range of possible applications.
Editor(s)
Chemin, Isabelle
Date Issued
2025-04-29
Date Acceptance
2025-03-27
Citation
PLoS One, 2025, 20 (4)
ISSN
1932-6203
Publisher
Public Library of Science (PLoS)
Journal / Book Title
PLoS One
Volume
20
Issue
4
Copyright Statement
© 2025 Žemaitis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/40299915
PII: PONE-D-24-57544
Subjects
Sequence Analysis, DNA
Nanopores
High-Throughput Nucleotide Sequencing
Gene Library
DNA
Nanopore Sequencing
Publication Status
Published
Coverage Spatial
United States
Article Number
e0318040
Date Publish Online
2025-04-29