Žemaitis Lukas, Palepšienė Rūta, Juzėnas Simonas, Alzbutas Gediminas, Burgi Pierre-Yves, Heinis Thomas, Charmet Jérôme, Angeloni Suter Silvia, Jost Martin, Raišutis Renaldas, Simmel Friedrich, Galminas Ignas
Department of DNA data storage, Genomika, Kaunas, Lithuania.
Ultrasound Research Institute, Kaunas University of Technology, Kaunas, Lithuania.
PLoS One. 2025 Apr 29;20(4):e0318040. doi: 10.1371/journal.pone.0318040. eCollection 2025.
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.
近年来，牛津纳米孔技术公司（ONT）因其相对于其他测序平台的独特优势，在核酸研究的各个领域获得了广泛关注。ONT技术最初是为长读长测序而开发的，随着技术的发展，近期的进展使其应用范围得到扩展，不再局限于长读长，还包括基于短的合成DNA的应用。然而，使用纳米孔技术对短DNA片段进行测序往往会导致数据质量较低，这可能是由于缺乏针对这些片段大小优化的方案。为应对这一挑战，我们对ONT标准文库制备方案进行了优化，以提高其对超短DNA靶点的性能。通过使用传统ONT工作流程所需的相同核心试剂，我们进行了有针对性的改进，以增强与更短片段长度的兼容性。然后，我们将这些调整与使用标准ONT方案制备的文库进行了对比测试。在此，我们提供了一个全面的、循序渐进的方案，不同技术水平的研究人员都可以使用，有助于对超短DNA片段进行高质量测序。该方案代表了使用ONT技术对短DNA序列进行测序质量的显著提升，拓宽了可能的应用范围。
