用于克隆爆发分析的开源纳米孔测序工作流程提供了短读水平的准确性。

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Journal of Clinical Microbiology Pub Date : 2025-08-13 Epub Date: 2025-07-18 DOI:10.1128/jcm.00664-25
Nick Vereecke, Thomas B Yoon, Ting L Luo, Brendan W Corey, Francois Lebreton, Patrick T Mc Gann, John P Dekker
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引用次数: 0

摘要

在这项工作中,我们提出了一个优化的纳米孔长读测序工作流程,用于使用开源工具构建克隆爆发的流行病学分析。选择一组不相关的临床铜绿假单胞菌分离株(n = 10)进行工作流程优化,并使用改进的快速条形码策略制备测序文库,该策略包含温度梯度,以提高高gc含量基因组的性能。测序数据用于对dorado套件(v0.9.1)的性能进行基准测试,包括其基调用器、预组装读取纠错和组装后抛光算法。所有的长读片段和核心基因组多位点序列分型(cgMLST)分别用Flye和pyMLST进行。结果与标准参考Illumina短读方法进行了比较,在核心和全基因组水平上确定了不一致的位置。dorado sup@v5.0.0碱基调用的最佳性能与dorado纠错和dorado抛光及其细菌模型。然后用四组回顾性医院爆发分离物验证了该工作流程,包括肺炎克雷伯菌(n = 12)、铜绿假单胞菌(n = 11)、屎肠球菌(n = 10)和金黄色葡萄球菌(n = 10)。与Illumina短读参考相比,从优化管道中获得的纳米孔组件显示出完全一致的基于cgmlst的最小生成树。在全基因组水平上,也观察到高度的一致性,与短读组装相比,每个基因组只有两个不一致的位置。这种优化的文库制备和开源计算工作流程使纳米孔克隆和爆发分析的性能可与Illumina短读测序相媲美,并将为医院感染控制做出重要贡献。重要性:在过去的十年中,细菌全基因组测序已经使用高精度短读测序进行。最近,使用牛津纳米孔技术(ONT)仪器的长读测序已经成为一种潜在的替代方法,它具有多种优势,包括更低的成本、便携性和速度。然而,该平台的基础调用错误率太高,无法用于临床微生物学的许多应用,包括疫情追踪。随着新的流动池化学物质和基调用算法的发布,准确性大大提高,使该方法适用于爆发调查。在这项工作中,我们优化了一个流线型的纳米孔工作流程,用于细菌病原体的流行病学分析。该工作流程与先前确定的四次临床爆发的分离株进行了验证,这些分离株具有不同的GC含量,与短读参考文献相比,显示出完全一致的cgMLST聚类。该工作流程将促进更广泛地实施ONT-only基因组和cgMLST分析,以协助全球医院暴发。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
An open-source nanopore-only sequencing workflow for analysis of clonal outbreaks delivers short-read level accuracy.

In this work, we present an optimized nanopore long-read only sequencing workflow for epidemiologic analysis of clonal outbreaks built with open-source tools. A set of unrelated clinical Pseudomonas aeruginosa isolates (n = 10) was chosen for workflow optimization, and sequencing libraries were prepared using a modified rapid barcoding strategy that incorporates temperature ramps to improve performance for high-GC content genomes. Sequencing data were used to benchmark the performance of the dorado suite (v0.9.1), including its basecaller, pre-assembly read error correction, and post-assembly polishing algorithms. All long-read assemblies and core genome multilocus sequence typing (cgMLST) were performed with Flye and pyMLST, respectively. Results were compared with a standard reference Illumina short-read approach, and discordant positions were determined at the core and whole-genome levels. Optimal performance was found with dorado sup@v5.0.0 basecalling with the inclusion of dorado error correction and dorado polish with its bacterial model. This workflow was then validated with four retrospective hospital outbreak isolate sets, including Klebsiella pneumoniae (n = 12), P. aeruginosa (n = 11), Enterococcus faecium (n = 10), and Staphylococcus aureus (n = 10). The nanopore-only assemblies obtained from the optimized pipeline demonstrated fully concordant cgMLST-based minimum spanning trees compared to the Illumina short-read reference. At the whole-genome level, high concordance was also observed, with as few as two discordant positions per genome compared to short-read assemblies. This optimized library preparation and open-source computational workflow enables nanopore-only clonality and outbreak analysis with performance comparable to that of Illumina short-read sequencing and will contribute critically to hospital infection control.

Importance: For the past decade, bacterial whole-genome sequencing has been performed using high-accuracy short-read sequencing. More recently, long-read sequencing with Oxford Nanopore Technologies (ONT) instruments has emerged as a potential alternative based on multiple advantages, including lower costs, portability, and speed. However, this platform has suffered from basecall error rates that were too high for many applications in clinical microbiology, including outbreak tracing. With the release of new flow cell chemistries and basecall algorithms, the accuracy has improved dramatically, making this approach feasible for outbreak investigations. In this work, we optimize a streamlined nanopore-only workflow for epidemiologic analysis of bacterial pathogens. The workflow was validated with isolates from four previously identified clinical outbreaks with varying GC content and demonstrated fully concordant cgMLST clustering as compared to short-read references. This workflow will facilitate the broader implementation of ONT-only genomes and cgMLST analysis to assist in hospital outbreaks worldwide.

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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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