当考虑到甲基化相关的错误时,牛津纳米孔技术R10测序能够准确地对脑膜炎奈瑟菌和肠炎沙门氏菌进行基于cgmlst的细菌爆发调查。

IF 5.4 2区 医学 Q1 MICROBIOLOGY
Bert Bogaerts, Margo Maex, Florian Commans, Nathalie Goeders, An Van den Bossche, Sigrid C J De Keersmaecker, Nancy H C Roosens, Pieter-Jan Ceyssens, Wesley Mattheus, Kevin Vanneste
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引用次数: 0

摘要

核心基因组多位点序列分型(Core-genome multi-locus sequence typing, cgMLST)是一种基于基因组学的细菌聚类检测和系统基因组分析方法。牛津纳米孔技术(ONT) R10测序的错误率降低,促使许多实验室探索将这项技术纳入他们的活动。然而,关于ONT R10测序用于cgMLST分析的性能存在矛盾的报道。本研究评估了ONT R10数据在cgMLST等位基因召唤和细菌爆发调查中聚类检测的适用性。对24株脑膜炎奈瑟菌和24株肠炎沙门氏菌进行ONT和Illumina测序。ONT采用快速条形码试剂盒(RBK)和快速PCR条形码试剂盒(RPB)检测。在两个物种中,ONT-only组合中调用的位点百分比都非常高。然而,对于使用RBK试剂盒的奈瑟菌ONT-only组装的杂交组合,错配等位基因的比例要高得多,导致错误的聚类分配。这些不匹配的等位基因大部分是由于甲基化位置的碱基调用错误造成的,这并不影响使用RPB试剂盒或任何沙门氏菌ONT-only组装产生的ONT数据。综上所述,ONT R10测序作为一种可行的cgMLST分析方法具有很大的潜力,但甲基化相关的错误可能会影响某些物种和菌株的性能。经过适当校正后,ONT R10与Illumina具有相同的cgMLST分析性能,并且两者可以互换使用。这些结果支持将ONT R10测序纳入常规公共卫生和临床工作流程。重要性:本研究评估了Oxford Nanopore Technologies R10测序在核心基因组多位点序列分型(cgMLST)中的适用性,cgMLST是(临床)暴发调查和细菌菌株追踪中广泛使用的方法。我们利用Illumina和ONT R10测序对24株脑膜炎奈瑟菌和24株肠沙门氏菌进行了测序,包括确诊的暴发病例,以评估cgMLST分析的性能。我们使用基于pcr和本地条形码协议进行ONT测序,这使我们能够证明甲基化相关错误对性能的实质性物种依赖影响。然而,我们证明,当这些错误得到适当解决时,ONT R10可以用于精确的基于cgmlst的聚类,包括与使用Illumina测序的菌株整合。我们的研究结果支持在常规公共卫生实践中将ONT R10作为替代Illumina测序的cgMLST分析方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Oxford Nanopore Technologies R10 sequencing enables accurate cgMLST-based bacterial outbreak investigation of Neisseria meningitidis and Salmonella enterica when accounting for methylation-related errors.

Core-genome multi-locus sequence typing (cgMLST) is a well-established and standardized method for genomics-based cluster detection and phylogenomic analysis of bacteria. The reduced error rate of Oxford Nanopore Technologies (ONT) R10 sequencing has prompted many laboratories to explore incorporating this technology into their activities. However, conflicting reports exist on the performance of ONT R10 sequencing for cgMLST analysis. This study evaluates the suitability of ONT R10 data for cgMLST allele calling and cluster detection for bacterial outbreak investigation. ONT and Illumina sequencing data were generated for 24 Neisseria meningitidis and 24 Salmonella enterica isolates. For ONT, both the rapid barcoding kit (RBK) and the rapid PCR barcoding kit (RPB) were used. The percentage of loci called in the ONT-only assemblies was very high for both species. However, the proportion of mismatched alleles to the hybrid assemblies was substantially higher for the Neisseria ONT-only assemblies with the RBK kit, resulting in incorrect cluster assignments. The large majority of these mismatched alleles were due to incorrect base calls at methylated positions, which did not affect the ONT data generated using the RPB kit or any of the Salmonella ONT-only assemblies. In conclusion, ONT R10 sequencing shows great potential as a viable method for cgMLST analysis, but methylation-related errors can affect performance for certain species and strains. When properly corrected for, ONT R10 had the same performance for cgMLST analysis as Illumina, and both could be used interchangeably. These results support the integration of ONT R10 sequencing into routine public health and clinical workflows.

Importance: This study evaluates the suitability of Oxford Nanopore Technologies R10 sequencing for core-genome multi-locus sequence typing (cgMLST), a widely used method in (clinical) outbreak investigation and bacterial strain tracking. We have sequenced 24 Neisseria meningitidis and 24 Salmonella enterica strains, including confirmed outbreak cases, using Illumina and ONT R10 sequencing to evaluate the performance for cgMLST analysis. We used a PCR-based and native barcoding protocol for the ONT sequencing, which enabled us to demonstrate a substantial species-dependent impact of methylation-related errors on the performance. However, we demonstrate that when these errors are properly addressed, ONT R10 can be used for accurate cgMLST-based clustering, including integration with strains sequenced using Illumina. Our findings support the use of ONT R10 as an alternative to Illumina sequencing for cgMLST analysis in routine public health practice.

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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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