Bogaerts Bert, Maex Margo, Commans Florian, Goeders Nathalie, Van den Bossche An, De Keersmaecker Sigrid C J, Roosens Nancy H C, Ceyssens Pieter-Jan, Mattheus Wesley, Vanneste Kevin
Transversal activities in Applied Genomics, Sciensano, Brussels, Belgium.
Bacterial Diseases, Sciensano, Brussels, Belgium.
J Clin Microbiol. 2025 Aug 22:e0041025. doi: 10.1128/jcm.00410-25.
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 and 24 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 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 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.
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 and 24 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.
核心基因组多位点序列分型（cgMLST）是一种成熟且标准化的方法，用于基于基因组学的细菌聚类检测和系统发育分析。牛津纳米孔技术公司（ONT）R10测序较低的错误率促使许多实验室探索将该技术纳入其工作流程。然而，关于ONT R10测序用于cgMLST分析的性能存在相互矛盾的报道。本研究评估了ONT R10数据用于细菌暴发调查的cgMLST等位基因分型和聚类检测的适用性。对24株和24株分离株分别生成了ONT和Illumina测序数据。对于ONT，使用了快速条形码试剂盒（RBK）和快速PCR条形码试剂盒（RPB）。仅使用ONT组装时，两个物种中被分型的位点百分比都非常高。然而，使用RBK试剂盒的仅ONT组装中，与混合组装相比，错配等位基因的比例要高得多，导致聚类分配错误。这些错配等位基因中的绝大多数是由于甲基化位点的碱基错误调用，这并不影响使用RPB试剂盒生成的ONT数据或任何仅ONT组装。总之，ONT R10测序作为一种可行的cgMLST分析方法显示出巨大潜力，但甲基化相关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在cgMLST分析中的性能与Illumina相同，两者可互换使用。这些结果支持将ONT R10测序整合到常规公共卫生和临床工作流程中。
本研究评估了牛津纳米孔技术公司R10测序用于核心基因组多位点序列分型（cgMLST）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。我们使用Illumina和ONT R10测序对24株和24株菌株进行了测序，包括确诊的暴发病例，以评估cgMLST分析的性能。我们对ONT测序使用了基于PCR和天然条形码方案，这使我们能够证明甲基化相关错误对性能有显著的物种依赖性影响。然而，我们证明，当这些错误得到妥善处理时，ONT R10可用于基于cgMLST的准确聚类，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使用ONT R10替代Illumina测序进行cgMLST分析。
