Eagle Shannon H C, Robertson James, Bastedo D Patrick, Liu Kira, Nash John H E
National Microbiology Laboratory, Public Health Agency of Canada, Guelph, Ontario, Canada.
National Microbiology Laboratory, Public Health Agency of Canada, Toronto, Ontario, Canada.
Access Microbiol. 2023 Feb 21;5(2). doi: 10.1099/acmi.0.000468.v3. eCollection 2023.
Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.
在诊断性公共卫生实验室环境中，用于疫情检测和监测活动的细菌病原体鉴定及特征分析方面，牛津纳米孔长读长测序相较于Illumina短读长测序具有优势。与Illumina相比，纳米孔测序对于小批量样本更具成本效益，资本成本更低，周转时间更快，此外还具备组装完整细菌基因组的能力。纳米孔测序所需的DNA数量和质量比Illumina测序更高，且推荐用于获取高分子量DNA的DNA提取方法与诊断实验室通常使用的方法不同。我们以一种先前已完成PacBio基因组封闭的肠杆菌科菌株作为模式生物，评估了五种商用DNA提取试剂盒的数量、质量和片段化情况。针对表现最佳的三种方法评估了纳米孔测序性能：Qiagen EZ1 DNA Tissue试剂盒、Qiagen DNeasy Blood and Tissue试剂盒以及改良后的内部版本MasterPure Complete DNA和RNA纯化试剂盒。为了评估提取后DNA纯化方法的效果，我们将从三种选定提取方法中提取的DNA分别用AMPure磁珠或乙醇沉淀法进行纯化，并将这些产物与未处理的DNA作为对照进行比较。所有方法均适用于常规全基因组测序（WGS），因为所有60个重复样本都具有非常高的基因组回收率，纳米孔测序读段覆盖了≥98%的参考基因组。对于85%的重复样本，使用Flye或Canu组装都能够产生完整的环状染色体。在大多数情况下，建议直接从提取进入测序，因为无论采用何种提取方法，未处理的DNA基因组封闭率最高。根据我们的评估标准，Qiagen DNeasy Blood and Tissue试剂盒因其成本低、能够从单管扩展到96孔板、产量和测序性能一致性高，被认为是总体最佳方法。
