Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories.
Shannon H C Eagle, James Robertson, D Patrick Bastedo, Kira Liu, John H E Nash
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引用次数: 1
Abstract
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 Salmonella 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.
在诊断公共卫生实验室环境中,牛津纳米孔长读测序在鉴定和鉴定细菌病原体的爆发检测和监测活动方面比Illumina短读测序具有优势。与Illumina相比,Nanopore在小批量生产中更具成本效益,资本成本更低,周转时间更快,此外还能组装完整的细菌基因组。纳米孔测序所需的DNA数量和质量都高于Illumina,并且推荐用于获得高分子量DNA的DNA提取方法与诊断实验室通常使用的方法不同。使用先前封闭的PacBio基因组的沙门氏菌分离物作为肠杆菌科生物模型,我们评估了五种商业DNA提取试剂盒的数量,质量和碎片化程度。对前三种方法的纳米孔测序性能进行了评估:Qiagen EZ1 DNA Tissue、Qiagen DNeasy Blood and Tissue和改良的MasterPure Complete DNA和RNA纯化。为了评估提取后DNA纯化方法的效果,我们将从三种提取方法中提取的DNA用AMPure珠或乙醇沉淀纯化,并将这些输出与未处理的DNA作为对照进行比较。所有方法都适用于常规的全基因组测序(WGS),因为所有60个重复都具有非常高的基因组回收率,绘制的纳米孔reads覆盖了≥98%的参考基因组。对于85%的重复,使用Flye或Canu组装能够产生完整的圆形染色体。在大多数情况下,建议直接从提取转移到测序,因为无论提取方法如何,未经处理的DNA具有最高的基因组闭合率。根据我们的评估标准,Qiagen DNeasy Blood and Tissue试剂盒被认为是最佳的整体方法,因为它成本低,能够从单管扩展到96孔板,产率和测序性能高度一致。