Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in antimicrobial resistance/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data have suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore [Oxford Nanopore Technologies (ONT)] flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 h of sequencing, most chromosomes and plasmids had circularized and there was no benefit associated with longer running times. Quality was similar at 12 h, suggesting that shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilizing shorter run times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per-isolate sequencing cost. Ultimately this will facilitate large-scale studies utilizing hybrid assembly, advancing our understanding of the genomics of key human pathogens.

中文翻译：

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优化使用 Oxford Nanopore 流通池进行混合组装

混合组件对肠杆菌科的研究非常有价值 由于它们能够完全解析移动遗传元件的结构，例如质粒，这些元件与临床重要基因的携带有关（例如，与抗菌素耐药性/毒力有关的基因）。该技术的广泛应用目前主要受成本限制。最近的数据表明，可以使用来自多路复用纳米孔 [Oxford Nanopore Technologies (ONT)] 流通池运行的总输出的一小部分来生产非劣质甚至优越的混合组件。在这项研究中，我们试图确定在获取混合组装读数时流动池的最佳最短运行时间。然后，我们评估了 ONT 清洗套件是否允许用户通过对每个流动槽的多个文库进行测序来利用更短的运行时间。测序 24 h 后，大多数染色体和质粒已经环化，并且没有与更长的运行时间相关的好处。12 小时时的质量相似，这表明较短的运行时间对于某些应用（例如质粒基因组学）可能是可以接受的。ONT 洗涤试剂盒在去除文库之间的 DNA 方面非常有效。即使在单个流动槽上连续使用相同的条形码，文库之间的污染似乎也不会影响后续的混合组装。利用更短的运行时间与库间核酸酶洗涤相结合，至少可以实现 36 ONT 洗涤试剂盒在去除文库之间的 DNA 方面非常有效。即使在单个流动槽上连续使用相同的条形码，文库之间的污染似乎也不会影响后续的混合组装。利用更短的运行时间与库间核酸酶洗涤相结合，至少可以实现 36 ONT 洗涤试剂盒在去除文库之间的 DNA 方面非常有效。即使在单个流动槽上连续使用相同的条形码，文库之间的污染似乎也不会影响后续的混合组装。利用更短的运行时间与库间核酸酶洗涤相结合，至少可以实现 36 每个流动池对肠杆菌科 分离株进行测序，显着降低了每个分离株的测序成本。最终，这将促进利用混合组装的大规模研究，促进我们对关键人类病原体基因组学的理解。