Background

The emergence of antibiotic-resistant bacterial pathogens in the environment has been increasing, posing a threat to public health. Next-generation sequencing technology, which is both low cost and large scale, was used to identify antibiotic-resistance genes or toxin genes in these pathogens. Short-read sequencing cannot fully reconstruct bacterial chromosomes and plasmids carrying toxin genes and antibiotic-resistance genes because of their location on the insertion sequences or repeat regions. Third-generation sequencing generated long reads that could cover the repetitive and/or insertion sequences, allowing for complete chromosome and plasmid reconstruction. However, the optimal protocols for whole-genome sequencing of antibiotic-resistance pathogenic bacteria, from DNA extraction to genome assembly, are still being researched.

Objective

To develop a pipeline of optimal methods for whole-genome sequencing of bacteria, we compared three commercial DNA extraction kits (column extraction, magnetic bead extraction, and precipitation extraction), two third-generation sequencing platforms (MinION and PacBio), and three assembly methods (flye, unicycler, and unicycler_hybrid). The assembly results were evaluated based on the number of contigs and the detection of anti-microbial-resistance genes.

Results

Magnetic bead extraction method generated longer N50 read lengths and greater read length distribution than the other two extraction methods. The Flye assembler in combination with magnetic bead extraction and MinION sequencing provided consistent successful plasmid assembly results and detected all antimicrobial-resistance gene in all datasets.

Conclusion

DNA extraction, sequencing platform, and assembly methods can all have an impact on the results of bacterial whole-genome sequencing. Our findings could be a practical protocol for researchers who use third-generation sequencing to perform bacterial whole-genome sequencing by consistently resolving small plasmids carrying antibiotic-resistance genes.

中文翻译：

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使用第三代测序平台对抗生素耐药病原菌进行全基因组测序的最佳标准方案

背景

环境中耐抗生素细菌病原体的出现不断增加，对公众健康构成威胁。利用低成本和大规模的二代测序技术来鉴定这些病原体中的抗生素抗性基因或毒素基因。短读长测序无法完全重建携带毒素基因和抗生素抗性基因的细菌染色体和质粒，因为它们位于插入序列或重复区域。第三代测序产生的长读长可以覆盖重复和/或插入序列，从而实现完整的染色体和质粒重建。然而，从 DNA 提取到基因组组装的抗生素耐药性病原菌全基因组测序的最佳方案仍在研究中。

目标

为了开发细菌全基因组测序的最佳方法管道，我们比较了三种商业 DNA 提取试剂盒（柱提取、磁珠提取和沉淀提取）、两个第三代测序平台（MinION 和 PacBio）和三个组装方法（flye、unicycler 和 unicycler_hybrid）。根据重叠群的数量和抗微生物基因的检测来评估组装结果。

结果

磁珠提取方法比其他两种提取方法产生更长的 N50 读取长度和更大的读取长度分布。Flye 组装器与磁珠提取和 MinION 测序相结合，提供了一致的成功质粒组装结果，并在所有数据集中检测到所有抗微生物基因。

结论

DNA 提取、测序平台和组装方法都会对细菌全基因组测序的结果产生影响。对于使用第三代测序通过持续解析携带抗生素抗性基因的小质粒来进行细菌全基因组测序的研究人员来说，我们的发现可能是一个实用的协议。