Peng Kai, Yin Yi, Li Yan, Qin Shangshang, Liu Yuan, Yang Xiaorong, Wang Zhiqiang, Li Ruichao
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
Institute of Comparative Medicine, Yangzhou University, Yangzhou, China.
Front Microbiol. 2022 Mar 23;13:778659. doi: 10.3389/fmicb.2022.778659. eCollection 2022.
Advancement of novel sequencing technologies facilitates modern life science and medicine unprecedentedly. Exploring complete genome sequences of bacteria by long-read sequencing technology is significant for microbial genomics research. However, third-generation long-read sequencing technologies are available with limited choices, which generate technological barrier to scientific research. Recently, a novel QitanTech nanopore long-read sequencing technology has emerged in China, but the potential application and performance were unexplored. Herein, we comprehensively evaluated the feasibility of the emerging sequencing technology in assembling complete genomes of MDR pathogens. The results showed that 500 Mbp QitanTech nanopore sequencing data could be generated within 8 h in one flow cell with the standard library preparation method. The mean read length, longest read length, and mean read-level accuracy of QitanTech sequencing data were 6,041 bp, 57,037 bp, and 81.50% (LAST)/81.40% (Minimap2), respectively. Two routine assembly strategies including long-read assembly and hybrid assembly enable the achievement of complete bacterial genomes. The accuracy of assembled draft bacterial genomes with QitanTech long-read data could be improved up to 99.9% dramatically by polishing using accurate short-read data. Furthermore, the assembled bacterial genomes cover accurate structures of complex resistance plasmids harboring critical resistance genes such as (X), , and , even the complex fusion MDR plasmid generated from homologous recombination. In conclusion, QitanTech nanopore sequencing, as a nanopore long-read sequencing technology launched in China, could be a good option for investigation of complex bacterial genomes. More potential applications based on this novel platform warrant investigations.
新型测序技术的进步前所未有地推动了现代生命科学和医学的发展。通过长读长测序技术探索细菌的完整基因组序列对微生物基因组学研究具有重要意义。然而，可用的第三代长读长测序技术选择有限，这给科学研究带来了技术障碍。最近，中国出现了一种新型的齐碳纳米孔长读长测序技术，但尚未探索其潜在应用和性能。在此，我们全面评估了这种新兴测序技术在组装多重耐药病原体完整基因组方面的可行性。结果表明，采用标准文库制备方法，在一个流动槽中8小时内可产生500 Mbp的齐碳纳米孔测序数据。齐碳测序数据的平均读长、最长读长和平均读级准确性分别为6,041 bp、57,037 bp和81.50%（LAST）/81.40%（Minimap2）。包括长读长组装和混合组装在内的两种常规组装策略能够实现完整细菌基因组的组装。通过使用准确的短读长数据进行校正，利用齐碳长读长数据组装的细菌基因组草图的准确性可显著提高至99.9%。此外，组装的细菌基因组涵盖了携带关键耐药基因如（X）、 和 的复杂耐药质粒的准确结构，甚至包括由同源重组产生的复杂融合多重耐药质粒。总之，齐碳纳米孔测序作为中国推出的一种纳米孔长读长测序技术，可能是研究复杂细菌基因组的一个不错选择。基于这个新平台的更多潜在应用值得研究。
