Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing

Frontiers in Microbiology

Microbiology

Zhenpeng Li¹  Bo Pang¹  Xin Lu¹  Yao Peng¹  Wenxuan Zhao²  Wei Zeng³  Biao Kan³  Jialiang Xu⁴  Ming Luo⁵ 

[1] National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China;National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China;Department of Health Statistics, School of Public Health, Shanxi Medical University, Jinzhong, Shanxi, China;National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China;School of Public Health, Shandong University, Jinan, China;School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing, China;Yulin Center for Disease Control and Prevention, Yulin, Shanxi, China;

关键词: ONT sequencing;    accuracy;    complete genome;    bacteria;    plasmid;   

DOI  :  10.3389/fmicb.2023.1179966

 received in 2023-03-05, accepted in 2023-04-27,  发布年份 2023

来源: Frontiers

【 摘 要 】

IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and DiscussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.

【 授权许可】

Unknown   
Copyright © 2023 Zhao, Zeng, Pang, Luo, Peng, Xu, Kan, Li and Lu.