摘要
Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak.The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads,portability,real-time accessibility of sequenced data,and very low initial costs.However,information is lacking on MinION sequencing of enterovirus genomes.Methods In this proof-of-concept study using Enterovirus 71(EV71) and Coxsackievirus A16(CA16) strains as examples,we established an amplicon-based whole genome sequencing method using MinION.We explored the accuracy,minimum sequencing time,discrimination and high-throughput sequencing ability of MinION,and compared its performance with Sanger sequencing.Results Within the first minute(min) of sequencing,the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains.In as little as 14 min,99% identity was reached for the single EV71 strain,and in 17 min(on average),99% identity was achieved for 10 CA16 strains in a single run.Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast,reliable and convenient method for routine use.
Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.
基金
supported by the National key research and development plan(2016TFC1202700,2016YFC1200900)
Beijing Municipal Science&Technology Commission project(grant numbers D151100002115003)
Guangzhou Municipal Science&Technology Commission project(grant numbers 2015B2150820)
