Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer

Abstract

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

High throughput rapid amplicon sequencing for multilocus sequence typing of M. ovipneumoniae using DNA obtained from clinical samples

“…Further investigation of R10.4.1 and Rapid Barcoding kit for multiplex amplicon sequencing could eliminate the need for manual homopolymer correction, as claimed by ONT. In this study we used a set runtime of 16 hours for ONT sequencing; however, a similar work ow for MLST of S. aureus stopped sequencing once there were 4000 reads per sample to ensure adequate coverage without oversampling (13). In that protocol, the authors used a single ow cell ve times (467 samples total), and fewer than 4 hours were required for adequate sequence data for the rst three runs, with successive run requiring 6-15 hours until the ow cell was depleted (13).…”

“…In this study we used a set runtime of 16 hours for ONT sequencing; however, a similar work ow for MLST of S. aureus stopped sequencing once there were 4000 reads per sample to ensure adequate coverage without oversampling (13). In that protocol, the authors used a single ow cell ve times (467 samples total), and fewer than 4 hours were required for adequate sequence data for the rst three runs, with successive run requiring 6-15 hours until the ow cell was depleted (13). This approach could decrease turnaround time and cost for our work ow.…”

“…Alternatively, Rapid Barcoding library preparation is faster and less expensive, however throughput and raw read accuracy are reported to be reduced ( 18). An amplicon-speci c protocol for Rapid Barcoding is not provided by ONT, though several studies have used that kit for sequencing multiplexed amplicons with a high degree of accuracy (12)(13)(14)(15). Based on these successes, Rapid Barcoding library preparation is expected to be well suited for diagnostic settings because of the short library preparation time, exible multiplexing options, accuracy, and low cost.…”

“…This method is laborious, and expensive if processing large numbers of samples (11). Oxford Nanopore Technologies (ONT) sequencing has recently been used for singleplex and multiplex MLST (12)(13)(14)(15)(16). This method uses a small, low-cost sequencing device which delivers result in real-time capable of multiplexing and high-throughput sequencing.…”

Optimization of Oxford Nanopore Technology Sequencing Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool

“…The sequencing approach has been accelerated by Nanopore-type technologies that require minimal sequencing infrastructure and the ability to sequence anything, anywhere, and anytime by virtually anyone. Even with these clear advantages, to date there have been only a handful of studies investigating the feasibility of AmpSeq or MetaSeq approaches in biodefense applications [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ].…”