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

Abstract

An optimized, well-tested and validated targeted genomic sequencing-based high-throughput assay is currently not available ready for routine biodefense and biosurveillance applications. Earlier, we addressed this gap by developing and establishing baseline comparisons of a multiplex end-point Polymerase Chain Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon sequencing to real time PCR and customized data processing. Here, we expand upon this effort by identifying the optimal ONT library preparation method for integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in Real-Time). ONT-DART is a dockerized, real-time, amplicon-sequence analysis workflow that is used to reproducibly process and filter read data to support actionable amplicon detection calls based on alignment metrics, within sample statistics, and no-template control data. This analysis pipeline was used to compare four ONT library preparation protocols using R9 and Flongle (FL) flow cells. The two 4-Primer methods tested required the shortest preparation times (5.5 and 6.5 h) for 48 libraries but provided lower fidelity data. The Native Barcoding and Ligation methods required longer preparation times of 8 and 12 h, respectively, and resulted in higher overall data quality. On average, data derived from R9 flow cells produced true positive calls for target organisms more than twice as fast as the lower throughput FL flow cells. These results suggest that utilizing the R9 flowcell with an ONT Native Barcoding amplicon library method in combination with ONT-DART platform analytics provides the best sequencing-based alternative to current PCR-based biodetection methods.

Recent Progress in Nanotechnology-Based Approaches for Food Monitoring

“…To date, nanopore sequencing is considered one of the most advanced techniques for the detection of foodborne microorganisms belonging to this sensing concept due to its high sensitivity, real-time, and low turnaround time [ 266 ]. The most recent nanopore sequencing technology, was improved and developed by Oxford Nanopore Technologies [ 274 , 275 , 276 ]. By embedding a nanohole in a thin membrane and recording the electrochemical signal, nanopore technology can investigate nucleic acids and other biomacromoleculars, which allows the identification of pathogens in food, such as bacteria, viruses, or toxins [ 274 ].…”

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms†

“…[ 54 ] ONT has continued to enhance its nanopore and motor proteins and has released eight system versions to date, ranging from R6 (June 2014) to R10.3 (January 2020). [ 52,55‐56 ] Additionally, ONT has incorporated a mutant CsgG and a new motive enzyme (whose origin is undisclosed) into the R9.4 system, resulting in higher sequencing accuracy (85%—94%, as reported in the literature [ 57‐63 ] ) and faster sequencing speeds (up to 450 bases per second [ 62 ] ).…”

“…[52, [55][56] Additionally, ONT has incorporated a mutant CsgG and a new motive enzyme (whose origin is undisclosed) into the R9.4 system, resulting in higher sequencing accuracy (85%-94%, as reported in the literature [57][58][59][60][61][62][63] ) and faster sequencing speeds (up to 450 bases per second [62] ).…”