Evaluation of Oxford Nanopore’s MinION Sequencing Device for Microbial Whole Genome Sequencing Applications

Abstract

The MinION sequencer (Oxford Nanopore Technologies) is a paradigm shifting device allowing rapid, real time long read sequencing of nucleic acids. Yet external benchmarking of this technologies’ capabilities has not been extensively reported, nor has thorough evaluation of its utility for field-based analysis with sub-optimal sample types been described. The aim of this study was to evaluate the capability of the MinION sequencer for bacterial genomic and metagenomic applications, with specific emphasis placed on the quality, yield, and accuracy of generated sequence data. Two independent laboratories at the National Microbiology Laboratory (Public Health Agency of Canada), sequenced a set of microbes in replicate, using the currently available flowcells, sequencing chemistries, and software available at the time of the experiment. Overall sequencing yield and quality improved through the course of this set of experiments. Sequencing alignment accuracy was high reaching 97% for all 2D experiments, though was slightly lower for 1D sequencing (94%). 1D sequencing provided much longer sequences than 2D. Both sequencing chemistries performed equally well in constructing genomic assemblies. There was evidence of barcode cross-over using both the native and PCR barcoding methods. Despite the sub-optimal nature of samples sequenced in the field, sequences attributable to B. anthracis the target organism used in this scenario, could none-the-less be detected. Together, this report showcases the rapid advancement in this technology and its utility in the context of genomic sequencing of microbial isolates of importance to public health.

Clinical metagenomics

“…In a published research study 126 , mNGS-based detection of Ebola and chikungunya virus infections on a nanopore sequencer was possible in <10 minutes of sequencing time and in <6 hours of sample-to-answer turnaround time overall. Research studies have also demonstrated the clinical potential of nanopore sequencing in targeted universal 16S ribosomal RNA (rRNA) bacterial detection 127 , microbiome analyses 128 , whole-genome sequencing of bacteria 129 and outbreak viruses 44,45,47 , RNA sequencing (RNA-seq) using standardized controls 130 and diagnosis of prosthetic joint 131 and lower respiratory infections 99 . Untargeted approaches such as mNGS or whole-transcriptome RNA-seq, however, may be limited by the lower throughput of nanopore sequencing relative to short-read sequencing such as with an Illumina instrument.…”

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

“…Fourth, the variable level of enrichment and coverage at a given nucleotide position achieved using MSSPE might preclude it from high-fidelity viral quasispecies analysis or whole-genome assembly at high coverage depth, especially on the nanopore sequencer given current sequencing error rates of ~5-10% (ref. 46 ). For these applications, MSSPE may be synergistic with complementary tiled multiplex PCR or capture probe enrichment approaches, as demonstrated here and previously 29 .…”

A rapid environmental DNA method for detecting white sharks in the open ocean

“…While the MinIon sequencer currently has a higher error rate than Illumina sequencers, a recent evaluation of 15 MinIon sequencing runs reported the average error rate to be 6% when using the same r9.4 flowcells as our study (Tyler et al, 2018). With a MinIon error rate of 6%, any species-level detections with blast % identities <95% should be interpreted with caution.…”