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.

Figure 1

DNA yield in number of reads which were analyzed further (pass) and reads which were excluded based on either internal Epi2Me filtering parameters or inability to demultiplex (Fail), on each of the flowcells tested. FAB-R9.4; FAF- R9.4 SpotOn. The first set of samples were assessed using Epi2Me, the second set by Albacore, and the third by MinKNOW direct basecalling. *This sample had a second top up run added 1 hour following the commencement of sequencing.

Figure 2

Mean quality per 2000 base pair window across each of the MinION runs included in this analysis calculated using fastqc. Lines extend further as the length of the read increases. Earlier runs were characterized by both shorter reads, and lower quality output data generated. L1-2D-FAB29623 is broken into pre and post, referring to read quality before and after the wash buffer was applied.

Figure 3

Boxplot depicting the mean genomic coverage obtained across each of the reference genomes included in this analysis. Sequenced organisms and the corresponding average levels of coverage for each are coloured as described.

Figure 4

Error characteristics of data generated in various sequencing runs included in this analysis. (A) Proportion of bases accurately matching mapped segment of the reference. (B) Insertions per 100 aligned bases. (C) Deletions per 100 aligned bases. (D) Substitutions per 100 aligned bases.

Figure 5

Assembly characteristics of contigs generated following subsampling of fastq reads generated with 1D sequencing and native barcoding. Dashed line is the reference genome size reported by NCBI.