Abstract
Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.
Highlights
The Oxford Nanopore MinION is a commercially available long read sequencer that connects to a personal computer through a USB port
Raw data on the E. kobei genome from a single flow cell was initially analysed using the Oxford Nanopore base calling software and defined as pass or fail based on a threshold set at approximately 85 % accuracy (Q9) and including only 2D reads, where data is generated from both the forward and reverse strand of DNA as it passes through the nanopore
MinION data alone could be used with the software described above to generate highly contiguous bacterial assemblies
Summary
The Oxford Nanopore MinION is a commercially available long read sequencer that connects to a personal computer through a USB port. The technology has shown promise for microbiological applications, including the delineation of position and structure of bacterial antibiotic-resistance islands (Ashton et al, 2014), assembly of bacterial genomes (Loman et al, 2015; Risse et al, 2015) and tracking of viral outbreaks (Quick et al, 2016; Zika Real time Sequencing Consortium, 2016). This has been supported by the development of analysis tools for MinION data. We consider factors key to medical microbiology including accuracy, time taken to generate assemblies and whether the assemblies were of sufficient quality to provide information on the presence and structure of plasmids carrying clinically relevant antimicrobial-resistance genes
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