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This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight  Enterobacteriaceae  isolates of six different species with plasmid populations of varying complexity. Species represented were  Escherichia coli ,  Klebsiella pneumoniae ,  Citrobacter freundii ,  Enterobacter cloacae, Serratia marcescens  and  Klebsiella oxytoca , with plasmid populations ranging from 1–11 plasmids with sizes of 2–330 kb. Isolates were sequenced using Illumina (short-read) and ONT’s MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70 % of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78 % recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in  Enterobacteriaceae  species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures.

microbial genomics

Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches