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

Abstract

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 <i>Enterobacteriaceae</i> isolates of six different species with plasmid populations of varying complexity. Species represented were <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, <i>Citrobacter freundii</i>, <i>Enterobacter cloacae, Serratia marcescens</i> and <i>Klebsiella oxytoca</i>, 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 <i>Enterobacteriaceae</i> 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.