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

Louise Pankhurst,Nicole Stoesser,Amy Mathers,Hang T T Phan,Chloe Eaton,Sophie George,Antonia Votintseva,Zamin Iqbal,Indre Navickaite,Derrick W Crook,Anna E Sheppard,Alasdair Hubbard,Rachel Norris

doi:10.1099/mgen.0.000118

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 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.

Highlights

Plasmids are extra-chromosomal genetic elements that are transmission vectors of many acquired antimicrobial resistance (AR) genes in Enterobacteriaceae [1]
In order to maximize the diversity of plasmid profiles, we chose eight isolates representing six Enterobacteriaceae species, namely Klebsiella pneumoniae (Kpne, n=2), Escherichia coli (Ecol, n=1), Klebsiella oxytoca (Koxy, n=2), Citrobacter freundii (Cfre, n=1), Enterobacter cloacae (Eclo, n=1) and Serratia marcescens (Smar, n=1)
MinION sequencing was beneficial in resolving plasmid structures for the Enterobacteriaceae species studied

Summary

Introduction

Plasmids are extra-chromosomal genetic elements that are transmission vectors of many acquired antimicrobial resistance (AR) genes in Enterobacteriaceae [1]. Plasmid tracking can be facilitated by the categorization of plasmids using plasmid classification schemes such as incompatibility (Inc), relaxase (MOB) and mating pair formation system (MPF) typing [3] These schemes lack resolution and fail to classify all known plasmids, limiting their application to plasmid transmission epidemiology. Plasmids often contain many smaller, mobile repeat structures including insertion sequences and transposable elements that enable AR genes to mobilize under evolutionary pressure. These repeat structures often extend beyond the current typical insert size of paired-end short-read sequencing (~300–500 bp), and inhibit complete

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