Genomic analyses of multidrug-resistant Salmonella Indiana, Typhimurium, and Enteritidis isolates using MinION and MiSeq sequencing technologies

Abstract

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in realtime using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the wholegenome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for

Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates

“…Promising results of Hyb ASM have facilitated the annotation of clinically relevant genomic elements. Interestingly, similar conclusions using Hyb ASM have previously been drawn for assemblies from environmental samples [18,61] and clinical samples [62,63].…”

Optimizing source tracking of Listeria monocytogenes with quasimetagenomics and integrated long and short read sequencing

“…Despite being of high enough quality to differentiate the strains, however, the polished genome assemblies had less accurate recovery of the cgMLST than contigs assembled using MiSeq Illumina short reads only. Another study similarly showed, for several strains of Salmonella enterica, that short read assembly followed by long read scaffolding more accurately reconstructed the genomes than either long read or short read assembly alone [10].…”

“…Here we provide the rst study we are aware of, that benchmarks the utility of integrated short and long read sequencing technologies for applied quasimetagenomic pathogen source tracking. Other studies have shown that integrating short read and long read information can reconstruct complete and accurate pathogen genome assemblies from pure culture isolates [9,10]. We sought to extend this work by testing whether we could reconstruct complete and accurate L. monocytogenes genomes from low species diversity quasimetagenomic samples.…”

Antimicrobial Resistance Profiling and Phylogenetic Analysis of Neisseria gonorrhoeae Clinical Isolates From Kenya in a Resource-Limited Setting

“…results (Lemon et al, 2017;Sanderson et al, 2020). Studies on comparison of assemblies derived from Nanopore-only, Illumina, and hybrid assemblies have shown that the AMR prediction is comparable across all three methods (Chen et al, 2020). Recent advances in Nanopore sequencing and analysis workflows have demonstrated the utility of ONT sequencers for metagenomic analysis (Sanderson et al, 2020;Street et al, 2020).…”