Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology

Abstract

18United States public health agencies are focusing on next-generation sequencing (NGS) to 19 quickly identify and characterize foodborne pathogens. Here, the MinION nanopore, long-read 20 sequencer was used to simultaneously sequence the entire chromosome and plasmids of Salmonella 21 enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. A rapid, random sequencing 22 approach, coupled with de novo genome assembly within a customized data analysis workflow, that can 23 resolve highly-repetitive genomic regions, was developed. In sequencing runs, as short as four hours, 24 using nanopore data alone, full-length genomes were obtained with an average identity of 99.87% for 25Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These long-26 read assemblies provided information on serotype, virulence factors, and antimicrobial resistance genes. 27Using a custom-developed, SNP-selection workflow, the potential of the nanopore-only assemblies 28 (after only 30 minutes of sequencing) for rapid phylogenetic inference, with identical topology 29 compared to the published dataset, was demonstrated. To achieve maximum quality assemblies, the 30 developed bioinformatics workflow employed additional polishing steps to correct the systematic errors 31 produced by the nanopore-only assemblies. Nanopore sequencing provided a shorter (10 hours library 32 preparation and sequencing) turnaround time compared to other NGS technologies. 33 34 Keywords: MinION, complete genome sequencing, plasmids, foodborne pathogen, long-reads, assembly 35 workflow, tracing. 36 basecalled using Albacore (ONT v 2.0.2b) and subsampled for assembly using Filtlong (v.0.2.0) 24 to a 132

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

“…Because high-quality reference genomes are not always available, the MiSeq assemblies were considered as the “gold standard” to assess the accuracy of the MinION and hybrid assemblies in our study [ 55 , 56 ]. Similarly, in the study of González-Escalona et al [ 55 ] on the MinION assemblies of three E .…”

“…Because high-quality reference genomes are not always available, the MiSeq assemblies were considered as the "gold standard" to assess the accuracy of the MinION and hybrid assemblies in our study [55,56]. Similarly, in the study of González-Escalona et al [55] on the MinION assemblies of three E. coli O26:H11 strains, the MiSeq or HiSeq assemblies of 155 E. coli O26:H11 strains were considered as the "gold standard" for accurate genome sequence determination and SNP analyses.…”

“…Similarly, in the study of González-Escalona et al [55] on the MinION assemblies of three E. coli O26:H11 strains, the MiSeq or HiSeq assemblies of 155 E. coli O26:H11 strains were considered as the "gold standard" for accurate genome sequence determination and SNP analyses. Taylor et al [56] also used the MiSeq assembly as the reference genome for the E. coli O157:H7 isolate lacking a published reference genome when evaluating Nanopore sequencing technology for rapid phylogenetic inference. Although most MinION assemblies were more contiguous than the MiSeq assemblies, SNPs remained problematic in de novo assemblies generated from our MinION long reads.…”

Draft Genome Assembly of Rhodobacter sphaeroides 2.4.1 Substrain H2 from Nanopore Data

“…Notably, PGAP reports a large number of pseudogenes. These are reportedly caused by indels leading to frameshifts, which are known to be an error source in Nanopore sequencing-only assemblies ( 13 ) and must be taken into account for downstream purposes, such as annotation. While the plasmid architecture is substantially divergent from the sequence reported by Kontur et al ( 3 ) (accession numbers ASM1290v2 and PRJNA56 ) ( Fig.…”

Applying Nanopore sequencing to a One-Health scenario for colistin resistance transmission among pigs, cows and the farmer

“…Long-read output can span repetitive regions either from bacteria, archaea or eukarya, thus facilitating the assembly of genomes and the characterization of plasmids, and the location and genomic context of resistance genes 13 . These strategies have been also applied for resistome and plasmidome characterization [14][15][16][17][18] since antimicrobial resistance has become a global concern.…”