Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water

Frontiers in Microbiology

Microbiology

Padmini Ramachandran¹  Meghan Maguire¹  Marc W. Allard¹  Eric W. Brown¹  Steven M. Musser¹  Sandra Tallent¹  Narjol González-Escalona¹  Mark K. Mammel² 

[1] Center for Food Safety and Applied Nutrition, Office of Regulatory Science, College Park, MD, United States;Office of Applied Research and Safety Assessment, Food and Drug Administration, College Park, MD, United States;

关键词: foodborne pathogens;    Escherichia coli;    nanopore sequencing;    short-read sequencing;    pre-harvest agricultural water;    metagenomics;    hybrid assembly;   

DOI  :  10.3389/fmicb.2023.1221668

 received in 2023-05-12, accepted in 2023-08-04,  发布年份 2023

来源: Frontiers

【 摘 要 】

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 μg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

【 授权许可】

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Copyright © 2023 Maguire, Ramachandran, Tallent, Mammel, Brown, Allard, Musser and González-Escalona.