Fishing in the Soup – Pathogen Detection in Food Safety Using Metabarcoding and Metagenomic Sequencing

Abstract

In food safety the detection of food contaminations with pathogenic microorganisms is a race against time and often outpaced by error-prone epidemiological approaches. For evidence-based outbreak investigations fast and reliable techniques and procedures are required to identify the source of infection. Metagenomics has the potential to become a powerful tool in the field of modern food safety, since it allows the detection, identification and characterization of a broad range of pathogens in a single experiment without pre-cultivation within a couple of days. Nevertheless, sample handling, sequencing and data analysis are challenging and can introduce errors and biases into the analysis. In order to evaluate the potential of metagenomics in food safety, we generated a mock community containing DNA of foodborne bacteria. Herewith, we compare the aptitude of the two prevalent approaches – 16S rDNA amplicon sequencing and whole genome shotgun sequencing – for the detection of foodborne bacteria using different parameters during sample preparation, sequencing and data analysis. 16S rDNA sequencing did not only result in high deviations from the expected sample composition on genus and species level, but more importantly lacked the detection of several pathogenic species. While shotgun sequencing is more suitable for species detection, abundance estimation, genome assembly and species characterization, the performance can vary depending on the library preparation kit, which was confirmed for a naturally Francisella tularensis contaminated game meat sample. The application of the Nextera XT DNA Library Preparation Kit for shotgun sequencing did not only result in lower reference genome recovery and coverage, but also in distortions of the mock community composition. For data analysis, we propose a publicly available workflow for pathogen detection and characterization and demonstrate its benefits on the usability of metagenomic sequencing in food safety by analyzing an authentic metagenomic sample.

German-wide interlaboratory study compares consistency, accuracy and reproducibility of whole-genome short read sequencing

“…Nevertheless, we found that all assemblies were overall very similar, with respect to assembly length, N50, GC and the number of CDSs, with a few notable exceptions. In particular, assemblies constructed from short read data of laboratories LC06 and LC08 (both using a MiSeq Illumina instrument) had much lower N50 values and a greater number of contigs, probably due their use of the Nextera XT DNA Library Preparation Kit, which was recently shown to have a strong GC bias (Grützke et al, 2019;Sato et al, 2019;Uelze et al, 2019) (also compare Supplementary File 7). This is a concern since a high number of contigs in a genome assembly may cause a fragmentation of genes at the contigs borders, thereby affecting gene annotation and multilocus sequence typing.…”

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producingEscherichia coliin irrigation water

“…Each metagenomic approach provides different levels of analysis. 16S rDNA has the most sensitive detection limit, requiring the lowest initial CFU, but the lowest resolution, reporting at the genus level because some species have almost identical 16S rDNA genes and the fragment of the 16S rDNA used is relatively small (39). Shotgun metagenomic WGS provides a less sensitive detection limit (above 10 3 CFU/ml), but provides information from species to a strain level, including many functional genes in the microbiome sample analyzed (33,36,40,41).…”

“…Metagenomic analyses can be made either using short or long sequencing reads technologies. Short-read shotgun metagenomics was most commonly used for microbiome analyses (30)(31)(32)35,39,39,42), while the use of long-read metagenomics is on the rise in the last few years (36)(37)(38)40) for numerous reasons.…”

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

“…Currently, 16S rDNA amplicon and shotgun metagenomic sequencing are the two common approaches used to address different aspects of gut microbiota in various pathological conditions including inflammatory bowel disease, diabetes, and obesity [10]. In comparison with 16S rDNA amplicon sequencing, shotgun metagenomic offers greater sensitivity, specificity, microbial abundance estimation, and greater resolution of microbes up to the species level [11]. Evidence support the fact that various pathogenic microbes can be transmitted from animals to humans or from humans to animals either through direct contact or while living with livestock in close contact and it has been exemplified by a recent report indicating the transmission of antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus, and various members of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae [12][13][14].…”