Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community

Abstract

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

Keywords: experimentally spiked samples; proficiency test; shotgun metagenomics; smoked salmon; wet-lab protocols.

Figure 1

Relative abundance of the microorganisms of the mock community quantified in the DNA metagenomic datasets obtained from spiked salmon. The first bar in the figure refers to the expected relative abundance for the microorganisms experimentally spiked in the salmon.

Figure 2

Heat map of Bray–Curtis dissimilarity and average linkage clustering dendrogram showing the similarity between the benchmark (expected) and the DNA metagenomic datasets obtained from spiked salmon, considering the microorganisms of the mock community. In the heat map, when a cell is colored in red, it indicates that the two associated samples (in the corresponding row and column) have an identical composition, while a white cell refers to two samples with the most different composition.