Towards profiling food microbiota and bacterial enrichments using a long-read 16S rRNA gene sequencing approach: an enoki mushroom pilot

Funding period: 2023-2025

Lead: Calvin Lau

Total GRDI funding: $60,000

Nanopore sequencing is a revolutionary technology that allows direct, real-time analysis of long DNA fragments (>10 b), and that it has been increasingly-adopted into molecular diagnostics. Currently, research labs at CFIA conduct culture-free, "omic"-based microbial profiling analysis, by combining a 16S-metabarcoding approach and the Illumina short-read sequencing platform, to characterize the comprehensive bacterial composition of food and cultural samples. In this research project, the overarching objective is to explore a full-length 16S rRNA gene amplicon nanopore sequencing approach in studying food microbiome and addressing the limited taxonomic resolution achieved by the short-read sequencing approach. Given the growing food-safety concern related to enoki mushroom and its frequent association with Listeria monocytogenes contamination, the herein developed long-read 16S sequencing workflow will be piloted to determine the indigenous bacterial microbiota of enoki mushroom, and, to explore the enrichment dynamics of L. monocytogenes and other co-enriched background bacteria from contaminated enoki mushroom samples undergoing the cultural method used to detect foodborne Listeria bacteria. The side-by-side performance comparison against the established short-read 16S sequencing method will provide practical insights on the real-world application of long-read 16S sequencing within the context of food microbiology research, while the overall microbiome findings from this study can ultimately be harnessed for the purposes of developing better Listeria isolation methods and exposing any unforeseeable complications associated with enoki mushroom inspection/investigation.