Impact of wet- and dry-lab workflows on genome assembly, typing and enterotoxin gene detection in whole-genome sequencing of foodborne Staphylococcus aureus.

Abstract

Whole genome sequencing (WGS) has revolutionised surveillance and outbreak investigation of pathogenic bacteria in food, but cross-laboratory comparability remains challenging. An Inter-laboratory Comparison Test (ILCT) on the application of whole genome sequencing (WGS) for typing and characterisation of foodborne Staphylococcus aureus isolates was organised by the European Reference Laboratory for Coagulase-Positive Staphylococci (EURL for CPS) from 2023 to 2025. The test involved 12 National Reference Laboratories (NRLs) and evaluated variability in sequencing output, genome assembly metrics, MLST/cgMLST allele calling, and Staphylococcal enterotoxin gene detection introduced at three critical stages of the WGS workflow - DNA extraction, sequencing, and bioinformatics. While DNA extraction protocols showed minimal effects on sequencing quality, major differences arose from bioinformatics pipelines, particularly in contamination detection, reference database use, and assembly tools. Independent sequencing revealed that genome complexity, rather than sequencing depth or assembly metrics alone, most influenced cgMLST concordance. This ILCT provided a comprehensive overview of the capabilities and inconsistencies in WGS workflows at that time and highlights the need for harmonised analytical pipelines, curated reference databases, and quality thresholds.