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 detec- tion, 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.