cgMLST typing reproducibility is maintained across diverse whole-genome sequencing workflows in a multi-country HERA2 surveillance pilot

Introduction Whole-genome sequencing (WGS) has become indispensable in infectious disease surveillance, yet its implementation across laboratories remains heterogeneous. Six laboratories from four European countries participated in 2023 in a pilot ring trial as part of the HERA2 project, using a standardized panel of 25 bacterial isolates representing five public health-relevant species. The aim was to assess the comparability and reproducibility of laboratory-specific WGS workflows and to determine whether workflow-related variability affected cgMLST-based interpretations relevant for routine surveillance and outbreak investigations. Methods Participating laboratories applied their established wet-laboratory workflows, including different sample-processing, DNA-extraction and library-preparation methods and sequencing platforms. Illumina sequencing was performed by all laboratories, while Oxford Nanopore Technologies (ONT) sequencing was available in two. To specifically evaluate variability introduced by wet-laboratory procedures, all raw sequencing data were centrally processed using the same bioinformatic pipeline. Reproducibility was primarily assessed using cgMLST allelic distances to the Lab 1 Illumina comparator assembly and concordance of cluster assignments, complemented by analyses of sequencing and assembly variability. Results Despite substantial differences in wet-laboratory workflows and technical sequencing characteristics, retained Illumina records showed only small cgMLST allelic differences across laboratories (0-2 alleles) and concordant cluster assignments. Sequencing and assembly metrics varied according to laboratory, species, and technical factors, but this variability did not prevent concordant Illumina-based cluster interpretation among records meeting predefined minimum quality requirements. ONT results were more species-dependent, and several records, particularly for Salmonella enterica, did not achieve the required cgMLST target completeness. These ONT findings should be interpreted in the context of the technology available at the time of the 2023 ring trial. Conclusions Our results suggest that complete standardization of wet-laboratory workflows may not be required to achieve comparable Illumina-based cgMLST results across laboratories. Rather, laboratories may retain validated local workflows provided that the resulting sequencing data meet appropriate minimum quality requirements. This supports a pragmatic approach to multi-laboratory genomic surveillance that prioritizes data quality and fitness-for-purpose over uniformity of individual wet-laboratory procedures.