cgMLST typing reproducibility is maintained across diverse whole-genome sequencing workflows in a multi-country HERA2 surveillance pilot
11 affiliations
- 1Institute for Surveillance and Infectious Disease Epidemiology, Austrian Agency for Health and Food Safety
- 2Institute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety
- 3Laboratory of Hygiene and Epidemiology, Faculty of Medicine, University of Thessaly
- 4Regional Public Health Laboratory (PEDY) of Thessaly
- 5Department of Molecular Epidemiology, Central Public Health Laboratory, National Public Health Organization
- 6Division of Microbiological Reference Laboratories, National Center for Public Health and Pharmacy
- 7Regional Laboratory of Public Health of Crete, School of Medicine, University of Crete
- 8Department of Statistics and Analytical Epidemiology, Austrian Agency for Health and Food Safety
- 9Institute for Veterinary Disease Control Mödling, Austrian Agency for Health and Food Safety
- 10Institute of Hygiene and Medical Microbiology, Medical University Innsbruck
- 11Croatian Institute of Public Health
Abstract
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.
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BibTeX
@article{rosel2026cgmlst,
title = {{cgMLST typing reproducibility is maintained across diverse whole-genome sequencing workflows in a multi-country HERA2 surveillance pilot}},
author = {Adriana Cabal Rosel and Achilleas P. Galanopoulos and Michalis Polemis and Judit Henczkó and Olga Pappa and Eleni Karligiotou and Maria Malliarou and Dimosthenis Chochlakis and Patrick Hyden and Beatriz Daza Prieto and Anna Stöger and Daniel Polzer and Erika Ungvári and Ákos Tóth and Werner Ruppitsch and Dragan Juric and Ivana Ferencak and Irena Tabain},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1930807},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1930807/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - cgMLST typing reproducibility is maintained across diverse whole-genome sequencing workflows in a multi-country HERA2 surveillance pilot
AU - Adriana Cabal Rosel
AU - Achilleas P. Galanopoulos
AU - Michalis Polemis
AU - Judit Henczkó
AU - Olga Pappa
AU - Eleni Karligiotou
AU - Maria Malliarou
AU - Dimosthenis Chochlakis
AU - Patrick Hyden
AU - Beatriz Daza Prieto
AU - Anna Stöger
AU - Daniel Polzer
AU - Erika Ungvári
AU - Ákos Tóth
AU - Werner Ruppitsch
AU - Dragan Juric
AU - Ivana Ferencak
AU - Irena Tabain
PY - 2026
DA - 2026/10/28
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1930807
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1930807/full
AB - 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.
SN - 1664-302X
ER -