title: High Interlaboratory Reproducibility and Accuracy of Next-Generation-Sequencing-Based Bacterial Genotyping in a Ring Trial.
abstract: Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-codedStaphylococcus aureusDNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of >75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods.
authors.affiliation: Institute of Hygiene, University Hospital Münster, Münster, Germany mellmann@uni-muenster.de.
authors.forename: Alexander
authors.identifier: 0000-0002-0649-5185
authors.initials: A
authors.lastname: Mellmann
authors.affiliation: Department of Microbiology and Infection Control, Statens Serum Institut (SSI), Copenhagen, Denmark.
authors.forename: Paal Skytt
authors.initials: PS
authors.lastname: Andersen
authors.affiliation: Institute of Hygiene, University Hospital Münster, Münster, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bletz
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Friedrich
authors.affiliation: Molecular and Experimental Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany.|German Center for Infection Research (DZIF), Borstel, Germany.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kohl
authors.affiliation: Department of Microbiology and Infection Control, Statens Serum Institut (SSI), Copenhagen, Denmark.
authors.forename: Berit
authors.initials: B
authors.lastname: Lilje
authors.affiliation: Molecular and Experimental Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany.|German Center for Infection Research (DZIF), Borstel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Niemann
authors.affiliation: Department of Periodontology and Restorative Dentistry, University Hospital Münster, Münster, Germany.
authors.forename: Karola
authors.initials: K
authors.lastname: Prior
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: John W
authors.initials: JW
authors.lastname: Rossen
authors.affiliation: Department of Periodontology and Restorative Dentistry, University Hospital Münster, Münster, Germany.
authors.forename: Dag
authors.initials: D
authors.lastname: Harmsen
mesh_terms: D016680:Genome, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000145:classification* / Q000235:genetics*
keywords: cgMLST; interlaboratory reproducibility; molecular subtyping; ring trial; whole-genome sequencing