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Next-generation sequencing  cluster typing; All single nucleotide polymorphisms are equal but some are more equal than others

R de Zwaan(1) E Ubbelohde(1,2) A Mulder(1) M Kamst(1) G de Vries(1) K Rebel(3) S NS Kautz(4) D van Soolingen(1) K Kremer(2) R M Anthony(1)

1:National Institute for Public Health and the Environment; 2:KNCV Tuberculosis Foundation; 3:Municipal Public Health Services, Amsterdam; 4:Municipal Public Health Services, Rotterdam

In the Netherlands both molecular typing and epidemiological data relating to transmission of tuberculosis (TB) is investigated. In this study we investigated a large Dutch TB cluster of more than 150 isolates over 25 years. Next-generation sequencing (NGS) data from 61 isolates (2003-2022) was available including one follow-up isolate and two cases of reactivationOur routine NGS pipeline calls isolates as potentially epidemiologically linked on the basis of a standard 12-single nucleotide polymorphism (SNP) distance cutoff. Identifying additional information in the NGS data could guide epidemiological investigations and improve our understanding of transmission chains.  We supplemented our automated analysis with a more detailed manual investigation of cluster “specific” SNPs and evaluated their confidence and the presence of minority SNP populations or unfixed SNPs. The SNP analysis was consistent with the epidemiological data and identified two nonsynonymous dnaA mutation (M348I, R400H) events that transitioned from an unfixed SNP to a fixed SNP. Mutations in dnaA have previously been observed by others as emerging among clustered isolates and are associated with reduced katG expression, potentially improving mycobacterial survival during isoniazid treatment. Thought most of the high confidence SNPs acquired within this cluster were unique in our Dutch database (>4,000 TB isolates) one of the high confidence mutations in the dnaA gene was present in eight unrelated isolates. The examination of high confidence SNPs and the presence of fixed and unfixed SNPs detected both inter- and intrapatient microevolution within this cluster, and provided additional resolution for the identification of epidemiologically linked isolates.

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