Multiplex amplicon sequencing for the comprehensive genotyping of <i>Mycoplasma pneumoniae</i>

Genotyping plays a central role in the molecular epidemiology of pathogenic bacteria, and many methods have been developed to identify prevalent lineages, infection routes, and antimicrobial resistance. Whole-genome sequencing generally provides most of the genetic information targeted by classic PCR-based schemes and has contributed to the construction of a simplified workflow for many bacterial species. However, several issues concerning the <i>Mycoplasma pneumoniae</i> genome, such as the presence of repetitive elements of the <i>p1</i> gene, prevent the collection of genotyping results from a single run of short-read shotgun sequencing. Herein, we describe a simplified workflow using amplicon sequencing that covers most of the major genotyping schemes for <i>M. pneumoniae</i>, including <i>p1</i> genotyping. The workflow effectively characterized <i>M. pneumoniae</i> clinical isolates. This workflow could help advance research on the molecular epidemiology of <i>M. pneumoniae</i> and the detection of novel genotypes.