Multiplex amplicon sequencing for the comprehensive genotyping of .

UNLABELLED

Major genotyping methods used to characterize strains are based on various experimental approaches that need to be implemented in parallel for each strain. In this study, we developed a comprehensive workflow based on amplicon sequencing using next-generation sequencing. This workflow comprised PCR amplification with seven tubes, collection into single tubes, shotgun sequencing, assembly separating each target into individual contigs, and genotyping. The results for , , multilocus sequence types, 23S ribosomal RNA gene mutations conferring macrolide resistance, and single-nucleotide polymorphisms identifying the type 1 lineage were obtained simultaneously. The genotyping accuracy was confirmed by comparing the sequences with the whole-genome sequences of 40 . isolates collected in Tokyo, Japan. The workflow described not only enables high-throughput comprehensive data collection but also enables the detection of novel genotypes with single-nucleotide resolution.

IMPORTANCE

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 genome, such as the presence of repetitive elements of the 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 , including genotyping. The workflow effectively characterized clinical isolates. This workflow could help advance research on the molecular epidemiology of and the detection of novel genotypes.