{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T01:35:15Z","timestamp":1787016915725,"version":"build-2736575974"},"reference-count":26,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22K07063"],"award-info":[{"award-number":["22K07063"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24K10230"],"award-info":[{"award-number":["24K10230"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003478","name":"Ministry of Health, Labour and Welfare","doi-asserted-by":"publisher","award":["24LA2003"],"award-info":[{"award-number":["24LA2003"]}],"id":[{"id":"10.13039\/501100003478","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007449","name":"Takeda Science Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007449","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Major genotyping methods used to characterize\n                      <jats:italic toggle=\"yes\">Mycoplasma pneumoniae<\/jats:italic>\n                      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,\n                      <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                      assembly separating each target into individual contigs, and\n                      <jats:italic toggle=\"yes\">in silico<\/jats:italic>\n                      genotyping. The results for\n                      <jats:italic toggle=\"yes\">p1<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">orf6<\/jats:italic>\n                      , multilocus sequence types, 23S ribosomal RNA gene mutations conferring macrolide resistance, and single-nucleotide polymorphisms identifying the\n                      <jats:italic toggle=\"yes\">p1<\/jats:italic>\n                      type 1 lineage were obtained simultaneously. The genotyping accuracy was confirmed by comparing the sequences with the whole-genome sequences of 40\n                      <jats:italic toggle=\"yes\">M<\/jats:italic>\n                      .\n                      <jats:italic toggle=\"yes\">pneumoniae<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic toggle=\"yes\">Mycoplasma pneumoniae<\/jats:italic>\n                      genome, such as the presence of repetitive elements of the\n                      <jats:italic toggle=\"yes\">p1<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">M. pneumoniae<\/jats:italic>\n                      , including\n                      <jats:italic toggle=\"yes\">p1<\/jats:italic>\n                      genotyping. The workflow effectively characterized\n                      <jats:italic toggle=\"yes\">M. pneumoniae<\/jats:italic>\n                      clinical isolates. This workflow could help advance research on the molecular epidemiology of\n                      <jats:italic toggle=\"yes\">M. pneumoniae<\/jats:italic>\n                      and the detection of novel genotypes.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02719-24","type":"journal-article","created":{"date-parts":[[2025,5,22]],"date-time":"2025-05-22T09:01:31Z","timestamp":1747904491000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Multiplex amplicon sequencing for the comprehensive genotyping of\n                    <i>Mycoplasma pneumoniae<\/i>"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6339-8568","authenticated-orcid":true,"given":"Hiroaki","family":"Kubota","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00w1zvy92","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of 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