{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T03:46:43Z","timestamp":1787802403325,"version":"build-2784847793"},"reference-count":69,"publisher":"Microbiology Society","issue":"10","license":[{"start":{"date-parts":[[2024,10,1]],"date-time":"2024-10-01T00:00:00Z","timestamp":1727740800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100023924","name":"Graduate School, Chulalongkorn University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100023924","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2024,10,1]]},"abstract":"<jats:p>\n                    Ribosomal RNA gene amplicon sequencing is commonly used to evaluate microbiome profiles in health and disease and document the impact of interventional treatments. Nanopore sequencing is attractive since it can provide greater classification at the species level. However, optimized protocols to target marker genes for bacterial and fungal profiling are needed. To achieve an increased taxonomic resolution, we developed extraction and full-length amplicon PCR-based approaches using Nanopore sequencing. Three lysis conditions were applied to a mock microbial community, including known bacterial and fungal species: ZymoBIOMICS lysis buffer (ML) alone, incorporating bead-beating (MLB) or bead-beating plus MetaPolyzyme enzymatic treatment (MLBE). In profiling of bacteria in comparison to reference data, MLB had more statistically different bacterial phyla and genera than the other two conditions. In fungal profiling, MLB had a significant increase of Ascomycota and a decline of Basidiomycota, subsequently failing to detect\n                    <jats:italic>Malassezia<\/jats:italic>\n                    and\n                    <jats:italic>Cryptococcus<\/jats:italic>\n                    . Also, a principal coordinates analysis plot by the Bray\u2013Curtis metric showed a significant difference among groups for bacterial (\n                    <jats:italic>P=<\/jats:italic>\n                    0.033) and fungal (\n                    <jats:italic>P=<\/jats:italic>\n                    0.012) profiles, highlighting the importance of understanding the biases present in pretreatment. Overall, microbial profiling and diversity analysis revealed that ML and MLBE are more similar than MLB for both bacteria and fungi; therefore, using this specific pipeline, bead-beating is not recommended for whole gene amplicon sequencing. However, ML alone was suggested as an optimal approach considering DNA yield, taxonomic classification, reagent cost and hands-on time. This could be an initial proof-of-concept study for simultaneous human bacterial and fungal microbiome studies.\n                  <\/jats:p>","DOI":"10.1099\/acmi.0.000754.v3","type":"journal-article","created":{"date-parts":[[2024,10,7]],"date-time":"2024-10-07T11:16:49Z","timestamp":1728299809000},"source":"Crossref","is-referenced-by-count":7,"title":["Optimization of a DNA extraction protocol for improving bacterial and fungal classification based on Nanopore sequencing"],"prefix":"10.1099","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6759-6003","authenticated-orcid":false,"given":"May","family":"Soe Thu","sequence":"first","affiliation":[{"name":"Center of Excellence in Immunology and Immune-Mediated Diseases, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand"},{"name":"Department of Infection Biology & Microbiomes, Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Liverpool, L69 3GE, UK"},{"name":"Joint Chulalongkorn University\u2013University of Liverpool Doctoral Program in Biomedical Sciences and Biotechnology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2805-6690","authenticated-orcid":false,"given":"Vorthon","family":"Sawaswong","sequence":"additional","affiliation":[{"name":"Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1914-1818","authenticated-orcid":false,"given":"Prangwalai","family":"Chanchaem","sequence":"additional","affiliation":[{"name":"Center of Excellence in Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, 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