{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T20:09:10Z","timestamp":1770840550606,"version":"3.50.1"},"reference-count":40,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T00:00:00Z","timestamp":1673395200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Faculty of Health at Witten\/Herdecke University","award":["IFF 2021-02"],"award-info":[{"award-number":["IFF 2021-02"]}]},{"name":"Faculty of Health at Witten\/Herdecke University","award":["2021-0246"],"award-info":[{"award-number":["2021-0246"]}]},{"DOI":"10.13039\/100010101","name":"Helios Kliniken GmbH","doi-asserted-by":"publisher","award":["IFF 2021-02"],"award-info":[{"award-number":["IFF 2021-02"]}],"id":[{"id":"10.13039\/100010101","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010101","name":"Helios Kliniken GmbH","doi-asserted-by":"publisher","award":["2021-0246"],"award-info":[{"award-number":["2021-0246"]}],"id":[{"id":"10.13039\/100010101","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In contrast to bacteria, microbiome analyses often neglect archaea, but also eukaryotes. This is partly because they are difficult to culture due to their demanding growth requirements, or some even have to be classified as uncultured microorganisms. Consequently, little is known about the relevance of archaea in human health and diseases. Contemporary broad availability and spread of next generation sequencing techniques now enable a stronger focus on such microorganisms, whose cultivation is difficult. However, due to the enormous evolutionary distances between bacteria, archaea and eukaryotes, the implementation of sequencing strategies for smaller laboratory scales needs to be refined to achieve as a holistic view on the microbiome as possible. Here, we present a technical approach that enables simultaneous analyses of archaeal, bacterial and eukaryotic microbial communities to study their roles in development and courses of respiratory disorders. We thus applied combinatorial 16S-\/18S-rDNA sequencing strategies for sequencing-library preparation. Considering the lower total microbiota density of airway surfaces, when compared with gut microbiota, we optimized the DNA purification workflow from nasopharyngeal swab specimens. As a result, we provide a protocol that allows the efficient combination of bacterial, archaeal, and eukaryotic libraries for nanopore-sequencing using Oxford Nanopore Technologies MinION devices and subsequent phylogenetic analyses. In a pilot study, this workflow allowed the identification of some environmental archaea, which were not correlated with airway microbial communities before. Moreover, we assessed the protocol\u2019s broader applicability using a set of human stool samples. We conclude that the proposed protocol provides a versatile and adaptable tool for combinatorial studies on bacterial, archaeal, and eukaryotic microbiomes on a small laboratory scale.<\/jats:p>","DOI":"10.3390\/ijms24021426","type":"journal-article","created":{"date-parts":[[2023,1,12]],"date-time":"2023-01-12T01:32:26Z","timestamp":1673487146000},"page":"1426","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Overlooked Microbiome\u2014Considering Archaea and Eukaryotes Using Multiplex Nanopore-16S-\/18S-rDNA-Sequencing: A Technical Report Focusing on Nasopharyngeal Microbiomes"],"prefix":"10.3390","volume":"24","author":[{"given":"Carolin","family":"Baehren","sequence":"first","affiliation":[{"name":"Laboratory of Experimental Pediatric Pneumology and Allergology, Center for Biomedical Education and Research, School of Life Sciences (ZBAF), Faculty of Health, Witten\/Herdecke University, 58455 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7952-1532","authenticated-orcid":false,"given":"Anton","family":"Pembaur","sequence":"additional","affiliation":[{"name":"Clinical Molecular Genetics and Epigenetics, Faculty of Health, Center for Biomedical Education & Research (ZBAF), Helios University Hospital Wuppertal, Witten\/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick P.","family":"Weil","sequence":"additional","affiliation":[{"name":"Clinical Molecular Genetics and Epigenetics, Faculty of Health, Center for Biomedical Education & Research (ZBAF), Helios University Hospital Wuppertal, Witten\/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nora","family":"Wewers","sequence":"additional","affiliation":[{"name":"Clinical Molecular Genetics and Epigenetics, Faculty of Health, Center for Biomedical Education & Research (ZBAF), Helios University Hospital Wuppertal, Witten\/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frank","family":"Schult","sequence":"additional","affiliation":[{"name":"Center for Child and Adolescent Medicine, Center for Clinical and Translational Research (CCTR), Helios University Hospital Wuppertal, Witten\/Herdecke University, 42283 Wuppertal, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stefan","family":"Wirth","sequence":"additional","affiliation":[{"name":"Center for Child and Adolescent Medicine, Center for Clinical and Translational Research (CCTR), Helios University Hospital Wuppertal, Witten\/Herdecke University, 42283 Wuppertal, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8562-3316","authenticated-orcid":false,"given":"Jan","family":"Postberg","sequence":"additional","affiliation":[{"name":"Clinical Molecular Genetics and Epigenetics, Faculty of Health, Center for Biomedical Education & Research (ZBAF), Helios University Hospital Wuppertal, Witten\/Herdecke University, Alfred-Herrhausen-Str. 50, 58448 Witten, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Malik","family":"Aydin","sequence":"additional","affiliation":[{"name":"Laboratory of Experimental Pediatric Pneumology and Allergology, Center for Biomedical Education and Research, School of Life Sciences (ZBAF), Faculty of Health, Witten\/Herdecke University, 58455 Witten, Germany"},{"name":"Center for Child and Adolescent Medicine, Center for Clinical and Translational Research (CCTR), Helios University Hospital Wuppertal, Witten\/Herdecke University, 42283 Wuppertal, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1007\/s42995-021-00092-0","article-title":"Towards enriching and isolation of uncultivated archaea from marine sediments using a refned combination of conventional microbial cultivation methods","volume":"3","author":"Hu","year":"2021","journal-title":"Mar. 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