{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T05:05:10Z","timestamp":1784005510723,"version":"3.55.0"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006013","name":"United Arab Emirates University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006013","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Formalin-fixed, paraffin-embedded (FFPE) tissues are yet underutilized resources for microbiome studies. Data on the mucosa-associated microbiota (MAM) of patients with Crohn\u2019s disease (CD) are scarce, due to several methodological limitations. In this study, we aimed to develop and validate a new optimized amplicon-based workflow to profile MAM from FFPE gut biopsies of pediatric CD patients.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We examined 68 FFPE samples, including 34 biopsies from treatment-na\u00efve patients with CD and 34 from healthy controls (HC). V3-V7 regions of the 16S rRNA gene were amplified and sequenced on the Oxford Nanopore platform. Two protocols were tested: Protocol 1 (P1), consisting of a single PCR amplification and purification step, and Protocol 2 (P2), including two sequential PCR amplifications with purification after each round. The second amplification and purification steps were introduced to increase sequencing yield and improve microbiota detection.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      P2 consistently outperformed P1, yielding significantly higher DNA concentration and purity, reducing human DNA contamination and sustaining pore performance. P2 also generated more microbial reads and recovered a richer, more taxonomically diverse community, including increased detection of species with low abundance. More taxa were enriched in P2 across all levels, enhancing species-level resolution. P2 enabled comprehensive detection of pathogenic genera, such as\n                      <jats:italic>Escherichia, Mycobacterium<\/jats:italic>\n                      , and\n                      <jats:italic>Klebsiella<\/jats:italic>\n                      , which were significantly enriched in the P2 samples compared to the P1 samples. Alpha diversity analysis showed increased richness and reduced evenness in P2 compared to P1, with a significant difference in beta diversity, while maintaining community structure in both CD and HC.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The optimized workflow with a two-step strategy improved sequencing performance and enhanced microbiota detection in FFPE tissues. This approach enabled successful profiling of MAM, providing a novel method for retrospective characterization of the microbiome from archival tissues and providing a scalable platform for clinical biomarker discovery.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2026.1885816","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T04:22:03Z","timestamp":1784002923000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimized protocol for profiling mucosa-associated microbiota from formalin-fixed paraffin-embedded gut tissues from treatment-na\u00efve pediatric patients with Crohn\u2019s disease"],"prefix":"10.3389","volume":"16","author":[{"given":"Noora","family":"Al-Ali","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haifa","family":"Al-Awadhi","sequence":"additional","affiliation":[{"name":"Department of Pediatric Gastroenterology, Tawam Hospital","place":["Al Ain, United Arab Emirates"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maya","family":"Hassane","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suhail","family":"Al-Salam","sequence":"additional","affiliation":[{"name":"Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]},{"name":"Zayed Center for Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farah","family":"Al-Marzooq","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]},{"name":"Zayed Center for Health Sciences, United Arab Emirates University","place":["Al Ain, United Arab Emirates"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,7,14]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/S2468-1253(19)30333-4","article-title":"The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories 1990\u20132017: a systematic analysis for the Global Burden of Disease Study 2017","volume":"5","author":"Alatab","year":"2020","journal-title":"Lancet Gastroenterol. 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