{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,2]],"date-time":"2026-11-02T01:27:13Z","timestamp":1793582833120,"version":"4.5.0"},"reference-count":22,"publisher":"Wiley","issue":"17","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["faseb.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FASEB Journal"],"published-print":{"date-parts":[[2026,9,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>The human gut mycobiome plays a critical role in host immunity, yet standardized workflows for fungal internal transcribed spacer (ITS) amplicon sequencing remain elusive. Unlike the bacterial 16S rRNA gene, where the impact of DNA versus RNA templates is well\u2010documented, such comparative benchmarks are lacking for fungal studies. Furthermore, fungal ITS regions exhibit significant length heterogeneity, posing unique challenges for denoising algorithms that were originally designed for conserved bacterial amplicons. We performed fungal ITS2 sequencing on six fecal samples, generating 12 matched DNA and RNA\u2010derived cDNA libraries for paired methodological benchmarking. We systematically compared three representative workflows, DADA2, Deblur, and VSEARCH, to evaluate read retention, feature recovery, and preservation of ITS2 length heterogeneity. To address dataset\u2010size limitation, we further added an independent public fecal fungal ITS2 validation cohort from PRJNA419104, including 32 baseline CDI samples and 21 metadata\u2010confirmed healthy controls, and reprocessed this dataset using the same comparative pipeline framework. AGI\u2010versus\u2010healthy comparisons in the internal cohort were treated as exploratory secondary analyses rather than disease\u2010biomarker discovery. DADA2 best preserved fungal ITS2 length heterogeneity while avoiding the fixed\u2010length truncation artifact observed with Deblur and the feature inflation typical of permissive OTU clustering. Paired DNA and RNA profiles showed concordant dominant community structure, with no ANCOM\u2010detected differential abundance between templates across major taxonomic levels. Exploratory AGI\u2010versus\u2010healthy comparisons suggested candidate differential\u2010abundance features, but these observations were considered hypothesis\u2010generating and were not interpreted as validated disease biomarkers. In this small\u2010sample paired methodological benchmark, DADA2 is the preferred workflow for fungal ITS2 amplicon analysis in this paired benchmark because it preserves variable\u2010length sequence information. DNA sequencing provides a practical primary layer for routine dominant\u2010community profiling, whereas RNA\u2010based sequencing can serve as an optional complementary layer when low\u2010abundance or activity\u2010oriented features are of specific interest. The results presented in this article are limited by the small sample size, and larger clinical cohorts are required for disease\u2010association or biomarker inference.<\/jats:p>","DOI":"10.1096\/fj.202601623rr","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T12:59:46Z","timestamp":1788267586000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Benchmarking Denoising Strategies and Nucleic Acid Templates for ITS2 Amplicon Sequencing of Gut Mycobiome"],"prefix":"10.1096","volume":"40","author":[{"given":"Ting","family":"Dai","sequence":"first","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangdong Basic Research Center of Excellence for Respiratory Medicine, GMU\u2010GIBH Joint School of Life Sciences, the Guangdong\u2010Hong Kong\u2010Macao Joint Laboratory for Cell Fate Regulation and Diseases Guangzhou Medical University  Guangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yupei","family":"Dai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangdong Basic Research Center of Excellence for Respiratory Medicine, GMU\u2010GIBH Joint School of Life Sciences, the Guangdong\u2010Hong Kong\u2010Macao Joint Laboratory for Cell Fate Regulation and Diseases Guangzhou Medical University  Guangzhou China"},{"name":"School of Artificial Intelligent Medicine Guilin Medical University  Guilin China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayi","family":"Bai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangdong Basic Research Center of Excellence for Respiratory Medicine, GMU\u2010GIBH Joint School of Life Sciences, the Guangdong\u2010Hong Kong\u2010Macao Joint Laboratory for Cell Fate Regulation and Diseases Guangzhou Medical University  Guangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenwen","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangdong Basic Research Center of Excellence for Respiratory Medicine, GMU\u2010GIBH Joint School of Life Sciences, the Guangdong\u2010Hong Kong\u2010Macao Joint Laboratory for Cell Fate Regulation and Diseases Guangzhou Medical University  Guangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8637-4833","authenticated-orcid":false,"given":"Ping","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, Guangdong Basic Research Center of Excellence for Respiratory Medicine, GMU\u2010GIBH Joint School of Life Sciences, the Guangdong\u2010Hong Kong\u2010Macao Joint Laboratory for Cell Fate Regulation and Diseases Guangzhou Medical University  Guangzhou China"},{"name":"Department of Gastroenterology, the Second Affiliated Hospital Guangzhou Medical University  Guangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nm.4517"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1000080107"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3869"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/mSystems.00191-16"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.2584"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0227434"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.096651.109"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-5-88"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6941.2012.01437.x"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0070837"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2023.1178744"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.3389\/fbinf.2022.864597"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0209-9"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.14806\/ej.17.1.200"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky1022"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-018-0470-z"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.5364"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-022-01449-y"},{"issue":"22","key":"e_1_2_12_20_1","first-page":"E2329","article-title":"Relating the Metatranscriptome and Metagenome of the Human Gut","volume":"111","author":"Franzosa E. A.","year":"2014","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1237-9"},{"key":"e_1_2_12_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2012.10.052"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0029-9"}],"container-title":["The FASEB Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.202601623RR","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/full-xml\/10.1096\/fj.202601623RR","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.202601623RR","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,11,2]],"date-time":"2026-11-02T00:51:23Z","timestamp":1793580683000},"score":1,"resource":{"primary":{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/10.1096\/fj.202601623RR"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":22,"journal-issue":{"issue":"17","published-print":{"date-parts":[[2026,9,15]]}},"alternative-id":["10.1096\/fj.202601623RR"],"URL":"https:\/\/doi.org\/10.1096\/fj.202601623rr","archive":["Portico"],"relation":{},"ISSN":["0892-6638","1530-6860"],"issn-type":[{"value":"0892-6638","type":"print"},{"value":"1530-6860","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]},"assertion":[{"value":"2026-04-01","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-06","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-01","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e72198"}}