{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T15:07:50Z","timestamp":1789398470575,"version":"build-2803163510"},"reference-count":33,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":13,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["U01 AT012970"],"award-info":[{"award-number":["U01 AT012970"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","award":["UL1 TR002345"],"award-info":[{"award-number":["UL1 TR002345"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Microbiome studies often seek to determine how the absolute abundances of individual taxa change across biological conditions, yet sequencing read counts are sample-specific scaled representations of those abundances. Because sampling depth can differ across samples, fold changes calculated directly from sequencing read counts do not generally represent absolute-abundance fold changes. Normalization methods attempt to account for these between-sample differences in sampling depth, but their accuracy depends on the reference used. In particular, total-sum scaling uses all taxa as the reference and can introduce compositional bias. Reference-based methods instead rely on taxa that are stable across conditions, but contamination of the reference set by differentially abundant (DA) taxa can distort sampling-depth estimation and downstream inference. Here, we present iterative reference selection (IRS), a robust normalization method that iteratively screens and refines a candidate reference set to exclude DA taxa. By deriving a clean reference set, IRS accurately captures between-sample differences in sampling depth and recovers absolute-abundance fold changes. Benchmarking using simulations and datasets with experimental absolute quantification shows that IRS outperforms standard scaling and existing reference-based methods in controlling false discovery rates while maintaining power.<\/jats:p>","DOI":"10.1093\/bib\/bbag495","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T11:38:08Z","timestamp":1787657888000},"source":"Crossref","is-referenced-by-count":0,"title":["IRS: iterative reference selection improves normalization of microbiome sequencing data"],"prefix":"10.1093","volume":"27","author":[{"given":"Yiming","family":"Shi","sequence":"first","affiliation":[{"name":"Institute for Informatics, Data Science and Biostatistics, Washington University School of Medicine , 660 S. Euclid Ave., MSC 8067-0013-05, St. Louis, MO 63110-1093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lili","family":"Liu","sequence":"additional","affiliation":[{"name":"Institute for Informatics, Data Science and Biostatistics, Washington University School of Medicine , 660 S. Euclid Ave., MSC 8067-0013-05, St. Louis, MO 63110-1093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Chen","sequence":"additional","affiliation":[{"name":"Division of Computational Biology, Department of Quantitative Health Sciences, Mayo Clinic , 200 First St. SW, Rochester, MN 55905 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristine M","family":"Wylie","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Washington University School of Medicine , 660 S. Euclid Ave., St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Todd N","family":"Wylie","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Washington University School of Medicine , 660 S. Euclid Ave., St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sung Hee","family":"Park","sequence":"additional","affiliation":[{"name":"Institute for Informatics, Data Science and Biostatistics, Washington University School of Medicine , 660 S. Euclid Ave., MSC 8067-0013-05, St. Louis, MO 63110-1093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruiwen","family":"Zhou","sequence":"additional","affiliation":[{"name":"Institute for Informatics, Data Science and Biostatistics, Washington University School of Medicine , 660 S. Euclid Ave., MSC 8067-0013-05, St. Louis, MO 63110-1093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9835-7662","authenticated-orcid":false,"given":"Yin","family":"Cao","sequence":"additional","affiliation":[{"name":"Division of Public Health Sciences, Department of Surgery, Washington University School of Medicine , 600 S. Taylor Ave., St. Louis, MO 63110 ,","place":["United States"]},{"name":"Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine , 660 S. Euclid Ave., Campus Box 8109, St. Louis, MO 63110 ,","place":["United States"]},{"name":"Division of Gastroenterology, Department of Medicine, Washington University School of Medicine , 660 S. Euclid Ave., Campus Box 8124, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie A","family":"Fritz","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Washington University School of Medicine , 660 S. Euclid Ave., St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Molly J","family":"Stout","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, University of Michigan Medical School , 1500 E. Medical Center Dr., Ann Arbor, MI 48109-0276 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Cristina","family":"Vazquez Guillamet","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine , 4990 Children\u2019s Place, St. Louis, MO 63110 ,","place":["United States"]},{"name":"Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine , 4490 Children\u2019s Place, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1844-338X","authenticated-orcid":false,"given":"Lei","family":"Liu","sequence":"additional","affiliation":[{"name":"Institute for Informatics, Data Science and Biostatistics, Washington University School of Medicine , 660 S. Euclid Ave., MSC 8067-0013-05, St. Louis, MO 63110-1093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"key":"2026091410301963400_ref1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-10656-5","article-title":"Establishing microbial composition measurement standards with reference frames","volume":"10","author":"Morton","year":"2019","journal-title":"Nat Commun"},{"key":"2026091410301963400_ref2","doi-asserted-by":"publisher","first-page":"3562","DOI":"10.1038\/s41467-021-23821-6","article-title":"Benchmarking microbiome transformations favors experimental quantitative approaches to address compositionality and sampling depth biases","volume":"12","author":"Llor\u00e9ns-Rico","year":"2021","journal-title":"Nat Commun"},{"key":"2026091410301963400_ref3","doi-asserted-by":"publisher","first-page":"3514","DOI":"10.1038\/s41467-020-17041-7","article-title":"Analysis of 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