{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T00:49:24Z","timestamp":1792025364210,"version":"4.4.0"},"reference-count":30,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T00:00:00Z","timestamp":1782950400000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100003509","name":"\u00d6rebro University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003509","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Microbiol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>16S rRNA gene sequencing is widely used for bacterial community profiling in both clinical and research contexts. The expanding availability of library preparation protocols and bioinformatic pipelines increases analytical flexibility but may also introduce method-dependent biases that affect inferred microbial composition and relative abundance estimates. The relative impact of library preparation protocol, amplicon region, and bioinformatic pipeline on species-level taxonomic inference and compositional agreement remains insufficiently characterised. We therefore compared the Illumina 16S Metagenomic Sequencing Library Preparation protocol (V3\u2013V4) and the Zymo Quick-16S Plus NGS Library Prep Kit (V1\u2013V2 and V3\u2013V4) in combination with two bioinformatic pipelines, nf-core\/ampliseq and TRANA. Performance was assessed using defined microbial community standards and human faecal and colonic biopsy samples.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Pipeline choice was the dominant driver of variation in inferred community composition, exceeding the effects of amplicon regions and library preparation protocols. Genus-level profiles were broadly concordant across methods. Species-level resolution and agreement with expected community composition differed systematically between pipelines, with TRANA demonstrating lower Bray\u2013Curtis dissimilarities to expected compositions than nf-core\/ampliseq.\u00a0Amplicon region had a secondary, pipeline-dependent effect, while protocol differences were minor. In clinical samples, inter-individual biological variation exceeded technical variation.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Bioinformatic processing substantially influenced species-level inference in short-read 16S sequencing, highlighting the importance of pipeline selection for microbiome study design and cross-study comparability.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12866-026-05344-6","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T15:35:36Z","timestamp":1782920136000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Comparison of library preparation protocols and bioinformatic pipelines in high-throughput 16S rRNA gene sequencing"],"prefix":"10.1186","volume":"26","author":[{"given":"Olivia","family":"Andersson","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Fagerstr\u00f6m","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Dannenberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Kekki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Rode","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ignacio","family":"Rangel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carl M\u00e5rten","family":"Lindqvist","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bianca","family":"Stenmark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,1]]},"reference":[{"key":"5344_CR1","doi-asserted-by":"publisher","unstructured":"Butler I, Turner O, Mohammed K, Akhtar M, Evans D, Lambourne J, et al. Standardization of 16S rRNA gene sequencing using nanopore long read sequencing technology for clinical diagnosis of culture negative infections. Front Cell Infect Microbiol. 2025;15. https:\/\/doi.org\/10.3389\/fcimb.2025.1517208.","DOI":"10.3389\/fcimb.2025.1517208"},{"key":"5344_CR2","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1186\/s12866-017-1101-8","volume":"17","author":"I Allali","year":"2017","unstructured":"Allali I, Arnold JW, Roach J, Cadenas MB, Butz N, Hassan HM, et al. A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome. BMC Microbiol. 2017;17:194. https:\/\/doi.org\/10.1186\/s12866-017-1101-8.","journal-title":"BMC Microbiol"},{"key":"5344_CR3","doi-asserted-by":"publisher","unstructured":"Pereira-Marques J, Hout A, Ferreira RM, Weber M, Pinto-Ribeiro I, van Doorn L-J et al. Impact of Host DNA and Sequencing Depth on the Taxonomic Resolution of Whole Metagenome Sequencing for Microbiome Analysis. Front Microbiol 2019;10-2019. https:\/\/doi.org\/10.3389\/fmicb.2019.01277.","DOI":"10.3389\/fmicb.2019.01277"},{"key":"5344_CR4","doi-asserted-by":"publisher","first-page":"4","DOI":"10.20517\/mrr.2023.02","volume":"3","author":"C Giulia Longhi","year":"2024","unstructured":"Giulia Longhi C, Argentini F, Fontana C, Tarracchini L, Mancabelli GA, Lugli, et al. Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses. Microbiome Res Rep. 2024;3:4. https:\/\/doi.org\/10.20517\/mrr.2023.02.","journal-title":"Microbiome Res Rep"},{"key":"5344_CR5","doi-asserted-by":"publisher","first-page":"310","DOI":"10.1186\/s12864-024-10213-5","volume":"25","author":"E Buetas","year":"2024","unstructured":"Buetas E, Jord\u00e1n-L\u00f3pez M, L\u00f3pez-Rold\u00e1n A, D\u2019Auria G, Mart\u00ednez-Priego L, De Marco G, et al. Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples. BMC Genomics. 2024;25:310. https:\/\/doi.org\/10.1186\/s12864-024-10213-5.","journal-title":"BMC Genomics"},{"key":"5344_CR6","doi-asserted-by":"publisher","first-page":"10590","DOI":"10.1038\/s41598-021-89881-2","volume":"11","author":"DM O\u2019Sullivan","year":"2021","unstructured":"O\u2019Sullivan DM, Doyle RM, Temisak S, Redshaw N, Whale AS, Logan G, et al. An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities. Sci Rep. 2021;11:10590. https:\/\/doi.org\/10.1038\/s41598-021-89881-2.","journal-title":"Sci Rep"},{"key":"5344_CR7","doi-asserted-by":"publisher","unstructured":"Isabel A-S, Matchado Monica S, Sandra R, Alina S, Zeno S, Jan B et al. Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing. mSphere. 2021;6: https:\/\/doi.org\/10.1128\/msphere.01202-20.","DOI":"10.1128\/msphere.01202-20"},{"key":"5344_CR8","doi-asserted-by":"publisher","first-page":"e1","DOI":"10.1093\/nar\/gks808","volume":"41","author":"A Klindworth","year":"2013","unstructured":"Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. 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Nature. 1949;163:688\u2013688. https:\/\/doi.org\/10.1038\/163688a0.","journal-title":"Nature"}],"container-title":["BMC Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12866-026-05344-6","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12866-026-05344-6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12866-026-05344-6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T02:34:58Z","timestamp":1782959698000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1186\/s12866-026-05344-6"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,1]]},"references-count":30,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["5344"],"URL":"https:\/\/doi.org\/10.1186\/s12866-026-05344-6","relation":{},"ISSN":["1471-2180"],"issn-type":[{"value":"1471-2180","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,1]]},"assertion":[{"value":"18 March 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 June 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"1 July 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. The use of clinical samples was approved by the Regional Ethical Review Board in Uppsala (Dnr 2018\/283). An amendment permitting the anonymised microbiota method comparison was approved by the Swedish Ethical Review Authority (Dnr 2025-05855-02). All participants provided written informed consent.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"O.A. and C.M.L. contributed to the development of the TRANA pipeline evaluated in this study.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"580"}}