{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T06:25:53Z","timestamp":1791440753402,"version":"4.3.3"},"reference-count":36,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T00:00:00Z","timestamp":1778284800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["#RGPIN\/4514-2018"],"award-info":[{"award-number":["#RGPIN\/4514-2018"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"award":["#RGPIN\/4514-2018"],"award-info":[{"award-number":["#RGPIN\/4514-2018"]}],"id":[{"id":"https:\/\/ror.org\/01h531d29","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Differences in gut microbiota composition related to diet have been reported in horses, but characterization of specific microbial taxa remains limited, particularly at the species level. The objective of this study was to use long-read sequencing of the 16S rRNA gene to provide additional taxonomic insight into the intestinal microbiota in horses. Fecal samples were collected from 12 horses on pasture and from 6 of them after switching to a hay diet. Sequencing yielded low read counts per sample, and the analysis failed to detect statistical differences in alpha- and beta-diversity among dietary groups (p &gt; 0.05). Species-level taxonomic resolution was not substantially enhanced using long-read sequencing, as only 3% of reads were assigned at the species level, and an additional 3% of reads were assigned at the genus level. The majority of reads (49%) were classified at the family level. Accordingly, in this dataset, long-read sequencing did not provide additional biological insight into diet-associated differences in the equine gut microbial community. This limited added value can be explained by the low sequencing depth obtained for several samples and the current incompleteness of reference databases for equine bacterial taxa, highlighting ongoing challenges in achieving high-resolution characterization of the equine gut microbiome.<\/jats:p>","DOI":"10.3390\/ani16101459","type":"journal-article","created":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T10:50:52Z","timestamp":1778496652000},"page":"1459","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Long-Read Sequencing for Species-Level Resolution of the Equine Gut Microbiota Reveals the Need for Improved Databases"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-6676-7137","authenticated-orcid":false,"given":"Laurence","family":"Leduc","sequence":"first","affiliation":[{"name":"Department of Clinical Sciences, Universit\u00e9 de Montr\u00e9al, Saint-Hyacinthe, QC J2S 2M2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-0552-6591","authenticated-orcid":false,"given":"Laurie","family":"Boucher","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biomedical Sciences, Universit\u00e9 de Montr\u00e9al, Saint-Hyacinthe, QC J2S 2M2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8001-6314","authenticated-orcid":false,"given":"Nuria","family":"Mach","sequence":"additional","affiliation":[{"name":"Interactions H\u00f4tes-Agents Pathog\u00e8nes, \u00c9cole nationale v\u00e9t\u00e9rinaire Toulouse, Institut national de recherche pour l\u2019agriculture, l\u2019alimentation et l\u2019environnement, Universit\u00e9 Toulouse, 31999 Toulouse, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3910-0370","authenticated-orcid":false,"given":"Mathilde","family":"Lecl\u00e8re","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Universit\u00e9 de Montr\u00e9al, Saint-Hyacinthe, QC J2S 2M2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5067-7501","authenticated-orcid":false,"given":"Marcio","family":"Costa","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biomedical Sciences, Universit\u00e9 de Montr\u00e9al, Saint-Hyacinthe, QC J2S 2M2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.tvjl.2019.05.005","article-title":"Methods and basic concepts for microbiota assessment","volume":"249","author":"Costa","year":"2019","journal-title":"Vet. J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5112","DOI":"10.1128\/AEM.01043-13","article-title":"Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform","volume":"79","author":"Kozich","year":"2013","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e13084","DOI":"10.7717\/peerj.13084","article-title":"Metagenomic investigation of the equine faecal microbiome reveals extensive taxonomic diversity","volume":"10","author":"Gilroy","year":"2022","journal-title":"PeerJ"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Ericsson, A.C., Johnson, P.J., Lopes, M.A., Perry, S.C., and Lanter, H.R. (2016). A Microbiological Map of the Healthy Equine Gastrointestinal Tract. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0166523"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1111\/jvim.15748","article-title":"Fecal microbiota in horses with asthma","volume":"34","author":"Leclere","year":"2020","journal-title":"J. Vet. Intern. Med."},{"key":"ref_6","first-page":"85","article-title":"Effects of concentrated fecal microbiota transplant on the equine fecal microbiota after antibiotic-induced dysbiosis","volume":"87","author":"Arroyo","year":"2023","journal-title":"Can. J. Vet. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1032","DOI":"10.1038\/s42003-022-03977-7","article-title":"Mining the equine gut metagenome: Poorly-characterized taxa associated with cardiovascular fitness in endurance athletes","volume":"5","author":"Mach","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1186\/s42523-019-0013-3","article-title":"The gut microbiome of horses: Current research on equine enteral microbiota and future perspectives","volume":"1","author":"Kauter","year":"2019","journal-title":"Anim. Microbiome"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Buetas, E., Jord\u00e1n-L\u00f3pez, M., L\u00f3pez-Rold\u00e1n, A., D\u2019Auria, G., Mart\u00ednez-Priego, L., De Marco, G., Carda-Di\u00e9guez, M., and Mira, A. (2024). Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples. BMC Genom., 25.","DOI":"10.1186\/s12864-024-10213-5"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Di Pietro, R., Arroyo, L., Leclere, M., and Costa, M. (2021). Species-Level Gut Microbiota Analysis after Antibiotic-Induced Dysbiosis in Horses. Animals, 11.","DOI":"10.3390\/ani11102859"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Raspa, F., Chessa, S., Bergero, D., Sacchi, P., Ferrocino, I., Cocolin, L., Corvaglia, M.R., Moretti, R., Cavallini, D., and Valle, E. (2024). Microbiota characterization throughout the digestive tract of horses fed a high-fiber vs. a high-starch diet. Front. Vet. Sci., 11.","DOI":"10.3389\/fvets.2024.1386135"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Leduc, L., Costa, M., and Lecl\u00e8re, M. (2024). The Microbiota and Equine Asthma: An Integrative View of the Gut\u2013Lung Axis. Animals, 14.","DOI":"10.3390\/ani14020253"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Weinert-Nelson, J.R., Biddle, A.S., Sampath, H., and Williams, C.A. (2023). Fecal Microbiota, Forage Nutrients, and Metabolic Responses of Horses Grazing Warm- and Cool-Season Grass Pastures. Animals, 13.","DOI":"10.3390\/ani13050790"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e70147","DOI":"10.1111\/asj.70147","article-title":"Complete Extruded Diet: How Does Equine Fecal Microbiota Change During Intake Adaptation?","volume":"97","author":"Franzan","year":"2026","journal-title":"Anim. Sci. J."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Costa, M.C., Arroyo, L.G., Allen-Vercoe, E., St\u00e4mpfli, H.R., Kim, P.T., Sturgeon, A., and Weese, J.S. (2012). Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0041484"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Al-Ansari, A.S., Duggan, V., Mulcahy, G., Yin, X., Brennan, L., Cotter, P.D., Patel, S.H., O\u2019Donovan, C.M., Crispie, F., and Walshe, N. (2025). Faecal microbiota and serum metabolome association with equine metabolic syndrome in connemara ponies. BMC Vet. Res., 21.","DOI":"10.1186\/s12917-025-04853-2"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1165\/rcmb.2017-0228OC","article-title":"Lung Microbiome Is Influenced by the Environment and Asthmatic Status in an Equine Model of Asthma","volume":"60","author":"Dickson","year":"2019","journal-title":"Am. J. Respir. Cell Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nmeth.3869","article-title":"DADA2: High-resolution sample inference from Illumina amplicon data","volume":"13","author":"Callahan","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"10-1128","DOI":"10.1128\/mBio.03656-20","article-title":"High-Resolution Differentiation of Enteric Bacteria in Premature Infant Fecal Microbiomes Using a Novel rRNA Amplicon","volume":"12","author":"Graf","year":"2021","journal-title":"mBio"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"006747","DOI":"10.1099\/ijsem.0.006747","article-title":"Setting new boundaries of 16S rRNA gene identity for prokaryotic taxonomy","volume":"75","author":"Hackmann","year":"2025","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1099\/ijs.0.059774-0","article-title":"Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes","volume":"64","author":"Kim","year":"2014","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1038\/nrmicro3330","article-title":"Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences","volume":"12","author":"Yarza","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mallick, H., Rahnavard, A., McIver, L.J., Ma, S., Zhang, Y., Nguyen, L.H., Tickle, T.L., Weingart, G., Ren, B., and Schwager, E.H. (2021). Multivariable association discovery in population-scale meta-omics studies. PLoS Comput. Biol., 17.","DOI":"10.1101\/2021.01.20.427420"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2437","DOI":"10.1111\/jvim.16206","article-title":"The effects of signalment, diet, geographic location, season, and colitis associated with antimicrobial use or Salmonella infection on the fecal microbio","volume":"35","author":"Arnold","year":"2021","journal-title":"J. Vet. Intern. Med."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Arnold, C., Pilla, R., Chaffin, K., Lidbury, J., Steiner, J., and Suchodolski, J. (2021). Alterations in the Fecal Microbiome and Metabolome of Horses with Antimicrobial-Associated Diarrhea Compared to Antibiotic-Treated and Non-Treated Healthy Case Controls. Animals, 11.","DOI":"10.3390\/ani11061807"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Arroyo, L.G., Rossi, L., Santos, B.P., Gomez, D.E., Surette, M.G., and Costa, M.C. (2020). Luminal and Mucosal Microbiota of the Cecum and Large Colon of Healthy and Diarrheic Horses. Animals, 10.","DOI":"10.3390\/ani10081403"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Zhu, Y., Wang, X., Deng, L., Chen, S., Zhu, C., and Li, J. (2021). Effects of Pasture Grass, Silage, and Hay Diet on Equine Fecal Microbiota. Animals, 11.","DOI":"10.3390\/ani11051330"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zaitseva, S., Dagurova, O., Radnagurueva, A., Kozlova, A., Izotova, A., Krylova, A., Noskov, S., Begmatov, S., Patutina, E., and Barkhutova, D.D. (2023). Fecal Microbiota and Diet Composition of Buryatian Horses Grazing Warm- and Cold-Season Grass Pastures. Microorganisms, 11.","DOI":"10.3390\/microorganisms11081947"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Garber, A., Hastie, P., McGuinness, D., Malarange, P., and Murray, J.-A. (2020). Abrupt dietary changes between grass and hay alter faecal microbiota of ponies. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0237869"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1038\/nbt.3935","article-title":"Shotgun metagenomics, from sampling to analysis","volume":"35","author":"Quince","year":"2017","journal-title":"Nat. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1038\/s41587-020-0501-8","article-title":"A complete domain-to-species taxonomy for Bacteria and Archaea","volume":"38","author":"Parks","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/s42523-020-0023-1","article-title":"Multi-kingdom characterization of the core equine fecal microbiota based on multiple equine (sub)species","volume":"2","author":"Edwards","year":"2020","journal-title":"Anim. Microbiome"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Thomas, A.M., and Segata, N. (2019). Multiple levels of the unknown in microbiome research. BMC Biol., 17.","DOI":"10.1186\/s12915-019-0667-z"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/s41586-023-06955-z","article-title":"Functional and evolutionary significance of unknown genes from uncultivated taxa","volume":"626","author":"Cantalapiedra","year":"2024","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2262","DOI":"10.2527\/jas.2015-0198","article-title":"Horse species symposium: The microbiome of the horse hindgut: History and current knowledge1","volume":"94","author":"Julliand","year":"2016","journal-title":"J. Anim. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"fix136","DOI":"10.1093\/femsec\/fix136","article-title":"A dysbiosis index to assess microbial changes in fecal samples of dogs with chronic inflammatory enteropathy","volume":"93","author":"AlShawaqfeh","year":"2017","journal-title":"FEMS Microbiol. Ecol."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/10\/1459\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T11:32:56Z","timestamp":1778499176000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/10\/1459"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,9]]},"references-count":36,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ani16101459"],"URL":"https:\/\/doi.org\/10.3390\/ani16101459","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,9]]}}}