Long-Read Sequencing for Species-Level Resolution of the Equine Gut Microbiota Reveals the Need for Improved Databases

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 > 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.