PacBio full-length 16S rRNA gene sequencing processed with Emu and GTDB provides the highest taxonomic resolution for rumen bacteriome profiling

Although full-length 16S rRNA gene sequencing has substantially improved taxonomic resolution compared to short-read approaches, a high proportion of unclassified taxa are reported in rumen microbiome studies. This limitation is largely driven by platform-specific analytical workflows and the insufficient representation of rumen-associated lineages in commonly used reference databases. Here, we identified the optimal combination of sequencing platform, analytical workflow, and reference database to improve rumen bacteriome classification. We analyzed short-read and full-length 16S rRNA gene sequences from rumen samples collected from two beef cattle populations. Short-read sequences were generated using Illumina NextSeq2000 and processed with QIIME2. Full-length sequences were generated using PacBio Revio (PacBio-16S) and Nanopore MinION (ONT-16S); PacBio-16S data were analyzed using QIIME2 and Emu, while Nanopore data were analyzed using EPI2ME and Emu. Five reference databases were evaluated across all analytical approaches: SILVA 138.2, SILVA 138.2 with Hungate1000 collection, NCBI, Greengenes2, and GTDB. The comparisons showed that PacBio-16S (Emu) achieved the highest proportion of classified reads among all platform-specific workflows, while GTDB consistently produced the highest number of non-redundant classified taxa. <i>Prevotella</i>, a dominant rumen genus, was abundant in Illumina and PacBio-16S datasets but was underrepresented in ONT-16S workflows. Species-level analyses further demonstrated that PacBio-16S (Emu) reliably provided more consistent and high-resolution identification of <i>Prevotella</i> species under GTDB across two beef populations. Overall, our results demonstrate that sequencing platform, workflow choice, and database selection strongly influence rumen bacteriome profiles. We recommend PacBio-16S (Emu) under GTDB as the most reliable workflow for achieving high-resolution taxonomic classification of rumen bacteriome.