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

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. Prevotella, 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 Prevotella 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.