Hypervariable region-specific detection of an avian gut pathobiont in multi-primer 16S rRNA metagenomics: the V9 region identifies Gallibacterium anatis undetected by conventional V3-V4 approaches.

Hypervariable region (V-region) selection critically determines which taxa are resolved in 16S rRNA amplicon surveys, yet most commercial poultry gut microbiome studies rely on the V3-V4 primer pair optimised for Illumina short-read platforms. The Ion GeneStudio S5 Prime with multi-primer 16S chemistry simultaneously amplifies six variable regions (V2, V3, V4, V67, V8, V9) from a single library, providing an unprecedented opportunity to benchmark region-specific taxonomic resolution in the same sample set without inter-library bias. 29 commercial broiler caecal samples (HEALTHY n = 10; DISEASED n = 19) were analysed per-V-region on the Ion GeneStudio S5 Prime using the Ion 16S Metagenomics Kit, yielding 46,542 classified reads distributed across six V-regions. From a total sequencing depth of 342,716-1,358,797 reads per sample. Independent ASV-level validation was performed using QIIME2 v2024.10 DADA2 (738 ASVs, SILVA 138), confirming all primary findings. V3 contributed the highest read volume (14,818 reads, 31.8%) and resolved the most genera (52 unique). V9 contributed the fewest reads (2831, 6.1%) but the highest number of region-exclusive genera (11), including the avian pathobiont Gallibacterium anatis. Critically, 121 of 220 total G. anatis reads (55%) were recovered exclusively via V9 primers; zero G. anatis reads were detected by V3 across all 29 samples.". In a parallel differential abundance analysis, G. anatis was the most significantly enriched taxon in diseased caecal microbiota (DESeq2 padj = 1.45 × 10-6), a finding that would have been entirely missed by a conventional V3-V4 workflow. In silico analysis of one of the samples from this set, found G. anatis (GenBank PX986441.1) confirmed absence of the 341F primer binding site. Mean sequence identity was uniformly high across all regions (98.74-99.05%), confirming that V9 underperformance is a coverage rather than quality issue. These findings demonstrate significant primer bias in single-region 16S workflows applied to poultry gut microbiome research, with direct implications for diagnostic assay design and pathobiont surveillance programmes.