From Field to Sequencer: Methodological Impacts of Sampling, Ethanol Storage, and DNA Extraction on Equine Fecal Microbiota (16S Amplicon Sequencing)

Structured abstract Background Profiling of the equine fecal microbiota with 16S amplicon sequencing is complicated by field--dependent sample preservation, identification of inhibitory compounds in feces, and determination of the degree of technical variation during DNA extraction and sequencing. The relative impacts of these methodological steps, however, remain poorly quantified in horses. This study evaluated how extraction kits, sequencing platforms, and long-term ethanol storage affect equine fecal microbiota characterization. Methods Fecal samples (n = 12; 4 horses × 3 biological replicates) were extracted via two commercial kits (QIAamp Fast DNA Stool Mini and DNeasy PowerFecal Pro), sequenced via Illumina NovaSeq and Ion S5, and processed via cutadapt, DADA2, VSEARCH, and SILVA v138.2. Storage effects were assessed on ethanol-preserved Fast Stool extracts stored at 4°C or room temperature for up to 13 months. Community variation was evaluated via Bray–Curtis dissimilarity, PCoA, PERMANOVA with appropriate permutation constraints, and betadisper. Results Horse identity explained the largest share of microbiota variation across all analyses (R² ≈ 0.26–0.34, p  ≤ 0.001). The sequencing platform contributed additional variation (R² = 0.099, p  ≤ 0.001), and Illumina yielded substantially greater read depth and OTU richness than Ion S5. The extraction kit introduced significant but heterogeneous compositional shifts (R² ≈ 0.07–0.08), including kit-specific OTU sets and multiple family-level differences. Long-term ethanol storage produced statistically detectable but modest shifts (R² ≈ 0.08), with no differences in dispersion across storage conditions or extraction dates. Across all analyses, inter-individual variation exceeded methodological effects. Conclusions In equine fecal 16S amplicon sequencing, biological individuality dominates over methodological variation. Nevertheless, the sequencing platform, extraction kit, and long-term ethanol storage each introduce measurable compositional differences. These findings underscore the importance of standardized extraction protocols and transparent reporting of preservation and sequencing parameters to improve reproducibility and comparability in equine microbiota studies.