Speaker
Description
The lack of standardized DNA extraction protocols in pig microbiome research limits reproducibility and cross-study comparability. This challenge becomes even more important in large-scale studies that generate substantial sequencing data and require coordinated, high-throughput computational analysis. To address this, we conducted a large-scale, multi-center validation study across six research facilities in Germany, evaluating three DNA extraction kits using both 16S rRNA gene amplicon sequencing and shotgun metagenomics on identical samples. Fecal samples from 200 male German Landrace × Pietrain pigs were processed independently at each site following harmonized protocols. Amplicon (V1–V2, V3–V4) and PCR-free metagenomic libraries were sequenced on Illumina NovaSeq platforms. A total of 7236 amplicon and 2700 metagenomic samples were generated, pooled and analyzed on the BinAC2 Cluster for bioinformatic processing. Analyses were performed using DADA2 and QIIME2 for amplicons and Trim Galore with moshpit distributions v2026.1 for metagenomes. Shannon entropy and Bray-Curtis distances were calculated as α- and β-diversity metrics.
Shannon entropy values derived from shotgun metagenomic profiles exhibited limited variability across facilities and extraction kits, with median values ranging from 3.42 to 4.09. For 16S amplicon data, entropy values were highly consistent across locations (median: V1-V2 6.93–7.80; V3-V4: 6.36–7.07), with narrow interquartile ranges and similar medians. Kruskal-Wallis tests for location were significant in nearly all comparisons (p<0.01), but numerical differences were small and not accompanied by clear shifts in diversity, indicating sensitivity to minor variation rather than biologically relevant effects. PERMANOVA on Bray-Curtis distances confirmed significant effects of location, extraction kit, and their interaction across all sequencing methods (p≤0.001 in all cases). Effect sizes were, however, minimal: location explained 1.25-2.4% of variance, extraction kit 3.6-10.1%, and their interaction with extraction kits ranged from 1.2-1.4%, with residual variation accounting for 87-93%. PERMDISP tests were largely non-significant, indicating that observed location effects predominantly reflect true differences in community composition (centroid position) rather than differences in within-group dispersion; a small number of kit/platform combinations showed significant dispersion effects and are interpreted with corresponding caution.
ANCOM-BC2 identified a subset of differentially abundant genera between locations within each kit and platform, consistent with expected facility-associated variation; the number and identity of these taxa varied by kit and sequencing method. This taxon-level variability did not translate into meaningfully altered overall community structure, underscoring that community-level consistency and individual-taxon sensitivity are compatible, not contradictory, findings.
Overall, despite minor statistically detectable compositional differences, microbial community structure was highly consistent across facilities and kits, with results showing that the selected DNA extraction kits, combined with the standardized protocol utilized, effectively reduce technical variance in multi-center pig microbiome studies. We propose this workflow as a standardized operating procedure for future porcine microbiome research, providing a reproducible framework that supports robust cross-study comparability and large-scale meta-analyses.