Towards reproducible pig gut microbiome profiling through standardized methodologies.

Abstract Reproducible microbiome profiling is essential for linking microbial communities to host health, yet methodological variation continues to undermine reproducibility across studies. This problem is acute in pig microbiome research, where no standardized DNA extraction protocols exist despite the species’ importance in agriculture and biomedicine. Here, we benchmark how 12 widely used extraction kits influence microbiome outcomes in 16S rRNA gene amplicon sequencing and shotgun metagenomics of pig fecal samples. We demonstrate that extraction choice biases 16S rRNA gene datasets, affecting DNA yield, diversity, community composition, and spike-in recovery, whereas metagenomic taxonomy and functional profiles are comparatively robust. Kit-dependent recovery of Gram-positive versus Gram-negative taxa revealed systematic biases with direct consequences for biological interpretation. By integrating spike-in controls, taxonomic resolution, and metagenome-assembled genomes, we establish a framework for evaluating DNA extraction methods in animal microbiome research. Our findings demonstrate that 16S rRNA gene amplicon sequencing is more susceptible to extraction-driven artifacts than metagenomics, highlighting the need for standardized protocols to ensure reproducibility and comparability across pig microbiome studies. Moreover, while shotgun metagenomics was comparatively robust to DNA extraction choice, the number of assembled good-quality MAGs recovered was strongly dependent on the extraction kit selection.