Towards standardization in pig microbiome research
Reproducible microbiome profiling is essential for linking microbial communities to host health, yet methodological variation continues to undermine reproducibility across studies. This problem is particularly acute in pig microbiome research, where no standardized DNA extraction protocols exist despite the species' importance in agriculture and biomedicine. Here, we provide the first systematic benchmark of 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 the choice of extraction method introduces profound biases in 16S rRNA gene datasets, affecting DNA yield, diversity, community composition, and spike-in recovery, whereas metagenomic profiles are comparatively robust to variations in protocol. Among hypervariable regions, the V4 region produced the most consistent results, while V1–V3 inflated artificial variability and chimeras. Kit-dependent recovery of Gram-positive versus Gram-negative taxa further 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 urgent need for standardized protocols to ensure reproducibility and comparability across pig microbiome studies.
Reproducible microbiome profiling is essential for linking microbial communities to host health, yet methodological variation continues to undermine reproducibility across studies. This problem is particularly acute in pig microbiome research, where no standardized DNA extraction protocols exist despite the species' importance in agriculture and biomedicine. Here, we provide the first systematic benchmark of 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 the choice of extraction method introduces profound biases in 16S rRNA gene datasets, affecting DNA yield, diversity, community composition, and spike-in recovery, whereas metagenomic profiles are comparatively robust to variations in protocol. Among hypervariable regions, the V4 region produced the most consistent results, while V1–V3 inflated artificial variability and chimeras. Kit-dependent recovery of Gram-positive versus Gram-negative taxa further 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 urgent need for standardized protocols to ensure reproducibility and comparability across pig microbiome studies.
UNIVERSITY HOHENHEIM