16The "One Health" framework emphasizes the ecological relationships between soil, plant, 17 animal and human health. Microbiomes play important roles in these relationships, as they 18 modify the health and performance of the different compartments and influence the transfer of 19 energy, matter and chemicals between them. Standardized methods to characterize 20 microbiomes along food chains are, however, currently lacking. To address this methodological 21 2 gap, we evaluated the performance of DNA extractions kits and commonly recommended 22 primer pairs targeting different hypervariable regions (V3-V4, V4, V5-V6, V5-V6-V7) of the 23 16S rRNA gene, on microbiome samples along a model food chain, including soils, maize roots, 24 cattle rumen, and cattle and human faeces. We also included faeces from gnotobiotic mice 25 colonized with defined bacterial taxa and mock communities to confirm the robustness of our 26 molecular and bioinformatic approaches on these defined low microbial diversity samples.