As the microbiome field moves from descriptive and associative research to mechanistic and interventional studies, being able to account for all confounding variables in the experimental design, which includes the maternal effect¹, cage effect², facility differences³, as well as laboratory and sample handling protocols⁴, is critical for interpretability of results. Despite significant procedural and bioinformatic improvements, unexplained variability and lack of replicability still occur. One underexplored factor is that the microbiome is dynamic and exhibits diurnal oscillations that can change microbiome composition^5,6,7. In this retrospective analysis of 16S amplicon sequencing studies in male mice, we show that sample collection time affects the conclusions drawn from microbiome studies and its effect size is larger than those of a daily experimental intervention or dietary changes. The timing of divergence of the microbiome composition between experimental and control groups is unique to each experiment. Sample collection times as short as only 4 hours apart can lead to vastly different conclusions. Lack of consistency in the time of sample collection may explain poor cross-study replicability in microbiome research. The impact of diurnal rhythms on the outcomes and study design of other fields is unknown but likely significant.

随着微生物组领域从描述性和关联性研究转向机械性和介入性研究，能够解释实验设计中的所有混杂变量，包括母体效应 ¹ 、笼子效应 ² 、设施差异 ³ 以及实验室和样品处理协议 ⁴ 对于结果的可解释性至关重要。尽管程序和生物信息学有了显着的改进，但仍然存在无法解释的变异性和缺乏可重复性。一个尚未充分探索的因素是微生物组是动态的，并且表现出可以改变微生物组组成的昼夜振荡 ^5,6,7 。在这项对雄性小鼠 16S 扩增子测序研究的回顾性分析中，我们发现样本采集时间会影响微生物组研究得出的结论，其影响大小大于日常实验干预或饮食改变的影响大小。实验组和对照组之间微生物组组成的差异时间对于每个实验来说都是独特的。样本采集时间间隔短至仅 4 小时，可能会得出截然不同的结论。样本采集时间缺乏一致性可能是微生物组研究中交叉研究可重复性差的原因。昼夜节律对其他领域的结果和研究设计的影响尚不清楚，但可能很重要。