Time of Sample Collection Critical for Microbiome Replicability

Although many aspects of microbiome studies have been standardized to improve experimental replicability, none account for how the daily diurnal fluctuations in the gut lumen cause dynamic changes in 16S amplicon sequencing. Here we show that sample collection time affects the conclusions drawn from microbiome studies and are larger than the effect size of a daily experimental intervention or dietary changes. The timing of divergence of the microbiome composition between experimental and control groups are unique to each experiment. Sample collection times as short as only four hours apart lead to vastly different conclusions. Lack of consistency in the time of sample collection may explain poor cross-study replicability in microbiome research. Without looking at other data, the impact on other fields is unknown but potentially significant.One-Sentence Summary If we are not controlling for host circadian rhythm time in microbiome studies when performing experiments, it is like trying to measure sea level rise while not knowing that tides or waves exist.### Competing Interest StatementAZ is a co-founder and holds equity in Endure Biotherapeutics. PCD is an advisor to Cybele and Co-founder and adviser to Ometa and Enveda with prior approval from UC-San Diego.