Sample Size Estimations Based on Human Microbiome Temporal Stability Over 6 Months: A Shallow Shotgun Metagenome...

Biological factors affect the human microbiome, highlighting the need for reasonably estimating sample sizes in future population studies.

We assessed the temporal stability of fecal microbiome diversity, species composition, and genes and functional pathways through shallow shotgun metagenome sequencing. Using intraclass correlation coefficients (ICC), we measured biological variability over 6 months. We estimated case numbers for 1:1 or 1:3 matched case-control studies, considering significance levels of 0.05 and 0.001 with 80% power, based on the collected fecal specimens per participant.

The fecal microbiome's temporal stability over 6 months varied (ICC < 0.6) for most alpha and beta diversity metrics. Heterogeneity was seen in species, genes, and pathways stability (ICC, 0.0-0.9). Detecting an OR of 1.5 per SD required 1,000 to 5,000 cases (0.05 significance for alpha and beta; 0.001 for species, genes, and pathways) with equal cases and controls. Low-prevalence species needed 15,102 cases, and high-prevalence species required 3,527. Similar needs applied to genes and pathways. In a 1:3 matched case-control study with one fecal specimen, 10,068 cases were needed for low-prevalence species and 2,351 for high-prevalence species. For ORs of 1.5 with multiple specimens, cases needed for low-prevalence species were 15,102 (one specimen), 8,267 (two specimens), and 5,989 (three specimens).

Detecting disease associations requires a large number of cases. Repeating prediagnostic samples and matching cases to more controls could decrease the needed number of cases for such detections.

Our results will help future epidemiologic study designs and implement well-powered microbiome studies.