4.6. Statistical Analysis of Microbiome Data
REGRESSION, EVENT
All statistical tests were run using R software (v. 4.0.3, R Core Team (2022)). Charts were generated using the ggplot2, ggrepel, pheatmap, ComplexHeatmap, survminer, and igraph R packages.Alpha diversity indexes and taxonomical relative abundances were compared between treatment groups using two-sided Wilcoxon–Mann–Whitney tests and Cliff’s delta as effect size, using the wilcox.exact function from the exactRankTests R package and the cliff.delta function from the effsize R package, respectively.To investigate the time to reach a given or later evolutionary microbiome state, the time of the event was defined as the age at the earliest visit where the infant was observed to have the FCT in question or a later FCT. As many infants were not observed in all clusters (due to in-frequent sampling or biology), the time of the event was defined as the age at the earliest visit where the infant was observed to have the FCT3 or later FCT, e.g., for ‘Proportion in FCT2 or lower’, time of event would be the earliest visit where the infant had an FCT3, FCT4, or FCT5 community type. Differences in FCT transition probabilities between treatment groups were tested for FCT2, FCT3, FCT4, and FCT5 using Cox proportional hazards regression and global statistical significance reported by the score log-rank test using the survival R package. Data were right- and interval censored, where the intervals between visits were long and of variable length (especially towards the end), resulting in uncertain estimates of the actual time of progression from one cluster to the next. The seven infants with only a V6 sample were excluded from this analysis.Finally, a taxon set enrichment analysis (TSEA) was performed at the family and genus level (full details are outlined in the 