2.8. Data Analysis
IBS
PCoA was used to visualize clustering of microbiota composition for the interventions (UniFrac distance metrics (unweighted and weighted)) with the R package Phyloseq (version 1.32.0). For supervised analysis, gut microbiota composition data was analyzed as relative abundance on genus level and filtered, such that each had to be present in at least 50% of the participants in any of the treatment or washout weeks. A pseudo count of 10Selected gut microbiota genera of interest, SCFAs in feces and plasma and diversity measures were further investigated using a mixed model design with participant ID as a random factor and intervention and period as fixed factors, with type 3 tests and pairwise comparisons. The factor IBS subtype and the interaction IBS subtype x intervention were initially included, but did not contribute meaningfully to any of the models and were therefore removed from the final modelling. All variables for genera and SCFA were log-transformed and results back-transformed. For all models, homoscedasticity and normality were assessed by residual and quantile–quantile plots. Partial Spearman correlation analysis (adjusting for age and sex) was performed for each intervention with specific variables from genera, SCFAs and metabolome and the underlying variables in IBS-SSS, the bowel emptying diary and the health and quality-of-life questionnaires. Data were analyzed as end of intervention. SCFA cross-correlations and correlations with questionnaires included datapoints from both interventions and preceding washout weeks, adjusted for age, sex, and identity. The bowel-emptying-diary variable, ‘whether medication was needed for bowel emptying’, was excluded, due to severe zero inflation (ninety-one percent zeros).Since the study was exploratory, there was no correction for multiple testing. However, due to the high number of tests in the correlation analysis, the stability of significant findings was evaluated according to the Benjamini–Hochberg false discovery rate procedure, considering the FDR-adjusted The study sample size was determined post hoc by assuming a difference of 50 points in the IBS-SSS with a power of 0.8, and a 20% dropout rate, using intra-individual SD for the interventions from this trial (SD = 111.6) [