2.6. Faecal Microbiome Analysis
EBOS
Faecal sample collection was performed in an OMNIgene GUT stool collection kit (DNA Genotek, Ottawa, ON, Canada) for sample preservation, according to instructions provided by the manufacturer, and using a disposable urine collection hat (EBOS Healthcare, Kingsgrove, NSW, Australia). Samples were returned to the laboratory within approximately 24 h of collection and stored at −80 °C until analyses. Faecal DNA extraction was performed by transferring 200 µL of the faecal slurry to a tube containing 200 µL of 1X phosphate buffered saline (PBS, pH 8.0) (Life Technologies, Waltham, MA, USA). The faecal slurry was mixed by vortexing and centrifuged at 13,000× Paired-end sequence reads were demultiplexed, and bioinformatics analysis was performed using the QIIME2 platform (release 2021) for microbiota profiling [Microbial alpha diversity measures of richness, evenness, and diversity (observed species, Pielou’s evenness, and Faith’s phylogenetic diversity, respectively), as well as weighted Unifrac distances for composition analysis, were computed using QIIME2. Raw sequence data are publicly accessible from the Sequence Read Archive repository (BioProject ID: PRJNA996323). 