Microbiome Analysis

Stool samples were collected and preserved using OMNIgene GUT microbial stabilisation kit (DNA Genotek, Ontario, Canada). Genomic DNA was extracted using DNeasy PowerLyzer PowerSoil DNA isolation kits (Qiagen, Hilden, Germany) as per manufacturer’s instructions. 16S rRNA sequencing was performed by first preparing the amplicon library using a previously described protocol (36), and sequenced on the Illumina Miseq sequencing platform using Illumina Miseq v3 kit with 2 x 300 bp cycle (Illumina Inc., CA, USA). Downstream processing of the amplicon sequencing reads was carried out as described (36). Briefly, reads were quality filtered and merged, followed by assigning operational taxonomic units (OTUs) using the Quantitative Insights into Microbial Ecology (37) software. No samples were eliminated following subsampling to the depth of 8,079 reads.