Gut Microbiome Profiling via Fecal Collection

Participants were instructed to use the investigator-provided stool collection kit to collect a stool sample at home. The sample was placed in a sterile conical tube, placed in a biohazard bag, and stored in the participant’s freezer until the date and time of the pre-determined pick-up from their home (not more than 3 days from time of collection). Samples were picked up by research personnel and immediately transported on ice to the University of Calgary Faculty of Kinesiology and stored at −80 °C degrees until analysis. Gut microbial profiling was conducted as previously described [47 (link),48 (link)]. Briefly, bacterial DNA was extracted from ~60 mg of fecal matter according to the manufacturer’s instructions, using the FastDNA Spin Kits for feces (MP Biomedicals, Lachine, QC, Canada). Bacterial DNA was then diluted to 4 ng/uL using the PicoGreen DNA quantification kit (Invitrogen, Carlsbad, CA, USA) and stored at −20 °C until sequencing. Microbial composition was assessed using the MiSeq Illumina platform, which amplified the V3 and V4 regions of the 16S rRNA gene (Illumina, San Diego, CA, USA). Extracted bacterial DNA samples were sequenced at the University of Calgary’s Centre for Health Genomics and Informatics (Calgary, AB, Canada).