Analyzing Gut Bacterial Communities

The OMNIgene-GUT collection kit (OMR-200, DNA Genotek, Ottawa, Canada) was used to collect stool samples. DNA extraction from faecal samples was performed within 1 month of storage using the MOBio PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. Amplification and sequencing were performed to analyse the bacterial communities as described previously [28 (link)]. Genomic DNA was amplified using fusion primers targeting the V3 and V4 regions of the 16S rRNA gene. Samples were pooled for sequencing on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions [29 (link)]. The DADA2 [11 (link)] plugin of the QIIME2 package (version 2019.7, https://qiime2.org) [30 (link)] was used to perform sequence quality control and construct a feature table of ASVs. For taxonomic structure analysis, taxonomy was assigned to ASVs using a pre-trained naïve Bayes classifier and the q2-feature-classifier plugin with the database Silva 132 release in the QIIME2 package.