For preparation of DNA samples for microbiome sequencing, fecal samples were defrosted to room temperature and the FastDNA Spin Kit for Soil DNA Extraction (MP Biomedicals) was used for DNA extraction; 16S ribosomal RNA (rRNA) gene sequencing was undertaken at Glasgow Polyomics for analysis of the microbiome. In brief, primers that were specific to the V3 and V4 regions were used to generate 16S amplicons from 12.5 ng of extracted DNA using polymerase chain reaction. The resulting amplicons had dual barcodes and adapters added using the Nextera XT v2 adapter sets (Illumina). The libraries were combined in equimolar ratios and sequenced on a MiSeq (Illumina) instrument using a paired end, 2 × 300–base pair (bp) sequencing run. Samples were sequenced with an average of 50,000 reads.

For data analysis, FastQ files were generated from the sequencing data and quality-filtered using cutadapt (43). Cutadapt removes adapter sequences from high-throughput sequencing reads with a minimum quality of 25 and a minimum length of 250 bp. Paired-end reads were combined using PANDAseq and collated into a single FASTA file using the Qiime package (44). Further processing and analysis were completed using the Qiime wrapper and software.

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