Rat Gut Microbiome Analysis by 16S Sequencing

Rat faecal samples were collected at the end of the study and the total genomic DNA was extracted using a Maxwell DNA Tissue Kit with automated extraction platform, Maxwell^® 16 Research System instrument (Promega, Madison, USA). The final equimolar pool was sequenced on the Illumina MiSeq platform using 341F (5′-CCTACGGGNGGCWGCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′) primers. PCR reactions and 16S sequencing were performed at the Molecular Research LP (MRDNA, Shallowater, Texas, USA). The MiSeq instrument (Illumina) was used for sequencing the 16S amplicons following the manufacturer’s instructions at MRDNA described by Garcia-Mazcorro et al. [21 (link)] with slight modifications. Raw 16S data were obtained from Illumina’s basespace as FASTQ files and analysed with the QIIME 2 pipeline using the procedure as described in the ‘moving pictures’ tutorial (https://qiime2.org/).