4.4. Gut Microbiota Analysis

Immediately after euthanasia and organ removal, two or three faecal pellets were collected from the colon of rats and stored at −80 °C in nuclease-free tubes. Total microbial community DNA was extracted from faecal samples using the DNeasy Powersoil Kit (Qiagen Australia, Chadstone, VIC, Australia) following the manufacturer’s instructions [45]. The bacterial gut microbiota was then characterised by amplifying and sequencing the 16S rRNA gene. The primers, 341F (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and 785R (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) were used to amplify the V3-V4 regions of the 16S rRNA gene, which was then sequenced on an Illumina MiSeq platform. Sequencing reads were processed to form zOTUs, which were taxonomically classified against the SILVA database.

The reaction mixture (50 μL total volume per sample) to amplify the 16S rRNA gene consisted of Econotaq® PLUS GREEN 2× Master Mix (Astral Scientific, Gymea, NSW, Australia) (25 uL), Ambion® nuclease-free water (17 μL), the primer pair 341F and 785R (1.5 μL of each; 10 μM) and DNA template (5 μL). The PCR program consisted of an initial denaturation at 94 °C (2 min), followed by 35 cycles of denaturation at 94^oC (30 s), annealing at 55^oC (30 s), extension at 72 °C (40 s) and a final extension at 72 °C (7 min). PCR products were then quantified using gel electrophoresis. Paired-end sequencing (2 × 300 bp) of the resulting 16S rRNA gene amplicons was performed at the Ramaciotti Centre for Genomics, University of New South Wales on an Illumina MiSeq platform following the MiSeq System User Guide [46]. For 16S rRNA gene sequencing analysis, sequence data were initially quality-filtered and trimmed using Trimmomatic version 0.36 truncating reads if the quality dropped below 20 in a sliding window of 4 bp [47]. USEARCH version 11.0.667 [48] was used for further processing [49] to merge and quality-filter sequencing reads, excluding reads with < 250 or > 550 nucleotides, in addition to reads with more than one ambiguous base or an expected error of more than 1. Filtered sequences were denoised and clustered into unique sequences (zero-radius operational taxonomic units; zOTUs) using the UNOISE algorithm [50] implemented in USEARCH. zOTUs represent unique bacterial entities and roughly are equivalent to species or strains. Chimeric sequences were removed de novo during clustering and subsequently in reference mode using UCHIME [51] with the SILVA database (https://www.arb-silva.de/browser/) (SILVA SSURef 132 NR) as a reference [52]. zOTUs were then taxonomically classified (i.e., assigned a likely taxonomic name) by BLASTN [53] against the SILVA database. All non-bacterial zOTUs were removed along with non-BLAST aligned and singleton zOTUs. Finally, processed sequences were mapped on zOTU sequences to calculate the distribution and counts of each zOTU in every sample. Only zOTUs occurring in more than two samples were considered for further statistical analysis.