2.4.8. DNA extraction and Illumina MiSeq sequencing
DNA from fecal contents was isolated following the procedure described by Rodríguez-Nogales et al . (Rodriguez-Nogales et al., 2017). Total DNA from stool samples was PCR amplified using primers targeting regions flanking the variable regions 4 through 5 of the bacterial 16S rRNA gene (V4-5), gel purified, and analyzed using multiplexing on the Illumina MiSeq machine. The amplification of a 600-bp sequence in the variable region V4-V5 of the 16S rRNA gene was performed using barcoded primers. PCR products were verified visually by running a high-throughput Invitrogen 96-well- E-gel. The PCR reactions from the same samples were pooled in one plate, then cleaned and normalized using the high-throughput Invitrogen SequalPrep 96-well Plate kit. The samples were then pooled to make one library to be quantified fluorometrically before sequencing.
The resulting sequences were completed, quality-filtered, clustered, and taxonomically assigned on the basis of 97% similarity level against the RDP (Ribosomal Database Project) (Wang, Garrity, Tiedje & Cole, 2007) by using the QIIME software package (Version 1.9.1) (Knight Lab, San Diego, CA, USA). Sequences were selected to estimate the total bacterial diversity of the DNA samples in a comparable manner and were trimmed to remove barcodes, primers, chimeras, plasmids, mitochondrial DNA and any non-16S bacterial reads and sequences <150 bp.