2.4.8. DNA extraction and Illumina MiSeq sequencing
DNA from fecal contents was isolated following the procedure described
by RodrÃguez-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.