2.4 Gut microbiota community analysis
DNA extraction was performed using the DNeasy Blood & Tissue Kit
(QIAGEN, Germany) according to the manufacturer’s instructions. PCR
amplification of bacterial 16S rRNA genes targeting the V4 region was
conducted using primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’; Caporaso et al.
2011) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’; Caporaso et al. 2011). The
PCR reactions were set up in a 25 μL volume with Platinum™ Taq
polymerase (Invitrogen, Waltham, MA, USA) and carried out in a Veriti™
96-Well Fast Thermal Cycler (Thermo Fisher Scientific, Waltham, MA,
USA). The PCR protocol included an initial denaturation step at 94℃ for
3 minutes, followed by 35 cycles of denaturation at 94℃ for 45 seconds,
annealing at 50℃ for 60 seconds, extension at 72℃ for 90 seconds, and a
final extension at 72℃ for 10 minutes. PCRs were performed in
triplicate, and the products were pooled and sequenced using the HiSeq
2500 System (Illumina, San Diego, CA, USA) with 2 × 250 bp paired-end
read configurations.
The amplicon sequencing results were analyzed using the QIIME2 pipeline,
adhering to established procedures for quality filtering,
demultiplexing, and denoising with DADA2 (Callahan et al. 2016).
Clustering into operational taxonomic units (OTUs) was performed at 97%
similarity, followed by taxonomy assignment and diversity analysis.
Taxonomic assignment of OTUs was conducted using the SILVA database
(release 138). Prior to community structure analysis, OTU reads were
normalized, and chloroplasts and mitochondria were removed.
Additionally, OTUs with a relative abundance of less than 0.1% were
filtered out. Paired t-tests and t-tests were performed using the R
package “vegan” (Oksanen et al. 2007).