The extraction of total DNA and construction of 16S rRNA V3-V4 hypervariable regions libraries were done using a FastDNA® Spin Kit for Soil (MP Biomedicals, USA) and NEXTflexTM Rapid DNA-Seq Kit (Bioo Scientific, USA), respectively. All raw reads sequenced by the Illumina MiSeq PE300 platform were quality-controlled, merged, and trimmed by fastp version 0.20.0 (Chen et al., 2018 (link)). Operational taxonomic units (OTUs) were clustered with a 97% similarity threshold in Uparse version 7.0.1090 (Edgar, 2013 (link)). The sequence taxonomy was identified by the RDP Classifier Bayesian algorithm against the Silva 16S rRNA database (version 1.3.8), with a default confidence threshold of 0.7. All raw reads were deposited in the NCBI Sequence Read Archive (SRA) database (BioProject ID: PRJNA1018001).
Alpha diversity indices (Sobs index, Simpson index, Shannoneven index, and Pd index) of the microbiome were estimated using Mothur (v1.30.2). Significant differences in alpha diversity indices were tested by Welch’s t-test at the OTU level. Beta diversity analysis results were visualized by the principal coordinates analysis (PCoA) clustered at the OTU level based on Bray-Curtis metrics. Species differences between adult and juvenile groups were analyzed by the Wilcoxon rank-sum test.
Alpha diversity indices (Sobs index, Simpson index, Shannoneven index, and Pd index) of the microbiome were estimated using Mothur (v1.30.2). Significant differences in alpha diversity indices were tested by Welch’s t-test at the OTU level. Beta diversity analysis results were visualized by the principal coordinates analysis (PCoA) clustered at the OTU level based on Bray-Curtis metrics. Species differences between adult and juvenile groups were analyzed by the Wilcoxon rank-sum test.
Free full text: Click here
