16S rRNA Amplicon Sequencing Analysis

Because the downloaded Coyote data were v4–v5 regions, we used USEARCH for tiling alignments after processing the sequences, and then used the plug-in “cutadapt” of QIIME2 to remove paired end reads from the primers and truncate the V4 region for subsequent analysis (Hall and Beiko, 2018 (link)). The plug-in “DADA2” was used to control sequence quality, correct amplicon errors, and generate ASVs (Callahan et al., 2016 (link)). Chimeras were filtered and the ASVs present in at least 2 samples were retained. Based on Silva_ 132 databases, trained a classification classifier against the bacterial V4 region of the 16S rRNA gene, and used this classifier to generate a classification map of out data. The resulting alignment was used for subsequent statistical analysis. Furthermore, the beta diversity distance matrices of the microbial community were calculated and performed by QIIME2.