2.5 | Total community clustering and analyses of non-metric multidimensional scaling
We concatenated all fasta files of the mitochondrial Cytochrome c oxidase subunit I (CO1) sequences prepared as described above. The mitochondrial CO1 gene contigs were then clustered using VSEARCH (Rognes et al. 2016: vsearch –cluster_fast combined.CO1.contig.fasta –strand both -id 0.90 –uc out.uc –centroids out.cent.fasta). Clustering was performed using a 90% similarity threshold (Machida et al. 2021). The Operational Taxonomic Unit (OTU) abundance table was created from .uc files generated as an outfile of the VSEARCH (standard UNIX commands were used for this process).
Non-metric multidimensional scaling (nMDS) analysis was performed using the prepared OTU abundance table as the infile. We used the metaMDS function from the Vegan v2.6-2 package (Oksanen et al., 2022), using the Bray-Curtis method for the distance matrix. Later, we extracted the “points” matrix from the output list (columns MDS1 and MDS2) to plot a point chart in ggplot2. All R commands are available in Methods S4 (R Core Team, 2017).