Total genomic DNA extraction was carried out using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, United States) according to the manufacturer’s instructions. For bacteria, the V3–V4 hypervariable region of the 16S rRNA gene was amplified by PCR with the universal primers of the forward 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse 806R (5′-GGACTACHVGGGTWTCTAAT-3′). For fungi, the ITS1 was amplified by PCR with the primers of ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS1 (5′- GCTGCGTTCTTCATCGATGC-3′). After the individual quantification, amplicons were pooled in equal quantities and subjected to high-throughput sequencing with a MiSeq Reagent Kit V3 (Personal, Shanghai, China) for pair-end 2 × 250 bp sequencing.
The sequences were analyzed with QIIME2 (2019.4), and the quality control of the sequences by using DADA2 (Callahan et al., 2016 (link)). Specifically, the default parameters of DADA2 were used for filtering and denoising. Secondly, the sequences were automatically calculated and spliced according to the clip length. Thirdly, the sequences with chimerism >8 were removed. Finally, the sequences with low abundance (reads ≤1) were removed. Each de-weighted sequence resulting from quality control using DADA2 is called ASV.
The sequences were analyzed with QIIME2 (2019.4), and the quality control of the sequences by using DADA2 (Callahan et al., 2016 (link)). Specifically, the default parameters of DADA2 were used for filtering and denoising. Secondly, the sequences were automatically calculated and spliced according to the clip length. Thirdly, the sequences with chimerism >8 were removed. Finally, the sequences with low abundance (reads ≤1) were removed. Each de-weighted sequence resulting from quality control using DADA2 is called ASV.
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