16SrRNA Seq of microbiome: human stool
The paired-end (2300 bp) sequencing by the MiSeq platform (Illumina, USA). Cutadapt (v3.2) for removing adapter and primer sequences from the raw data, with forward and reverse reads trimmed to 250bp and 200bp. To generate Amplicon Sequence Variants (ASV) sequences, the reads were performed error-correction, merging, and denoising processes with DADA2 (v1.18.0). Following error correction, paired-end reads were merged by overlapping. Chimeric sequences eliminated using the consensus method with the removeBimeraDenovo function in DADA2. ASVs with lengths shorter than 350bp were filtered out using R (v4.0.3). The resulting ASVs were utilized for downstream analysis using QIIME (v1.9.0). Each ASV was aligned to the organism with the highest similarity in the corresponding Reference Database(NCBI_16S), using algorithms such as Bayesian classifier (DADA2_v1.18.0, confidence value: 50)