feces metagenome Genome sequencing

Fresh faeces from the rectum were collected under anaesthesia, immediately frozen in liquid nitrogen, and stored until analysis. Total genomic DNA was extracted using a DNA Extraction Kit following the instructions. The V3-V4 regions of bacterial DNA were amplified with the universal 343F and 798R primers. After purification with AMPure XP beads (Agencourt) again, the final amplicon was quantified using a Qubit dsDNA assay kit. Equal amounts of purified amplicon were pooled for subsequent sequencing. The raw sequencing data were collected in FASTQ format. Paired-end reads were then preprocessed using cutadapt software to detect and cut off the adapter. After trimming, paired-end reads were filtered for low-quality sequences, denoised, merged and detected, and chimera reads were cut off using DADA2 with the default parameters of QIIME2 (2020.11). Finally, the software output the representative reads and the ASV abundance table. The representative read of each ASV was selected using the QIIME2 package. All representative reads were annotated and blasted against the Silva database Version 138 using q2-feature-classifier with the default parameters.