Qiaamp rapid dna mini kit

High-throughput sequencing technology was used to count the microorganisms in AnGS. About 20 g samples were collected before and after the reaction and sent to Majorbio (Shanghai, China) for DNA extraction and amplicon sequencing. The first step was to isolate total genomic DNA from sludge samples using the Qiagen QIAamp Rapid DNA mini kit. In the second step, the primers 341F, 805R, Ar Ba515F and Arch806R of bacteria, archaea and methanogens were used for PCR amplification of extracted DNA. Finally, all PCR products were directly observed on an Agarose gel (2% TAE buffer) and Bandage with the AxyPrep DNA Gel Extraction Kit. Paired-end amplicon library was constructed, and Illumina MiSeq platform was used for sequencing.^{22 (link)}Cluster analysis was performed on samples using Uparse software, and the similarity between microorganisms was greater than 97%, which was defined as an OTU (operational taxonomic unit). The Simpson index, Shannon index, the Converge index, Ace index and Chao index of microorganisms were calculated using QIIME software. Statistical analysis of microbial data using R language tools.
The PICRUSt was used to calculate the metabolic function of flora and microbial gene pathway.^{23 (link)}

The QIAamp Rapid DNA Mini Kit (Qiagen, United States) was used to extract total DNA from the intestinal contents samples according to the manufacturer’s procedures. After determining the DNA concentration and integrity, an amplicon sequencing library was constructed based on the PCR-amplified V3–V4 variable region of 16S rRNA. Then, use qualified libraries on the Illumina MiSeq platform for paired-end sequencing according to the manufacturer’s instructions. Use Trimmomatic, FLASH, and QIIME software to filter the original sequencing data. Then, UPARSE software with a 97% threshold was used to cluster the clean readings into operational taxonomic units (OTUs). Use the QIIME package to select the representative read from each OTU. Use the ribosome database item classifier v.2.2 to annotate and classify representative OTU sequences. The datasets generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP299194).