1
After the DNA extraction kit corresponding to various samples (please consult the corresponding technical support for details) is used to extract genomic DNA, the purity and concentration of DNA is detected by Nanodrop One (Thermo Fisher Scientific, MA, USA). Using genomic DNA as the template, PCR amplification was performed using specific primers with barcode and Premix Taq according to the selection of sequencing region (the enzyme used in different fragment selection may be different, the actual enzyme used is subject to the amplification report). The fragment length and concentration of PCR products are detected by 1% agarose gel electrophoresis, and samples with main band lengths in the normal range (e.g. 16S V4:290-310bp /16S V4-V5:400-450bp, etc.) can be used for further experiments. After comparing the concentration of PCR products, the required volume of each sample was calculated according to the principle of equal mass, and the PCR products were mixed. The E.Z.N.A. Gel Extraction Kit (Omega, USA) gel extraction kit was used to recover PCR mixed products and TE buffer eluted to recover target DNA fragments. The Library was constructed according to the ALFA-SEQ DNA Library Prep Kit standard process, and the size of the library fragments was evaluated on the Qsep400 high-throughput nucleic acid protein analysis system (Hangzhou Houze Biotechnology Co., LTD., China). The concentration of the library was measured using Qubit4.0 (Thermo Fisher Scientific, Waltham, USA). The constructed amplicon libraries were sequenced by PE250 using Illumina or MGI platforms. The original image data files obtained by sequencing were converted into Raw Reads by Base Calling analysis. The results were stored in FASTQ(fg) file format, which contains sequence information of Reads and corresponding sequencing quality information.