基因V3-V4可变区PCR扩增及测序方法

Using extracted DNA as a template, the V3-V4 variable region of the gene was amplified by PCR using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying Barcode sequences (PCR instrument: ABI GeneAmp® 9700). The amplification program was as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturation (95°C, 30 s), annealing (55°C, 30 s), and extension (72°C, 30 s), and a final extension at 72°C for 10 min. The reaction mixture for PCR consisted of 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM) and downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and water to make up a final volume of 20 μL. Each sample was replicated 3 times. The PCR products from the same sample were then mixed and recovered using a 2% agarose gel. The AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) was used for purification of the recovered products, followed by 2% agarose gel electrophoresis and quantification using the Quantus™ Fluorometer (Promega, USA). The purified PCR products were then library-prepped using the NEXTFLEX® Rapid DNA-Seq Kit: 1) adapter ligation, 2) removal of self-ligated fragments by magnetic bead selection, 3) PCR amplification for enrichment of library templates, and 4) magnetic bead recovery of PCR products to obtain the final library. Sequencing was performed on the Illumina NovaSeq PE250 platform.