PCR amplification

Based on the sequenced region, specific primers with index sequences were synthesized to amplify the V4 region of the sample, and the amplified primer sequences were as follows: Primer 5′-, −3′: 515 F (5′-gtgyCAGCMGCCGCGGTAa-3′) and 806 R (5′-ggactachvGGGTWTCTAat-3′). PCR test was conducted under the action of TOYOBO KOD-Plus-Neo DNA Polymerase (KOD-401B), which was performed on Applied Biosystems® PCR System 9700. The entire PCR system was listed in Table 3. PCR expansion conditions are as follows: pre-generation in 94 °C for 1 min in 1 cycle; transgender 94 °C for the 20 s, and then annealing 54 °C last 30 s and extension to 72 °C for 5 min in 1 cycle; and insulation thermal in 4 °C. Repeat the PCR technology 3 times per sample, and collect the equivalent mixture of linear PCR products used for subsequent library construction.

The composition of the PCR system.