Amplicon of environment: L02
Sample-specific 7-bp barcodes were incorporated into the primers  for multiplex sequencing. The PCR components contained 5 l of buffer (5), 0.25 l of Fast pfu DNA  Polymerase (5U/l), 2 l (2.5 mM) of dNTPs, 1 l (10 uM) of each Forward and Reverse primer, 1 l of DNA  Template, and 14.75 l of ddH2O. Thermal cycling consisted of initial denaturation at 98 C for 5 min,  followed by 25 cycles consisting of denaturation at 98 C for 30 s, annealing at 53 C for 30 s, and extension  at 72 C for 45 s, with a final extension of 5 min at 72 C. PCR amplicons were purified with Vazyme  VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen  dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were  pooled in equal amounts, and pair-end 2250 bp sequencing was performed using the Illlumina NovaSeq  platform with NovaSeq 6000 SP Reagent Kit (500 cycles)