Metabarcoding GH
The V3 hypervariable region of the bacterial 16S rRNA gene was amplified using the provided gDNA template and primers 341F (CCTACGGGNGGCWGCAG) and 518R (ATTACCGCGGCTGCTGG)  [29]. To incorporate inline barcodes, an additional 5 base pairs were added to the 5' end of each primer [30].  To incorporate inline barcodes, an additional 5 base pairs were added to the 5' end of each primer [30]. PCR amplification was performed using Solar Bio PCR master mix (Solar Bio, China) with the following cycling conditions: 95C for 3 minutes, followed by 30 cycles of 95C for 15 seconds, 50C for 20 seconds, and 72C for 10 seconds. Samples were amplified using different primer combinations to generate barcoded amplicons. Amplicon quality was assessed on a gel, and products were normalized and pooled based on intensity. PCR amplification was considered successful only when samples exhibited a distinct band of the expected size, and the negative control remained negative.  The pooled amplicons were purified using 0.8x volume of SPRI beads and subjected to library preparation using the NEB Ultra II Library Prep Kit to incorporate Illumina adapters and dual-index barcodes. Library quantification was performed using a Denovix high-sensitivity assay, and sequencing was conducted on a NovaSeq6000 platform (Illumina, San Diego) using 2x150 paired-end reads.