bacteria
The bacterial 16S rRNA V3V4a region was amplified by PCR using barcoded primers. The forward primer was F (5-barcode + ACTCCTACGGGAGGCAGCA) and the reverse primer was R (GGACTACHVGGGTWTCTAAT). PCR was performed in a 25 L reaction volume using NEB Q5 High-Fidelity DNA Polymerase with 5 Q5 reaction buffer and 5 Q5 High-Fidelity GC buffer. Thermal cycling consisted of an initial denaturation at 98 C for 5 min, followed by 2530 cycles of 98 C for 30 s, 52 C for 30 s, and 72 C for 45 s, and a final extension at 72 C for 5 min, with samples held at 12 C. After purification and quantification of PCR products, sequencing libraries were prepared and sequenced on an Illumina platform using paired-end 2250 bp reads.