16S rRNA Gene Amplification and Sequencing

The V3-V4 region of the 16S rRNA gene of all samples was amplified by PCR using the primer set 341f (5′-CCTACGGGNGGCWGCAG-3′) and 805r (5′-GACTACHVGGGTATCTAATCC-3′) (47 (link)) with 30 different barcodes (Table S1). Amplification was performed using TEMPase Hot Start 2× Master Mix (Ampliqon), 10 μM forward primer, and 10 μM reverse primer. The PCR program consisted of an initial denaturation step of 15 min at 95°C; 30 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 60°C, and elongation for 30 s at 72°C; and finally an elongation step at 72°C for 5 min. PCR products were pooled in equal amounts according to the barcodes as determined by measurement with the HS assay kit and a Qubit 2.0 Fluorometer and sequenced on an Illumina NovaSeq 6000 instrument with paired-end 250-bp reads (Novogene). To obtain enough reads per sample, the amplicons were sequenced in several lanes on an Illumina Flow Cell, resulting in two additional technical replicates per sample.