16s amplicon: biofloc
The bacterial 16S rRNA V3 hypervariable region was amplified using the primers 341F:CCTACGGGNGGCWGCAG and 518R:ATTACCGCGGCTGCTGG . An additional 5 bases of inline barcode were incorporated at the 5 end of the primers to enable inline barcoding . Different samples were amplified using different combinations of the forward and reverse inline primers. PCR was performed using WizBio HotStart PCR mastermix (WizBio, Korea). The barcoded amplicons were subsequently visualized on gel, normalized and pooled based on their intensity and purified with 1.5x volume of SPRI bead. The purified pooled amplicons were subsequently processed with the NEB Ultra II Library preparation kit to incorporate Illumina adapter and dual-index barcodes. The constructed library was quantified with Denovix high sensitivity assay and sequenced on an Illumina iSeq100 sequencer for 2x 150 paired-end sequencing.