16S rRNA V3-V4 amplicon sequencing of constructed wetland substrate biofilm (sewage sludge biochar, 0.5 mg/L PP-MP, lower layer, subsample 3)
Bacterial 16S rRNA gene V3-V4 region amplified with primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) by high-fidelity PCR in triplicate; sample-specific index sequences added by a second PCR; products purified with AMPure XP beads, quantified by Qubit, pooled in equimolar ratio and sequenced paired-end (2 x 300 bp) by a commercial provider. Submitted FASTQ files are raw demultiplexed reads that still contain the PCR primer sequences.