Miseq v3 technology

Total genomic DNA was extracted by repeated bead beating (Mini-Bead-beater 8, BioSpec Inc., Bartlesville, United Kingdom) plus column purification method (Yu and Morrison, 2004 (link)). The yield and quality of extracted DNA was determined using a NanoDrop spectrophotometer (VWR International BVBA, Leuven, Belgium). Extracted DNA was used for bacterial 16S rRNA gene amplicon sequencing and quantitative real time PCR (qPCR).
For amplicon sequencing, extracted gDNA was submitted to Macrogen Sequencing Service (Macrogen, Seoul, South Korea) for library preparation and bacterial 16S rRNA gene amplicon sequencing (V3–V4 region, primers: 344F and 806R; Klindworth et al., 2013 (link)). Preparation of the amplicons barcoded library was based on the Illumina 16S metagenomic sequencing library preparation protocol¹ and the sequencing was performed using Illumina MiSeq V3-technology (2 × 300 bp).

DNA extraction was carried out using the Powersoil DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions, using 250 mg of biofilm, pellet from washing water or activated sludge. The washing water samples were centrifuged at 10 000 rpm for 1 min. The precipitate was scraped off and if not enough was present, supernatant was used to reach 250 mg sample. DNA quantity and quality was measured using the NanoDrop ND‐1000 (Thermo Scientific, Wilmington, NC, USA) and the Quantus double‐stranded DNA assay (Promega, Madison, WI, USA).
The taxonomic profiles of bacterial communities were determined using amplicon sequencing of the V3–V4 variable region of the 16S rRNA gene. The library preparation was based on the Illumina 16S metagenomic sequencing library preparation protocol (Illumina, 2013; De Mulder et al., 2016). The amplicon PCR was performed with the primers described by Klindworth et al. (2013). The final barcoded library was sequenced using Illumina MiSeq V3‐technology (2 × 300 bp, paired‐end) by Oklahoma Medical Research Foundation (Oklahoma City, OK, USA).