title: 16S barcoding from IS2 PHB enrichment culture
design: Prior to DNA extraction, the enrichment cultures (9 mL each) were vortexed and biomass was collected by centrifugation at 10,000 rcf for 10 min at 4C. The pellets were resuspended in 250 L of sterile phosphate-buffered saline (PBS, pH 7.5) and transferred to 1.5 mL tubes. High molecular weight DNA was obtained using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, Ca, USA) in accordance with manufacturers instructions. Finally, DNA was eluted with 50 L of nuclease free water. The quality of extracted DNA was assessed by gel electrophoresis, and DNA concentration was estimated using Qubit 4.0 Fluorometer dsDNA BR Assay Kit (Life Technologies, USA).  The Illumina-compatible libraries of hypervariable V4 region of 16S rRNA gene were prepared by single PCR with dual-indexing primer system with heterogeneity spacer as described previously (37). Modified forward primer F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 prokaryotic primer (5-GGACTACHVGGGTWTCTAAT-3) were used. PCR reactions were performed using MyTaq Red DNA Polymerase (Bioline) in a Bio-Rad thermocycler with the following program: 95 C for 2 min for denaturation followed by 30 cycles at 95 C for 45 s, 50 C for 60 s, 72 C for 30 s, with a final elongation at 72 C for 3 min. PCR products of approximately 440 bp were visualised by gel electrophoresis and gel-purified using the QIAEX II Gel Extraction Kit (QIAGEN). The purified barcoded amplicons were quantified by Qubit dsDNA BR Assay Kit (Life Technologies, USA), pooled in equimolar amounts and sequenced on Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) using paired-end 250 bp reads at the Centre for Environmental Biotechnology (Bangor, UK). Sequencing reads were processed and analysed as previously described