Pcrbio ultra polymerase red mix

Both DNA and cDNA extracts were PCR amplified using a set of primers (515F-806R) specific to the V4 hypervariable region of the bacterial 16 s rRNA gene¹⁰⁸ . The PCR reaction mixture contained (per 25 µL): 1 × PCRBIO Ultra Polymerase red mix (PCR BIOSYSTEMS, United Kingdom), 0.4 μM forward primer and 0.4 μM reverse primer. The PCR conditions were an initial denaturation at 95 °C for 5 min, followed by 30 cycles of: denaturing 30 s at 95 °C, annealing at 1 min 56 °C, elongation at 1 min 72 °C and final elongation for 5 min at 72 °C. PCR products were precipitated as previously decribed¹⁰⁷ , and re-suspended in 20 µl of molecular grade water. Purified PCR products were quantified using Qubit fluorometric quantitation (ThermoFisher) and sequenced using the Ion Torrent PGM platform by the company Molecular Research LP (Texas, USA).

DNA was extracted using a modified variant of the Griffiths technique [23 (link)] from 5 mL aliquots of each replicate from the endpoint of the enrichment. Nuclease free water was processed through the extraction as a negative extraction control. The V4-V5 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction using the primers com1 and com2 [24 (link)]. The PCR reaction mixture contained (per 25 µL): 1 × PCRBIO Ultra Polymerase red mix (PCR Biosystems, London, United Kingdom), 0.4 μM forward primer and 0.4 μM reverse primer. The PCR products were sequenced using the Illumina MiSeq Platform by Molecular Research LP, (Shallowater, TX, USA). The data were processed by Mr DNA using a customized pipeline [25 (link),26 (link)]. All pair-end sequences were merged, chimeras removed and sequences less than 150 bp and/or with ambiguous base calls were removed. The sequences were clustered at 97% similarity and phylogeny was assigned using a curated database from GreenGenes, RDPII and NCBI [27 (link)]. Contaminant sequences identified in negative controls were eliminated from the datasets [28 (link)].