Amplicon sequencing of SSU biomarkers
Molecular ecosystem profiles of the samples were captured using an amplicon sequencing approach targeting the genes coding for the small ribosomal subunits 16S (prokaryotes) and 18S (eukaryotes) simultaneously using a previously described universal primer set: 926F (5’-AAACTYAAAKGAATTGACGG-3’) and 1392R (5’-ACGGGCGGTGTGTRC-3’) (Engelbrektson et al., 2010). Amplicon library PCR was performed on 10 ng of extracted DNA per 25 µL PCR reaction (1X Platinum High Fidelity buffer (Thermo Fisher Scientific, USA), 2 mU Platinum Taq DNA polymerase HF (Thermo Fisher Scientific, USA), 1.5 mM MgSO4, 400 nM of each dNTP and 400 nM of each primer), using the following PCR programme: 2 minutes initial denaturation at 95 °C, 30 cycles of 20 seconds at 95 °C, 30 seconds at 52 °C, 60 seconds at 72 °C, final elongation for 5 minutes at 72 °C. PCR reactions were performed in duplicates, pooled afterwards and subsequently purified using AMPure XP bead protocol (Beckmann-Coulter, USA) using a sample:bead ratio of 5:4. Amplicon quality was assessed using TapeStation 2200 and D1000 ScreenTapes (Agilent, USA), and quantity was determined using Quant-IT High Sensitivity DNA assay kit (Thermo Fisher Scientific, USA). The amplicon libraries were subsequently barcoded in accordance with the Nextera XT barcoding protocol (Illumina, USA). Equimolar concentrations of the libraries were sequenced on a MiSeq platform using reagent kit v3 (2x300 PE) (Illumina, USA), with a final library pool concentration of 4 pM and 20 % PhiX spike-in.