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.