DNA metabarcoding of bacteria: sample 2
For bacteria, we amplified the V4 region of the 16S rRNA gene using the forward primers 515F (5-GTGYCAGCMGCCGCGGTAA-3) and reverse primer 806R (5-GGACTACNVGGGTWTCTAAT-3) (Walters et al. 2016). The forward primers were designed so that they consist of 515F, an adapter and a barcode. Each PCR reaction consisted of 12.5 L Phusion Plus Master Mix (2X, Thermo Fisher Scientific, USA), 1.0 L forward primers (5 M), 0.5 L reverse primers (10 M), 1.0 L genomic DNA (approx 15 ng/L) and 10.0 L PCR-grade water. The thermocycler program consisted of an initial denaturation temperature of 98C for 30 s, followed by 30 cycles each with 98C for 10 s, 50C for 10 s, and 72C for 15 s, followed by a final 72C for 5 min. Amplicons were pooled and DNA yield was quantified fluorometrically with a Qubit 3.0 fluorometer (Invitrogen, USA) and purified using AMPure XP (Beckman Coulter, USA). For preparation of the libraries (end-repair, adaptor ligation and nick repair) Ion Plus Fragment Library Kit was used (Thermo Fisher Scientific, USA) according to the manufacturers instructions. The Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific, USA) was used to quantify the libraries before pooling. The final library got further preparation by Ion Chef System before sequencing on Ion GeneStudio S5 (Thermo Fisher Scientific, USA).