Nextera index kit v2 adapters

PCR amplification was performed on the extracted DNA using the 16S rRNA gene primers 515f and 806r to amplify bacterial sequences (Caporaso et al., 2012) (link), Internal Transcribed Spacer (ITS) ITS3 and ITS4 primers for fungi (White et al., 1990) , and the 18S rRNA gene primers Euk1391 and EukBr (Amaral-Zettler et al., 2009; (link)Stoeck et al., 2010) (link) for microbial eukaryotes. Primers were modified with Illumina Nextera Index Kit v2 adapters. PCRs were conducted using a Multigene Optimax thermal cycler (Labnet, USA), in a volume of 25 µl containing 15 ng DNA, Q5 R Hot start High Fidelity 2× Master Mix (New England Biolabs) and 0.5 µM of each primer. Thermocycling included an initial denaturation at 98 • C for 30s followed by 25 cycles (16S and 18S rRNA) or 30 cycles (ITS) of 98 • C for 10s, 50/57 • C (for 16S and 18S rRNA/ITS, respectively) for 15s and 72 • C for 20s. The final extension was at 72 • C for 5 min. PCR products were purified using the AMPure XP beads (Beckman Coulter, Germany) according to the manufacturer's instructions and the DNA concentrations were measured using the Qubit 2.0 Fluorometer (Life Technologies, USA) and diluted to 4 nM. The libraries were pooled and sequenced using the Illumina MiSeq Reagent Kit v300 for 2 × 300 bp sequencing.