16S rRNA sequencing of marine sediment
DNA was extracted using a Qiagen PowerSoilDNA Isolation Kit (CA, USA). The 16S hypervariable region V4-V5 was PCR amplified in triplicate reactions using the primer set 515F-Y and 926R (Parada et al., 2016; Quince et al., 2011) modified for Ion Torrent sequencing using a 1 step PCR strategy (IonCode Barcode Adapters, Thermo Fisher Scientific). For each sample amplicons were generated in triplicate in 20 L reaction volumes of 4.8 mM dNTP (Invitrogen, CA, USA), 1  PCR buffer, 120 mM MgCl2 (Invitrogen), 4 mM forward and reverse primer (Integrated DNA Technologies, Inc, IA, USA), 1U Taq DNA polymerase (Invitrogen), and 3 ng total genomic DNA) with the following conditions: 3 min initial denaturation at 94 C, followed by 30 cycles of 94 C for 45 sec, 50 C for 1 min, and 72 C for 1.5 min, and a final elongation step at 72 C for 10 min. Triplicate reactions were pooled and PCR amplicon products normalised using a SequalPrep Normalization Plate Kit (Invitrogen). Amplicons were sequenced using Ion Torrent Personal Genome Machine (PGM) DNA sequencer chemistry with an Ion 318v2 chip (Life Technologies) at the University of Waikato DNA sequencing facility (Hamilton, New Zealand).