16S rRNA amplicon sequencing of a seawater microbial community
Filtering of water samples and extraction of DNA using the standard protocol for Dneasy PowerWater Kit (Qiagen, Germany). Duplicate amplification of the 16S rRNA gene variable region 4 (abV4C): [515FB] GTGYCAGCMGCCGCGGTAA and [806RB] GGACTACNVGGGTWTCTAAT. PCRwas done with the following program: Initial denaturation at 95 C for 2 min, 30 cycles of amplification (95 C for 15 s, 55 C for 15 s, 72 C for 50 s) and a final elongation at 72 C for 5 min. The purified sequencing libraries were pooled in equimolar concentrations and diluted to 2 nM. The samples were pairedend sequenced (2x300 bp) on a MiSeq (Illumina, USA) using a MiSeq Reagent kit v3 (Illumina, USA) following the standard guidelines for preparing and loading samples on the MiSeq. > 10 % PhiX control library was spiked in to overcome low complexity issues often observed with amplicon samples.