18S sequencing of coral reef metagenome: ARMS sessile fraction
The DNA underwent PCR amplification targeting a 272-bp segment from the V1V3 region of the 18S rRNA gene using Euk7F (AACCTGGTTGATCCTGCCAGT) tagged with a unique 8-bp identifier barcode, and Euk570R (GCTATTGGAGCTGGAATTAC) primers. PCR reactions were carried out in 20 l total volume containing 10 l of the QIAGEN HotStarTaq Plus Master Mix Kit (Qiagen, USA), 9 l of water, and 1 l of template.  The PCR program consisted of an initial denaturation of 3 min at 94C followed by 28 cycles of 30 s at 94C, 40 s at 53C, and 60 s at 72C, and a final extension of 5 min at 72C. PCR product quality was assessed by gel electrophoresis in 2% agarose gels, and amplification success and proportions for sample pooling were informed by the band length and intensity. The uniquely tagged PCR products were pooled together in equal proportions based on DNA concentrations and electrophoresis band characteristics, and purified using Ampure XP beads (Agencourt Bioscience, USA) with a ratio of beads to PCR products of 0.75x following Illumina guidelines. They were then ligated to Illumina adapters using the Illumina TruSeq protocol, and sequenced on an Illumina MiSeq platform using a paired-end 2 x 300bp procedure. Only forward reads were retained for analysis.