16S rRNA gene amplicons of Acropora tenuis: recruits inoculated with adult coral mucus
DNA was extracted from the samples following slight modifications of the protocol reported in Wilson et al. 2002. The hypervariable region V4 of the 16S rRNA gene was amplified using revised versions of the primers 515F [5Õ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3Õ] (Apprill et al. 2015) and 806R [5Õ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT-3Õ] (Parada et al. 2015) containing Illumina adapters. The PCRs were conducted in triplicate 10 µL reactions using the Qiagen Multiplex Mastermix and 0.2 µM of each primer. The amplification cycles were set as following: 95¡C for 15 min; 28 cycles each at 94¡C for 30 sec, 57¡C for 1 min, 72¡C for 30 sec; a final extension at 72¡C for 10 min. The PCR products were then pooled and sent to Ramaciotti Centre for Genomics (UNSW, Sydney) for library preparation and sequencing on the Illumina MiSeq system. Amplicon sequences were processed using the QIIME pipeline v2.7.9 (Caporaso et al. 2010). Poor quality and short sequences (< 170 bp) were removed using Trimmomatic. The module PEAR v0.9.6 (Zhang et al. 2014) was used to assemble reads.
SUB2775992
Gal.17D