title: V4 16S amplicon sequencing of reef bacterioplankton: bottle samples
design: 515F and 806R Earth Microbiome Project primers were used with barcodes on the 515F primer. Amplicons were generated from a single round of PCR using primers that include Illumina spacers, Illumina adapters, index sequences (on the forward primers), and 16S rRNA gene template region. PCR reagents included 7.2 l DNase free water, 10 l PLAT II MM (2X) (Invitrogen, Platinum II Hot-Start PCR Master Mix, Catalog No: 14000012), 0.4 l forward primer (10 M), 0.4 l reverse primer (10 M), and 2 l DNA template. PCR was performed on an Applied Biosystems SimpliAmp (Catalog No: A24811) system using an initial denature of 94 C for 2:00 min followed by 35 cycles of 94 C for 15 s, 54 C for 15 s, 68 C for 7 s, followed by a final extension at 68 C for 3 min. Total amplicons per sample were normalized to between 12.5 and 15 ng using Charm Biotech Just-a-Plate PCR purification and normalization kit. Amplicons were pooled and sequenced using an Illumina MiSeq V3 600 paired-end cycle run at the University of Hawaii at Mnoa Advanced Studies in Genomics, Proteomics and Bioinformatics facility.