title: 16S amplicon sequencing: control samples
design: Bacteria 16S rRNA gene amplicon libraries were created following the Earth Microbiome Project protocol, without the extra step of pooling triplicate PCR technical replicates (Marotz et al., 2019). The 16S V4 region was amplified using the 515F and 806R primer set (Apprill, McNally, Parsons, & Weber, 2015). PCR reactions were prepared in a sterile cell culture hood, and consisted of 20 L of Platinum Hot Start PCR Master Mix, 1 L each of the forward and reverse primers, 2 L of template DNA, and 26 L of PCR-grade water for a total reaction volume of 50 L. Thermocycler conditions consisted of a 3 minute at 94C initial denaturation, then 35 PCR cycles of 45 seconds at 94C, 60 seconds at 50C, 90 seconds at 72C followed by a final extension for 10 minutes at 72C before holding at 4C. PCR amplifications were also completed using the ZymoBIOMICS microbial community standard DNA and only PCR-grade water as a positive and negative control, as well as unfiltered and 0.2 m-filtered seawater samples from the first as last day of the experiment. PCR amplicons were checked for quality and size distribution on a 1.5% TE-agarose gel, and amplicon DNA concentration was determined using a Qubit fluorimeter and Qubit dsDNA BR kit (ThermoFisher Q32853). 16S amplicons from all samples and controls were purified using AMPure XP beads and 80% ethanol washes to remove contaminants, then final DNA amplicon products were normalized to 4 nM concentration and pooled before submission to the University of Miami Center for Genome Technology (CGT) for 300 bp paired-end sequencing on the Illumina MiSeq instrument.