16s rRNA amplicon sequencing of oyster and water microbiomes
DNA was extracted from water samples using the Qiagen PowerSoil kit, and from oyster tissues using the Qiagen DNEasy blood and tissue kit (Qiagen, Hilden, Germany). Digestive gland and gill composites, which were homogenized at the time of collection, were digested with Proteinase K prior to DNA extraction. The V4-V5 region of the 16S rRNA gene was amplified using 515F-926R primers (27). Libraries were assessed for quality using an Agilent 2200 TapeStation (Agilent, Santa Clara, CA). Since these primers also produce amplicons from 18S rRNA, BluePippin size selection was used to enrich for ~550bp amplicons prior to sequencing (Sage Science, Beverly, MA, USA). Additionally, 3 blanks (lysis buffer only, taken through extraction protocol, 2 extracted with tissue samples and 1 run with water samples) and 2 samples of DNA from mock bacterial communities (ZymoBIOMICS Microbial Community DNA Standard, Zymo Research, Irvine CA) were prepared and sequenced, with all samples included in the same sequencing run. After library construction, samples were sequenced using the Illumina MiSeq 2x300 (kit v.3) with custom adapters and dual barcode indices at the University of California Davis Genome Center (https://genomecenter.ucdavis.edu/).