title: 16S amplicon sequencing of estuarine water: Central Eau Gallie River June 2017 replicate 1
design: Based upon Research and Testing's (Lubbok, TX) procedure: Research and Testing constructed the forward primer with (5-3) the Illumina i5 sequencing primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG) and the modified 515F primer, it also constructed the reverse primer  with (5-3) the Illumina i7 sequencing primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) and the modified 806R primer (Caporaso et al., 2012; Earth Mirobiome Project. 16S rRNA Amplification Protocol., 2019). Amplifications were performed in 25 ul reactions with Qiagen HotStar Taq master mix (Qiagen Inc, Valencia, California), 1ul of each 5uM primer, and 1ul of template. Polymerase chain reactions (PCRs) were performed on ABI Veriti thermocyclers (Applied Biosystems, Carlsbad, California) under the following thermal profile: 95C for 5 min, then 35 cycles of 94C for 30 s, 54C for 40 s,72C for 1 min, followed by one cycle of 72C for 10 min and 4C hold. A second PCR was performed on the products from the first amplification based on qualitatively determined concentrations for only 10 cycles but with the same conditions. The primers for this PCR were designed based on the Illumina Nextera PCR primers as follows: Forward  AATGATACGGCGACCACCGAGATCTACAC[i5index]TCGTCGGCAGCGTC and Reverse  CAAGCAGAAGACGGCATACGAGAT[i7index]GTCTCGTGGGCTCGG. They used the Amplification products were visualized with eGels (Life Technologies, Grand Island, New York) and then pooled at equimolar concentrations. The SPRIselect Reagent (BeckmanCoulter, Indianapolis, Indiana) set at a 0.75 ratio was used to size select each pool in two rounds. A Qubit 4 Fluorometer (Life Technologies) was used to quantify these pools which were then loaded onto an Illumina MiSeq (Illumina, Inc. San Diego, California) 2x300 flow cell at 10pM.