title: 16S amplicon sequencing of fish feed pellet: 100% Fish Meal replicate 3
design: Based upon RTL Genomic's procedure: Samples underwent DNA extraction with the Zymo ZR-96 magbead kit (Irvine, CA, USA) on the Thermo Scientific KingFisher FLEX instruement (Waltham, MA, USA) using a modified version of the manufacters instructions which included a mechanical lysis step with a Qiagen TissueLyser (Hilden, Germany).  Samples were amplified for sequencing in a two-step process. The forward primer was constructed (5-3) with the forward Illumina overhang adapter (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG) added to the 28F (GAGTTTGATCNTGGCTCAG) primer. The reverse primer was constructed (5-3) with the reverse Illumina overhang adapter (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) added to the 519R (GTNTTACNGCGGCKGCTG) primer. 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. Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, California) under the following thermal profile: 95C for 5 min, then 10 cycles of 94C for 30 s, 50C for 90 s (+0.5C per cycle), 72C for 1 min, followed by 25 cycles of 94C for 30 s, 54C for 90 s, 72C for 1 min, and finally, one cycle of 72C for 10 min and 4C hold. Products from the first stage amplification were added to a second PCR based on  qualitatively determine concentrations. Primers for the second PCR were designed based on the Illumina Nextera PCR primers as follows: Forward - AATGATACGGCGACCACCGAGATCTACAC[i5index]TCGTCGGCAGCGTC and Reverse - CAAGCAGAAGACGGCATACGAGAT[i7index]GTCTCGTGGGCTCGG. The second stage amplification was run with the following thermal profile: 95C for 5 min, then 10 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. Amplification products were visualized with eGels (Life Technologies, Grand Island, New York). Products were then pooled equimolar and each pool was size selected in two rounds using SPRIselect Reagent (BeckmanCoulter, Indianapolis, Indiana) in a 0.75 ratio for both rounds. Size selected pools were then quantified using the Qubit 4 Fluorometer (Life Technologies) and loaded on an Illumina MiSeq (Illumina, Inc. San Diego, California) 2x300 flow cell at 10pM.