18S rRNA amplicon sequencing of water from Tennessee River Chattanooga: C0205
The DNA amplification was performed on a target region of the eukaryote 18s ribosomal RNA gene sequence. Each sample underwent two rounds of amplification with PCR following the protocol described by Miya et al. (Miya et al., 2015). The primers used were 18s-euk forward GTACACACCGCCCGTC and 18s-euk reverse TGATCCTTCTGCAGGTTCACCTAC (Amaral-Zettler et al., 2009; Stoeck et al., 2010). The first round of PCR was carried out with 35 cycles with a reaction volume of 12 l. The reaction mixture contained 6.0 l 2 x KAPA HiFi HotStart ReadyMix, 0.6 l of each primer, and 2.0 l of the template DNA (d-water for the control) added to 2.8 l of distilled water to reach the total reaction volume. The thermal cycle began with 3 minutes at 95 C for DNA denaturation. Then, 35 cycles of the following three steps were performed: 20 seconds at 98 C for denaturation, 15 seconds at 57 C for annealing, and 15 seconds at 72 C for the extension. The PCR was completed with 5 minutes at 72 C for final DNA extension. This PCR product was then examined by gel electrophoresis.The first PCR product was diluted ten times with HyPure Molecular Biology Grade water and 1.0 l of this diluted PCR product was added to the reaction volume in addition to 6.0 l 2 x KAPA HiFi HotStart ReadyMix, 5.0 l of TruSeq indexes, for a total volume of 12.0 l. This second PCR reaction was run with the following thermal cycle profile: 3 minutes at 95 C for DNA denaturation, 15 cycles of 20 seconds at 98 C for denaturation, 15 seconds at 72 C for annealing and extension, and 5 minutes at 72 C for a final extension.Once the second PCR was complete, the sample products were pooled into one library, with a maximum of 10 samples per pool. Each library was then purified using a Qiagen kit DNA purification and sequenced at a private sequencing facility with Illumina High Throughput 2500 platform.