18S marker sequencing of micro-algae and fish parasites from environmental samples
For the initial round of amplification, each PCR was conducted in a 25l reaction volume containing 10ng of template DNA, 0.5M of each primer and 12.5 units of Q5 Hot Start High-Fidelity 2X Master Mix . The following primers were used in the PCR reactions to amplify the 18S V9 region: forward primer 1391_F and reverse primer 1560_R with Illumina adapter sequences attached in the PCR cycle to amplify sequences for the MiSeq sequencing run. The following PCR profile was then used to amplify fragments for MiSeq sequencing: one initial cycle of fifteen minutes at 95C; 35 cycles of 45s at 95C, 45s at 58C, and 60s at 72C; and one final cycle of ten minutes at 72C. The PCR profile of the fragment amplification for Ion Torrent sequencing only differed from this protocol in the duration of the different temperature phases within the 35-cycle loop, which were 30s for each of the three phases. The PCR products were then run on a 2% agarose gel for quality control using SYBR safe.