RIK_104_201606DD_HLSU
Large subunit haptophyte-specific fusion primers (HAP_LSU_F: GGURUCGGAGARGGUGAGAAUCC and LHapto20R_bis: TCAGACTCCTTGGTCCGTGTTTCT) containing Illumina-specific adaptors with 6 unique inline barcodes for the forward primer and 8 indices for the reverse primer were used. Triplicate PCR reactions were carried out for each genomic DNA sample with a 33  cocktail of 1.0 U Platinum High-Fidelity DNA Polymerase (Life Technologies, Carlsbad, CA): with 1X Hi-Fidelity buffer, 200  dNTP mix, 1.5 mM MgSO4, and 0.2  of each primer. 5-20 ng of template DNA were added to each PCR and included a no-template control for the run. PCR conditions were as follows: 2 min at 94, 30 s at 94, 30 s at 55, 3 min 68, with repetition from step 2 for 35 cycles followed by 5 min at 68.  Resulting PCR tripiclate reactions were pooled and products were verified using a Caliper LabChip GX. Amplicons were cleaned with Agencourt AMPure XP magnetic beads and quantified with Quant-iT PicoGreen dsDNA Assay Kit. The amplicons were equimolarly pooled and the size was verified using the Agilent Bioanalyzer DNA 1000 chip. In addition, the concentration of the amplicon pool was checked with qPCR. The pool of amplicon libraries was diluted to 13 pM prior to clustering, and PhiX was added as a diversity control for the MiSeq sequencing run.
SUB4309658
RIK_104_201606DD_HLSU