NSL_046_20160625_HSSU
Small subunit haptophyte-specific fusion primers (528F_long_HAP_454: GCGGTAATTCCAGCTCCA and PRYM01+7_HAP454: GATCAGTGAAAACATCCCTGG) 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: 30 s at 94, 15 s at 94, 30 s at 55, 60 s at 68 with repetition at step 2 for 30 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.
SUB4302425
NSL_046_20160625_HSSU