DNA extraction and PCR amplification
All samples were centrifuged at 11,000 xg for 30 minutes to harvest
diatom cells. Then, supernatant was discarded and pellet was resuspended
in 200 µL of nuclease-free water. DNA was isolated using the Power Soil
DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, California, US)
following the manufacturer instructions and its concentration was
measured with Nanodrop 1000 (NanoDrop Technologies, Wilmington,
Delaware, US). A 312 bp fragment of rbc L gene
(ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) was
amplified by PCR using an equimolar mix of degenerate primers with
overhang adapters for Illumina sequencing (Table 2) (Rivera et
al., 2017). For all samples, we performed six PCR reactions on 50 µL of
reaction mixture containing 10-20 ng/µL of extracted DNA, 2 U of
Platinum II Taq Hot-Start DNA Polymerase (Invitrogen, Grand Island, New
York, US), 10 µL of 5X Platinum II PCR Buffer, 0.5 µM of each primer, 5
µL of dNTP mix (2mM each), 10 µL of Platinum GC Enhancer and 9.6 µL of
nuclease-free water. PCR conditions included an initial denaturalization
step at 94 °C for 4 min followed by 40 cycles of denaturalization at 94
°C for 30 s, annealing at 55 °C for 30 s and extension at 68 °C for 30
s, and a final extension step at 68 °C for 10 min. PCR products were
visualized with ultraviolet light in a 1.5% agarose gel stained with
ethidium bromide. Bands targeting the rbc L barcode gene were
excised off from agarose gel and then DNA extracted with Clean-Easy
Agarose Purification Kit (Canvax Biotech, Córdoba, Spain) following the
manufacturer instructions, except for elution volume, which was 15 µL.