Amplification and Sequencing of Eukaryotic 18S rRNA

DNA was extracted using a PowerWater DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). Extracted DNA samples were processed by Research and Testing Laboratories (Lubbock, TX, USA). The V-4 fragments of 18S rRNA genes were amplified with TAReuk454FWD1 and TAReukREV3 (see Table S1 in the supplemental material). Amplifications were performed in 25-μl reaction volumes with recombinant Hot Start Taq polymerase (Qiagen HotStarTaq master mix; Qiagen, Inc., Valencia, CA, USA), 1 μl of each 5 μM primer, and 1 μl of template on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, CA, USA) under the following thermal profile: 95°C for 5 min, followed by 10 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 1 min and then 25 additional cycles of 94°C for 30 s, 45°C for 45 s, and 72°C for 1 min, and a final 2-min extension at 72°C (51 (link)). As the reverse primer TAReukREV3 poorly targets haptophytes, we additionally sequenced samples with high haptophyte abundance (23 May to 30 July) using the reverse primer HaptoR1 (Table S1) under the following thermal profile: 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min, and a final 2-min extension at 72°C (55 (link)). The amplicons were sequenced using the Roche 454 GS FLX Titanium platform with an average sequencing depth of 10,000 raw reads per sample.