DNA-seq of Emiliania huxleyi virus encoded serine palmitoyltransferase
Semi-quantitative qPCR reactions were performed in order to characterize PCR kinetics for each North Atlantic sample and identify a threshold cycle (Ct) for which amplicons would most accurately reflect the relative template abundance in environmental nucleic acid samples. The primers  (FW primer: 5‚Äô TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3‚Äô; RV primer: 5‚Äô GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3‚Äô) were anchored within the flanking large (LCB2) and small (LCB1) SPT subunits, spanned the linker region, and included overhangs for sequencing and library preparation, based on manufacturer protocols. These reactions used reagents from the GoTaq Promega kit and consisted of 1 ¬µl  template of total extracted nucleic acid, 1 √ó PCR reaction buffer, 2.5 mM MgCl2, 0.04 U ¬µl-1 Taq DNA polymerase, 10 pmol of each primer (IDT Oligo, US), 0.2 mM dNTPs, 5% DMSO, 0.5 ¬µg ¬µl-1 BSA, 2X SYBR Green mix and dH2O to a final reaction volume of 25 Œºl, in a Mx3000P thermocycler (Stratagene). Thermocycler conditions included an initial cycle at 95¬∞C for 5 min, followed by 40 cycles at 95¬∞C for 30 sec, 53¬∞C for 60 sec, and 74¬∞C for 90 sec, finishing with a dissociation curve. Dissociation curves and visual inspection on agarose gels confirmed desired amplicon lengths. Once the threshold cycle was determined for each sample, subsequent PCR reactions were performed (as described above) without SYBR green and DMSO for each sample at the empirically determined cycle number. Amplicon products were purified using the Agencourt¬Æ AMPure ¬ÆXP beads DNA purification kit (Beckman Coulter) and used for downstream library preparation of NAVICE virus SPT linker amplicons. Amplicons were used to prepare dual-index libraries using the Nextera XT Index Kit, according to manufacturer instructions20. Libraries were pooled and run on an Illumina MiSeq sequencer with a paired-end (2x250 bp) configuration.