Pleurochrysis carterae CCMP645 cells in mid-exponential growth phase were transferred (10% v/v) into flasks containing either F/2 (Guillard, 1975) [1] or L1 (Guillard and Hargraves, 1993) [2] media, in duplicate. Virus production in each flask was monitored by digital PCR (dPCR) for a 106-day period. The frequency of sampling varied throughout the period of the study as indicated in the excel data file. At each sampling point, 0.5 ml samples were collected and stored at -80C without the addition of any fixatives for further quantification of viral abundances. Prior to analysis, the samples were thawed at room temperature, diluted 1:1 in nuclease-free water. Cellular debris was removed by centrifugation at maximum speed for 5 seconds. Two microliter aliquots were taken from the supernatant and put directly into digital PCR (dPCR) reactions to quantify the abundance of co-infecting virus genotypes. Specifically, probes and primers were designed to target genotypes: P. carterae endemic virus genotypes 2 and 1b (PsEV2 and PsEV1b, respectively; dsDNA viruses); P. carterae Polinton-like viruses (PleuroPLV; dsDNA viruses); and P. carterae CRESS viruses (PcCV1, ssDNA viruses). For the latter, we designed probes for both the REP and the CAP genes to discriminate viral particles with partial (i.e., amplification for only the REP or the CAP markers from a single dPCR intact drop) or complete genomes (i.e., amplification with both molecular markers from a single dPCR intact drop). For more information about primers used in these experiments see the Virus dPCR assay primer dataset.
Specific primer/probe assays for all samples were multiplexed as follows:
* Multiplex Assay 1 – PsEV2 and PsEV1b probes were TET-labelled and multiplexed with PcCV-1 Rep and Cap probes (FAM labelled).
* Multiplex Assay 2 – For the second dPCR multiplex assay, the same samples were thawed again, diluted and spun out as before, and 2 ul of the supernatant was put into multiplex reaction with PleuroPLV assay (probe was FAM-labelled) and same 2 PsEV assays as an internal control to see how the repeated freeze-thaw cycles impacted the counts.
dPCR reactions contained 900 nM of each primer set and 200 nM probe, except for PsEV1b-TET and PcCV1-REP-FAM, which contained 450 nM primers and 100 nM probe. PCR reaction volumes were parsed into 5 million droplets per sample (RainDrop Source, RainDance Technologies) and then amplified in a C1000 Touch deep-well thermal cycler (Bio-Rad) with the following thermal protocol: 95C for 10 min; 95C for 15 s and then 60C for 60 s, with a ramping rate of 0.5C s−1 for 50 cycles; and final in-activation at 98C for 10 min. The droplets were enumerated on the RainDrop Sense (RainDance Technologies).
Reactions were run on a Bio Rad icycler and data was analyzed following Bio Rad’s Instruction Manual, Catalog Number 170-8740.
References
[1] Guillard, Robert RL. "Culture of phytoplankton for feeding marine invertebrates." Culture of marine invertebrate animals. Springer US, 1975. 29-60.
[2] Guillard, R. R. L., and P. E. Hargraves. "Stichochrysis immobilis is a diatom, not a chrysophyte." Phycologia 32.3 (1993): 234-236. https://doi.org/10.2216/i0031-8884-32-3-234.1
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