Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

This dataset contains temporal dynamics information for viruses co-infecting Pleurochrysis carterae, a coccolithophorid alga.  Parameters included in this dataset are the number of virus droplets used, number of virus genomes per milliliter of culture, droplet normalization factors used, and the normalized number virus droplets used.

Related datasets:
* Pleurochrysis carterae growth
*  Virus dPCR assay primers
* Accession numbers (P. carterae viruses and field samples)
* TEM Pleurochrysis carterae thin section images
* TEM Pleurochrysis carterae virion images

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

Standard procedures based on Cq value were followed to quantify gene copies in each sample. We followed general guidance from Bio Rad’s Instruction Manual, Catalog Number 170-8740. 

BCO-DMO Data Manager Processing Notes:
* Added date column from start date 2015-10-07 and date information in sample ID.
* added a conventional header with dataset name, PI name, version date
* modified parameter names to conform with BCO-DMO naming conventions
* blank values replaced with no data value 'nd'
* Date format converted to ISO Date format

Description from NSF award abstract:
Viruses are prevalent in every part of the environment of our living planet, and yet our understanding of type, distribution, and function is the least well-known aspect of biodiversity. In recent years we have developed an increased appreciation for the role viruses play in driving host evolution in the environment, but fundamental knowledge about the mechanisms involved remain lacking. Additionally, viruses may influence diversity indirectly through "kill the winner" scenarios, as well as through cell lysis and subsequent release of dissolved nutrients, which facilitate restructuring of microbial communities. The majority of research on marine viruses to date has focused on combinations of acutely susceptible host strains with highly virulent virus isolates. However, it is likely that marine viruses also employ a persistent infection life strategy, arguably preferring it to the more widely recognized lytic cycle. The objective of this project is to demonstrate that persistent virus infections occur in marine phytoplankton, and that these are a crucial component of ocean ecosystem function and a key evolutionary driver in primary producers. Using a range of persistent virus:host systems, this project will investigate:
1) how pervasive persistent virus infections are in marine systems; and
2) the role of non-coding RNAs in maintaining host:virus symbiosis.

This is a high risk-high pay research as it involves a radically different approach to the analysis of viruses in marine systems. The investigators plan to apply a suite of molecular (transcriptomics, genomics and development of novel diagnostic markers) techniques to include the analysis of microRNAs to determine the functional importance of persistent viruses in the ocean. The results of this project will be potentially transformative for our understanding of virus-driven phytoplankton evolution and its potential impact on biodiversity in marine phytoplankton, a vital component of the global carbon cycle.

Note: William Wilson (Bigelow Laboratory) was the Former Principal Investigator on this project award.