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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net

Proper citation: David Dunigan and Irina Agarkova 2016. Isolation Of Total DNA From NC64A Chlorella. protocols.io https://dx.doi.org/10.17504/protocols.io.ethbej6 Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Reverse Cross-linking Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.c6bzam Copy   

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Authors: Amy Zimmerman
Group: VERVE Net, Worden Lab
Summary: Purpose: To quantify infectious viruses and evaluate the infectivity of a viral sample.50 µL of serially-diluted (10-3 to 10-10) virus sample is added to 150 µL of exponentially growing host cells in triplicate 96-well microplates and incubated at normal growth conditions for ~2 weeks. Cell lysis is assessed every few days qualitatively by visual inspection and quantitatively by measuring optical density on a microplate reader. The MPN of infective viruses in each concentrate is estimated from the proportion of virus-positive (i.e., lysed) wells using the MPN_ver4.xls Excel spreadsheet from Jarvis, B., Wilrich, C., and P.-T. Wilrich (2010), Journal of Applied Microbiology 109 (2010), 1660 – 1667. Percent infectivity is then calculated by comparing the MPN-estimated abundance of infective viruses to the abundance of virus-like particles (VLPs) determined by flow cytometry analysis.

Proper citation: Amy Zimmerman 2017. MPN (Most Probable Number) assay for infectivity of algal viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.gunbwve Copy   

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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net

Proper citation: David Dunigan and Irina Agarkova 2016. Viral DNA Miniprep Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.eq6bdze Copy   

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Authors: Edmond Dion
Group: VERVE Net
Summary: This mock protocol is a fun description of the steps one might undertake to train for a one-way mission to the planet Mars.  The One-Way mission concept is gaining popularity due to it's simpler approach and lower cost.  Rather than expend effort to bring back the Marstronauts, the plan calls for leaving them there and ultimately colonizing the red planet.

Proper citation: Edmond Dion 2016. Train for a One-Way Mission to Mars. protocols.io https://dx.doi.org/10.17504/protocols.io.etqbemw Copy   

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Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviRI Purification From XZ-6E Virus Infected NC64A Chlorella.

Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. 1X CviRI Assay Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.es2bege Copy   

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Authors: Jennifer Brum
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This protocol describes how to negatively or positively stain viruses that have been deposited onto TEM grids so that they can be imaged using transmission electron microscopy (TEM). Positive staining stains the virus itself such that the virus is dark against a lighter background (Figure 1A). This is the easiest staining to do and yields results suitable for analyzing the morphological diversity of viruses from aquatic samples. Negative staining relies on some stain remaining around the edges of a virus such that the details of the virus are clearly defined (Figure 1B). Negative staining is more difficult and is used to obtain publication quality micrographs of viruses.Note: To deposit viruses onto the grids prior to staining, use either the “Adsorbing Viruses onto TEM Grids” protocol or the “Quantitatively Depositing Viruses onto TEM Grids” protocol. Grids should be stained immediately after depositing viruses onto them.

Proper citation: Jennifer Brum 2016. Positive and Negative Staining of Viruses on TEM Grids. protocols.io https://dx.doi.org/10.17504/protocols.io.day2fv Copy   

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Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviRI Purification From XZ-6E Virus Infected NC64A Chlorella.

Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. CviRI Storage Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.eszbef6 Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol provides a method for analysis of the relative abundance of potential antibiotic resistance genes found in the virome. We use the Comprehensive Antibiotic Resistance Database (CARD) as our reference for potential antibiotic resistance genes.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script P12: Antibiotic Resistance. protocols.io https://dx.doi.org/10.17504/protocols.io.ehwbb7e Copy   

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Authors: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith
Group: VERVE Net
Summary: This protocol is based on Schleper et al. (1992) as modified by Stedman et al. (2003).This is a protocol from: Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7Please see the published manuscript for additional information.

Proper citation: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith 2015. Spot-on-lawn (halo) assay for screening enrichment cultures and isolates for viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.eagbabw Copy   

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Authors: Jennifer Brum
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This protocol describes how to analyze a natural aquatic virus sample using transmission electron microscopy (TEM). The purpose is to obtain the capsid diameter distributions of the viral assemblage, tail length distributions of the viral assemblage, and percentages of each viral morphotype in a sample.Note: Prior to using this protocol, deposit viruses from your sample onto a TEM grid using the protocol “Quantitatively Depositing Viruses onto TEM Grids” then stain the viruses using the protocol “Positive and Negative Staining of Viruses on TEM Grids”.

Proper citation: Jennifer Brum 2016. Analysis of Viral Morphological Characteristics. protocols.io https://dx.doi.org/10.17504/protocols.io.dde23d Copy   

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Authors: Amy Zimmerman
Group: VERVE Net, Worden Lab
Summary: Purpose: To quantify infectious viruses and evaluate the infectivity of a viral sample.50 µL of serially-diluted (10-3 to 10-10) virus sample is added to 150 µL of exponentially growing host cells in triplicate 96-well microplates and incubated at normal growth conditions for ~2 weeks. Cell lysis is assessed every few days qualitatively by visual inspection and quantitatively by measuring optical density on a microplate reader. The MPN of infective viruses in each concentrate is estimated from the proportion of virus-positive (i.e., lysed) wells using the MPN_ver4.xls Excel spreadsheet from Jarvis, B., Wilrich, C., and P.-T. Wilrich (2010), Journal of Applied Microbiology 109 (2010), 1660 – 1667. Percent infectivity is then calculated by comparing the MPN-estimated abundance of infective viruses to the abundance of virus-like particles (VLPs) determined by flow cytometry analysis.

Proper citation: Amy Zimmerman 2016. MPN (Most Probable Number) assay for infectivity of algal viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.fcbbisn Copy   

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Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: Phage lysis of host bacteria can be visualized by plaque formation on lawns of host cells in soft agar overlayed on agar plates (Adams, 1959; Sambrook et al. 1989). This principle can be used for the detection and subsequent isolation of specific lytic phages in environmental samples (e.g., Carlson 2005). Spotting environmental samples on lawns of a target host cell would thus reveal the presence of a lytic phage for that particular host cell in the given sample.

Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Bacteriophage isolation by spotting on target host cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dps5nd Copy   

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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net

Proper citation: David Dunigan and Irina Agarkova 2016. Isolation and Purification of DNA from Chlorella Viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.eq5bdy6 Copy   

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Authors: James Thornton
Group: VERVE Net

Proper citation: James Thornton 2016. TFB buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.eq9bdz6 Copy   

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Authors: Grieg F. Steward and Alexander I. Culley
Group: VERVE Net
Summary: This protocol is a minor modification of that reported by Culley and Steward (2007). As the starting point for this protocol, we assume that viruses have been collected on an aluminum oxide 0.02-µm syringe-tip filter (Anotop, Whatman), but other filters capable of capturing viruses may be substituted.This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7Please see the published manuscript for additional information.

Proper citation: Grieg F. Steward and Alexander I. Culley 2015. Extracting nucleic acids from viruses on a filter. protocols.io https://dx.doi.org/10.17504/protocols.io.dsm6c5 Copy   

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Authors: Amy Zimmerman
Group: VERVE Net, Worden Lab
Summary: Purpose: To reproducibly generate fresh preparations of virus for independent experiments.Fresh virus sample for an experiment is generated by a primary infection of exponentially growing host cells from a master stock of virus. The infected host is allowed to lyse until the culture is cleared. The lysate is filtered to remove any large cellular debris, then the viral-size fraction is concentrated from the filtered lysate and washed with buffer using a centrifugal concentrator.  The viral concentrate is stored at 4°C protected from light and should be used for an experiment within 1-2 days. On the day of (or the day before) the experiment, an MPN assay should be set up to assess infectivity of the fresh viral concentrate.

Proper citation: Amy Zimmerman 2017. Propagation of marine eukaryotic viruses (Prasinoviruses). protocols.io https://dx.doi.org/10.17504/protocols.io.gujbwun Copy   

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Authors: Curtis Huttenhower
Group: VERVE Net, Huttenhower Lab
Summary: In this protocol we describe some approaches to graphically represent single profiled samples or a merged table of relative abundances.

Proper citation: Curtis Huttenhower 2016. GraPhlAn visualization of single and multiple samples. protocols.io https://dx.doi.org/10.17504/protocols.io.d9s96d Copy   

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Authors: Amy Zimmerman, Susanne Wilken
Group: VERVE Net, Worden Lab
Summary: Purpose: To evaluate the influence of nutrient availability on cellular elemental composition (quotas and stoichiometry) in marine picoeukaryotes.Elemental quotas and ratios are assessed under nutrient replete and deplete conditions at the same time to minimize potential variation between experiments. Cells from an exponentially growing culture are concentrated by centrifugation, washed and re-suspended in a small volume of nutrient deplete media. These concentrated cells are used to inoculate triplicate replete and deplete culture flasks at a starting density corresponding to early-exponential growth. Culture growth is monitored daily. When the mean growth rate of the nutrient deplete treatment is half or less of the replete treatment (GRDEP/GRREP

Proper citation: Amy Zimmerman, Susanne Wilken 2017. Nutrient deplete/replete algal culture for elemental analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.gukbwuw Copy   

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Authors: Li Deng, Bonnie T. Poulos, Matthew B. Sullivan
Group: VERVE Net, Sullivan Lab
Summary: AbstractOcean viruses are abundant, ubiquitous, and play important roles in global biogeochemical cycles through mortality, horizontal gene transfer and manipulation of host metabolism. However, the ability to link viruses to their hosts in a high-throughput manner bottlenecks our ability to understand virus-host interactions in complex communities. Here, we present viral tagging (VT), a method that combines mixtures of isotope labeled host cells and fluorescent viruses with flow cytometry. In a single experiment, we can screen 107 uncultivated ocean viruses with a single strain of Synechococcus. These viruses can then be sequenced to quantitatively link objectivelydefined environmental viral populations, and their genomes, to their hosts.

Proper citation: Li Deng, Bonnie T. Poulos, Matthew B. Sullivan 2016. Obtaining targeted metagenomes from uncultivated environmental Synechococcus using flow cytometry-based viral tagging. protocols.io https://dx.doi.org/10.17504/protocols.io.c6zzf5 Copy   

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