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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Checking DNA Concentration with Agarose Gel. protocols.io https://dx.doi.org/10.17504/protocols.io.c35yq5 Copy   

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Authors: Curtis Huttenhower
Group: VERVE Net, Huttenhower Lab
Summary: In this protocol we will show how to taxonomically profile 20 metagenomic shotgun samples Human Microbiome Project (HMP). Specifically, we will look at 10 samples from the buccal mucosa and 10 from the tongue dorsum. Please note these 20 samples are not necessarily an unbiased selection of microbiomes from the over 200 buccal mucosa and tongue dorsum samples generated by the HMP.REQUIREMENTS: BowTie2 installed and in the system path, Mercurial, basic unix commands (wget, tar).The size of the 20 samples (60GBs) can make their download quite slow. For this reason we are also providing the overall output of this protocol (few KBs) available for download so that one can start the tutorial from MetaPhlAn output merge and visualization if needed.

Proper citation: Curtis Huttenhower 2016. MetaPhlAn profiling of 20 HMP samples. protocols.io https://dx.doi.org/10.17504/protocols.io.d9e93d Copy   

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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net
Summary: For use in Polyacrylamide Gel System For Electrophoresis Of Proteins.

Proper citation: David Dunigan and Irina Agarkova 2016. 30.0% Acrylamide stock solution (30.0:0.8). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2bdye Copy   

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Authors: Janice E. Lawrence and Grieg F. Steward
Group: VERVE Net
Summary: OptiPrep gradients, as described for purifying viral isolates, may also be used for purifying marine viral assemblages. There are as yet, however, few descriptions of this application in the literature. CsCl gradients, on the other hand, have been used extensively for purifications of viral isolates and natural viral assemblages. Given the continued popularity of CsCl gradients, and their well-characterized performance, we present the following two-part purification and fractionation protocol for viruses in CsCl gradients. The first part involves relatively quick sedimentation through a step-gradient to remove the bulk of the contaminants. This is followed by a higher resolution continuous gradient to separate the viruses from residual contaminants and to separate viruses having differing buoyant densities from one another. We assume the starting material for the following procedure to be a concentrated suspension of viruses (whether filtered to remove cells or not).

Proper citation: Janice E. Lawrence and Grieg F. Steward 2015. Purification of viral assemblages from seawater in CsCl gradients. protocols.io https://dx.doi.org/10.17504/protocols.io.d2s8ed Copy   

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Authors: Nick Tang
Group: VERVE Net, Upton-Lab
Summary: Viral Bioinformatic Resource CentreProvide databases of viral genomic information. Please check the Organisms menu to see which viruses we support: we’re now focusing on large DNA viruses The VOCs (Virus Orthologous Clusters) database is at the heart of our system. The database links directly to integrated tools for comparative analyses. VOCs sorts genes into ortholog clusters (e.g. RNA polymerase) to simplify data retrieval. Provide easy access to the genes, gene families, and genomes of the different virus families. via a unique series of powerful Java tools that support multiple computer platforms (see VBRC Tools menu). design and build software to tackle specific bioinformatics/virology problems, often in collaboration with virologists. Rally the research community to provide expert curation of these viral genomes by: Adding value to GenBank sequences through enhancing and updating genome annotations Linking to research reviews/papers for the research community. Collaborate with researchers to help on specific bioinformatics problems, e.g. Custom searches of the databases Building new features into our tools Help with genome annotation

Proper citation: Nick Tang 2016. VBRC Base By Base: Export an image of the alignment. protocols.io https://dx.doi.org/10.17504/protocols.io.ecnbave Copy   

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Authors: Bonnie Poulos
Group: VERVE Net, Sullivan Lab
Summary: Purpose: to measure the infective titer (number of infective elements per mL) of from 1-5 viral samples in a quick and efficient manner.

Proper citation: Bonnie Poulos 2016. Most Probable Number (MPN) Counts of Cyanophages. protocols.io https://dx.doi.org/10.17504/protocols.io.ddb22m Copy   

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Authors: Karen D. Weynberg, Elisha M. Wood-Charlson, Curtis A. Suttle, and Madeleine J. H. van Oppen
Group: VERVE Net
Summary: Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

Proper citation: Karen D. Weynberg, Elisha M. Wood-Charlson, Curtis A. Suttle, and Madeleine J. H. van Oppen 2016. Generating viral metagenomes from the coral holobiont. protocols.io https://dx.doi.org/10.17504/protocols.io.ejgbcjw Copy   

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Authors: Amy Zimmerman, Camille Poirier
Group: VERVE Net, Worden Lab
Summary: Purpose: To quantify virus-like particles in culture or environmental samples.Host cells and virus-like particles are enumerated from the same glutaraldehyde-fixed sample. For virus counts, an aliquot is diluted in 0.02-µm-filtered TE buffer prior to staining with SYBR Green I nucleic acid dye to improve staining efficiency and ensure an appropriately low event rate in the instrument. An unstained aliquot of sample is run for host cell counts.

Proper citation: Amy Zimmerman, Camille Poirier 2017. Enumerating algal viruses by flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.guibwue Copy   

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Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviJI Purification From IL-3A Virus Infected NC64A Chlorella.

Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. CviJI Assay Conditions. protocols.io https://dx.doi.org/10.17504/protocols.io.esqbedw Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: For Transcriptomics During One-Step Growth Curves for Cellulophaga Phages protocol.

Proper citation: Matthew Sullivan 2016. RNA Samples Steps. protocols.io https://dx.doi.org/10.17504/protocols.io.dev3e5 Copy   

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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net

Proper citation: David Dunigan and Irina Agarkova 2016. Polyacrylamide Gel System For Electrophoresis Of Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.ep7bdrn Copy   

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Authors: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John
Group: VERVE Net, Sullivan Lab
Summary: For use in Wet-mount Method for Enumeration of Aquatic Viruses.

Proper citation: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John 2016. Ascorbic Acid Antifade Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.c87zzm Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: For use in DNase I Treatment protocol for DNase inactivation

Proper citation: Matthew Sullivan 2015. EGTA tetrasodium salt. protocols.io https://dx.doi.org/10.17504/protocols.io.c3pymm Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines the analysis used to plot MetaPhlAn taxonomic assignments. Based on methods from the following publication:Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script R8: Plotting Bacterial Taxonomy from MetaPhlan. protocols.io https://dx.doi.org/10.17504/protocols.io.ejbbcin Copy   

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Authors: André M. Comeau and Rachel T. Noble
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: The following procedure is an adaptation of the original FLVP protocol from Hennes et al. (1995) for use with SYBR Green I stain. Normally, this protocol will take 2 days given that the softening of the viral pellet is an overnight step. However, if the viral pellet redissolves quickly, it can be accomplished in 1 day.

Proper citation: André M. Comeau and Rachel T. Noble 2016. Viral and bacterial isolates, propagation and preparation of stocks. protocols.io https://dx.doi.org/10.17504/protocols.io.dzs76d Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: This mixture is is used with Cesium Chloride Dialysis for Viruses

Proper citation: Matthew Sullivan 2016. MTN100 with 1x NaCl. protocols.io https://dx.doi.org/10.17504/protocols.io.def3bm Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol provides a method for identifing CRIPR repeats using the program PilerCR on assembled contigs. Based on the methods from the following publication: Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script P10: CRISPR Identification. protocols.io https://dx.doi.org/10.17504/protocols.io.ehdbb26 Copy   

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Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net
Summary: For use in "Isolation of cyanophages by plaque assays"

Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Preparing target indicator cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dqi5ud Copy   

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Authors: Shannon Williamson
Group: VERVE Net, Sullivan Lab
Summary: This protocol describes the use of a sucrose cushion to purify viruses. It is the Matthew Sullivan Lab adaptation of the Shannon Williamson protocol.DNase I treatment, CsCl purification, and sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012.Citation: Hurwitz, B. L., Deng, L., Poulos, B. T., & Sullivan, M. B. (2012). Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics. Environmental Microbiology. doi:10.1111/j.1462-2920.2012.02836.x

Proper citation: Shannon Williamson 2015. Purifying Viruses Using Sucrose Cushion. protocols.io https://dx.doi.org/10.17504/protocols.io.c3wypd Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Screening Recombinant Clones by PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.c48yzv Copy   

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