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Authors: Benjamin Bolduc
Group: Sullivan Lab, iVirus
Summary: Identifying putative viral sequences from SPAdes-assembled data from the Ocean Sampling Day (2014) metagenomic datasets using VirSorter.

Proper citation: Benjamin Bolduc 2016. Identifying Viral Sequences Using VirSorter (Cyverse). protocols.io https://dx.doi.org/10.17504/protocols.io.eyjbfun Copy   

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Authors: Ms Alex Aitken
Group: VERVE Net, Sullivan Lab
Summary: TE is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation: 10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA

Proper citation: Ms Alex Aitken 2016. TE Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.c5uy6v Copy   

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Authors: Cristina Moraru & Elke Allers
Group: VERVE Net, Sullivan Lab
Summary: This protocol combines phage gene detection with rRNA detection for the identification of host cells and detection of free phage particles.

Proper citation: Cristina Moraru & Elke Allers 2016. phageFISH Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.c9iz4d Copy   

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Authors: Benjamin Bolduc
Group: VERVE Net, Sullivan Lab, iVirus
Summary: A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses actual reads from Ocean Sampling Day (2014) and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.Quality trimming of reads is important!

Proper citation: Benjamin Bolduc 2016. Quality Control of Reads Using Trimmomatic (Cyverse). protocols.io https://dx.doi.org/10.17504/protocols.io.gvybw7w Copy   

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Authors: Jennifer Brum
Group: VERVE Net, Sullivan Lab
Summary: This protocol describes how to concentrate viruses in liquid samples using an Amicon or Nanosep centrifugal ultrafiltration device. We use Amicons to concentrate medium volumes of samples (10s to 100s of mls) down to a final volume of ~4 ml. We use Nanoseps to concentrate smaller volumes of sample (

Proper citation: Jennifer Brum 2016. Concentrating Viruses with an Amicon or Nanosep Centrifugal Ultrafiltration Device. protocols.io https://dx.doi.org/10.17504/protocols.io.c54y8v Copy   

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Authors: Matt Sullivan Lab
Group: VERVE Net, Sullivan Lab
Summary: Must be made fresh before experiment because of the Sucrose. For 20 mL solutions.

Proper citation: Matt Sullivan Lab 2017. Lysis Buffer (20 mL). protocols.io https://dx.doi.org/10.17504/protocols.io.c4gytv Copy   

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Authors: Alexander Culley, Grieg Steward
Group: VERVE Net, Sullivan Lab
Summary: Culley Protocol (from Culley & Steward (2007), New genera of RNA viruses in subtropical seawater, inferred from polymerase gene sequences, Applied & Environmental Microbiology 73(18):5937-5944).

Proper citation: Alexander Culley, Grieg Steward 2015. Can Crusher-Culley Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.c3jykm Copy   

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Authors: Li Deng
Group: VERVE Net, Sullivan Lab
Summary: Case 1: Quick check of viral concentration. Use this protocol when you can count your samples in a few days.For when long term storage of slide is required, see Case 2.

Proper citation: Li Deng 2016. SYBR Gold Staining for Viral Enumeration (Case 1). protocols.io https://dx.doi.org/10.17504/protocols.io.c69zh5 Copy   

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Authors: Jiarong Guo, Dean Vik, Akbar Adjie Pratama, Simon Roux, Matthew Sullivan
Group: Sullivan Lab, iVirus
Summary: This protocol shows how to use VirSorter2, checkV, DRAMv and some manual curation criteria for viral sequence identification.

Proper citation: Jiarong Guo, Dean Vik, Akbar Adjie Pratama, Simon Roux, Matthew Sullivan 2021. Viral sequence identification SOP with VirSorter2. protocols.io https://dx.doi.org/10.17504/protocols.io.btv8nn9w Copy   

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Authors: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer
Group: VERVE Net, Sullivan Lab
Summary: This protocol gives a method for treatment and preservation of seawater samples that will be subject to fluorescence in situ hybridization (FISH). Samples are used with fluorescent probes to determine what populations of bacteria or other microbes are present. The filters may also be used to simply count the number of microbes in a sample. Requires varying amounts of 10µm or 200µm filtrate (up to 11.5L for open ocean depth profile) as shown in Table I.

Proper citation: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer 2015. Treatments and Preservation of Seawater Samples for FISH. protocols.io https://dx.doi.org/10.17504/protocols.io.c5by2m Copy   

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Authors: Chisholm Lab
Group: VERVE Net, Sullivan Lab

Proper citation: Chisholm Lab 2016. NATURAL SEAWATER-BASED PRO99 MEDIUM. protocols.io https://dx.doi.org/10.17504/protocols.io.c7zzp5 Copy   

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Authors: Benjamin Bolduc
Group: Sullivan Lab, iVirus
Summary: Preparing data for use in vContact by using VirSorted Ocean Sampling Day (2014) contigs, using tools available in Cyverse. This protocol creates a BLAST DB, BLASTs sequences, and creates a gene-to-contig mapping file. Results from this protocol are suitable for vContact-PCs.

Proper citation: Benjamin Bolduc 2017. Preparing Data for vContact from Proteins (Cyverse). protocols.io https://dx.doi.org/10.17504/protocols.io.gwdbxa6 Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Plating Prochlorococcus and Synechococcus strains in top agarose for plaque assays. protocols.io https://dx.doi.org/10.17504/protocols.io.c3vyn5 Copy   

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Authors: Howard Ochman
Group: VERVE Net, Sullivan Lab
Summary: Modified from H. Ochman protocol: Environ. Micro. 2009 Kunin, Engelbrekson Ochman & Hugenholtz

Proper citation: Howard Ochman 2016. 16s Pyrotag PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.c5iy4d Copy   

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Authors: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer
Group: VERVE Net, Sullivan Lab
Summary: This protocol consists of several methods for the preservation of seawater samples to later perform various techniques in the lab. It is meant as a sampling treatment in the field and is written as comprehensive; however, depending on needs and scientific questions, any or all of the methods may be used.Flow Cytometry (FCM): Samples are used for counting bacteria and for sorting different groups of microbes (eg, sorting synechococcus and prochlorococcus and non-fluorescent bacteria). Glutaraldehyde fixes microbes and renders them unculturable whereas microbes preserved in DMSO and Betaine can usually be cultured after thawing and rinsing. Betaine was developed for single cell sorting and may be a better preservative than DMSO.SYBR staining: Samples are used for counting bacteria (unfiltered or pre-filtered seawater) and viruses (unfiltered, pre-filtered or 0.2um filtered seawater) microscopically after staining with SYBR-Gold (or SYBR-Green). Transmission Electron Microscopy (TEM): Samples are used for determining frequency of visibly infected cells (FVIC) and determining types of microbes or viruses present, and can be quantitative.Quantitative PCR (qPCR): Samples are used for quantitative PCR of microbes. They can also be used for quantitative PCR of viruses.Viral qPCR: Samples are used for quantitative PCR of viruses.Culturing Viruses: Samples are used for cultivating viruses by solid or liquid plaque assay on susceptible hosts. Samples can also be used for viral tagging (using flow cytometry) and most probable number (MPN) assays.Fluorescence in situ hybridization (FISH): Samples are used with fluorescent probes to determine what populations of bacteria or other microbes are present. The filters may also be used to simply count the number of microbes in a sample. For detailed FISH procedure see here.

Proper citation: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer 2015. Treatments for the Preservation of Seawater Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.c29yh5 Copy   

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Authors: Benjamin Bolduc
Group: VERVE Net, Sullivan Lab, iVirus
Summary: A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses actual reads from Ocean Sampling Day (2014) and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.

Proper citation: Benjamin Bolduc 2019. Applying vContact to Viral Sequences and Visualizing the Output (Cyverse). protocols.io https://dx.doi.org/10.17504/protocols.io.x5xfq7n Copy   

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Authors: Jonathan King
Group: VERVE Net, Sullivan Lab
Summary: This mixture is used in the CsCl Step Gradient to Purify Phage Protocol

Proper citation: Jonathan King 2016. CsCl Step Gradient Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.dmj44m 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: Purpose: This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method. Briefly, fluorescently-stained samples are wetmounted directly onto slides for epifluorescence microscopy after an optional chemical flocculation concentration step used for samples with anticipated virus concentrations of 7 viruses mL-1 (samples with >5×107 viruses mL-1 do not require this concentration step prior to analysis). Virus concentration in the wet-mounted sample is determined from the ratio of viruses to microsphere beads, which are added at a known concentration. This wet-mount method for enumerating viruses is significantly less expensive than the filter-mount method (i.e., the cost of microsphere beads per sample is ~500-fold lower than the cost of one filter per sample), and is appropriate for rapid, precise and accurate enumeration of aquatic viruses over a wide range of viral concentrations encountered in field and laboratory samples. The only limitation of this method is that samples with virus concentrations ≤1×106 viruses mL-1 cannot be enumerated, as the abundance of viruses is too low for efficient enumeration.Figure 1. Overview of the 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. Wet-mount Method for Enumeration of Aquatic Viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.c8pzvm Copy   

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Authors: Lauren Chittick
Group: Sullivan Lab

Proper citation: Lauren Chittick 2016. 1M Citrate and Magnesium Resuspension Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.exybfpw Copy   

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Authors: Ross Fu and Matt Sullivan
Group: VERVE Net, Sullivan Lab
Summary: Purpose: to determine the concentration of DNA-containing particles in a given sample; useful in finding cell counts or phage titers.

Proper citation: Ross Fu and Matt Sullivan 2016. Protocol for SYBR Counts of Cyanophages and Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.c7wzpd Copy   

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