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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: protocols.io team
Group: VERVE Net, protocols.io news, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Protocols.io offers extensive flexibility in entering and presenting detailed methods. However, simple suggestions in here can help to make your protocol more useful to you and others. Importantly, easy steps outlined here can greatly increase the visibility of your protocol and ensure that scientists who need can find it.If you have any questions, please feel free to reach out to the protocols.io team by e-mailing us at  info [at] protocols.io. 

Proper citation: protocols.io team 2019. How to make your protocol more reproducible, discoverable, and user-friendly. protocols.io https://dx.doi.org/10.17504/protocols.io.g7vbzn6 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: Lenny Teytelman
Group: VERVE Net
Summary: This is a highly reproducible method for creating hit science videos. Guaranteed to lead to millions of views, fame, faculty position, tenure, and stable funding.* Legal disclaimer: This is not a peer reviewed protocol. Therefore, all claims should be taken with as much skepticism as anything that is peer reviewed.

Proper citation: Lenny Teytelman 2016. Making a hit science video. protocols.io https://dx.doi.org/10.17504/protocols.io.emqbc5w 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: David Dunigan and Irina Agarkova
Group: VERVE Net
Summary: This medium is for growing Chlorella variabilis NC64A, Chlorella heliozoae SAG 3.83, and Coccomyxa C-169.

Proper citation: David Dunigan and Irina Agarkova 2016. Formulation of MBBM (Modified Bold's Basal Medium). protocols.io https://dx.doi.org/10.17504/protocols.io.etwbepe Copy   

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Authors: Curtis Huttenhower
Group: VERVE Net, Huttenhower Lab
Summary: In this protocol, we will merge the 20 profiled metagenomes in a table of relative abundances and visualize the table with a heatmap.

Proper citation: Curtis Huttenhower 2016. MetaPhlAn output merge and visualization. protocols.io https://dx.doi.org/10.17504/protocols.io.d9q95v Copy   

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Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: K-mer-based approaches to determining sequence similiarity can be faster than traditional use of BLAST.  This application uses Jellyfish to index host FASTA files, then uses the mode value (default 2) of the number of matching k-mers (default k=20) from the input sequence to determine whether the sequence is similar enough to the host sequence to be rejected.  The output of the app is:"screened" directory containing the sequences from each file that were found to be dissimlar to the host"rejected" directory containing the sequence from each file that were too similar to the host"jf" directory containing Jellyfish indexes of each "host" file (useful for later runs with other files to skip recreating)"kmer" directory containing k-mers of query sequences and ".loc" file showing the number of ".kmer" lines associated to each sequence Code is freely available at Github.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. K-mer-based host screening. protocols.io https://dx.doi.org/10.17504/protocols.io.ehjbb4n Copy   

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Authors: William H. Wilson and Declan Schroeder
Group: VERVE Net
Summary: Generation of DNA fragments by DNase digestion involves digesting DNA for a range of times, then picking the time that gives optimal-sized DNA fragments (typically 1000–4000 bp).

Proper citation: William H. Wilson and Declan Schroeder 2016. Generation of DNA fragments by DNase digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.dy47yv Copy   

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Authors: Irina Agarkova
Group: VERVE Net, The Chlorovirus Group

Proper citation: Irina Agarkova 2016. Pulse Field Gel Electrophoresis (PFGE) Protocol for separation of Chlorella chromosomal DNA and Chlorella virus DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.essbeee Copy   

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Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: Hurwitz, Bonnie L., and Matthew B. Sullivan. "The Pacific Ocean Virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology." PLoS One 8.2 (2013): e57355.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. Running PCPipe (protein clustering) in iPlant. protocols.io https://dx.doi.org/10.17504/protocols.io.ef8bbrw 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: More than 40 years ago, Safferman and Morris (1963) used the plaque assay method to isolate the first cyanophage that infects a freshwater filamentous cyanobacterium, Plectonema boryanum. Since then, this approach has been used successfully to detect and isolate a number of different phages infecting marine Synechoccocus and Prochlorococcus (e.g., Suttle and Chan 1993; Waterbury and Valois 1993; Wilson et al. 1993; Sullivan et al. 2003).The advantages of this method are that results are easily interpreted as plaques formed on pigmented lawns can be easily identified. Since a plaque is the result of a single infection event, the virus can be easily purified and cloned. The first part of the procedures described as follows: (1) preparation of base plates and (2) preparation of top agar/agarose are applicable also for bacteriophage plaque assays.

Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Isolation of cyanophages by plaque assays. protocols.io https://dx.doi.org/10.17504/protocols.io.dqg5tv 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: Ruth-Anne Sandaa, Steven M. Short, and Declan C. Schroeder
Group: VERVE Net
Summary: The viral concentrate used for PFGE analysis must be molded into plugs, followed by lysis of the virus particles to release their DNA. It is possible to run solution-based preparation of viral DNA for PFGE (Steward 2001); however, large DNA molecules (>100 kb) are extremely sensitive to mechanical shearing in aqueous solution (Bouchez and Camilleri 1997). The consensus is that lysis inside viral plugs prevents mechanical shearing of the DNA, resulting in more discrete PFGE bands. Intact viral genomes are then separated by size by PFGE. After separation, the banding pattern is visualized by staining with a fluorescent DNA stain. This banding pattern provides a visual record of the genome size distribution that can be used for qualitative and quantitative comparisons between samples.

Proper citation: Ruth-Anne Sandaa, Steven M. Short, and Declan C. Schroeder 2016. Fingerprinting aquatic virus communities using pulsed field gel electrophoresis (PFGE). protocols.io https://dx.doi.org/10.17504/protocols.io.dy27yd Copy   

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Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: Long-standing questions in marine viral ecology are centered on understanding how viral assemblages change along gradients in space and time. However, investigating these fundamental ecological questions has been challenging due to incomplete representation of naturally occurring viral diversity in single gene- or morphology-based studies and an inability to identify up to 90% of reads in viral metagenomes (viromes).  In this protocol, I describe how to use an annotation- and assembly-free strategy for comparative metagenomics that combines shared k-mer and social network analyses (regression modeling). This robust statistical framework enables visualization of complex sample networks and determination of ecological factors driving community structure.  This tutorial describes a protocol to reproduce work from the Pacific Ocean virome comprised of 32 viromes from diverse sites in the Pacific Ocean. "Modeling ecological drivers in marine viral communities using comparative metagenomics and network analyses" (July 7, 2014, doi: 10.1073/pnas.1319778111, PNAS July 22, 2014 vol. 111 no. 29 10714-10719)Code is freely available at Github.

Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. Modeling ecological drivers in marine viral communities using comparative metagenomics and network analyses. protocols.io https://dx.doi.org/10.17504/protocols.io.efgbbjw 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: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines the analysis used to plot contig coverage statistics, as well as sequence count and length stats. We begin with visualizing contig length vs coverage. We then visualize the distributions of sequence counts per sample as a probability density plot (similar idea as a histogram), and then do the same for median sequence length. 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 R1: Virome Contig and Sequencing Statistics. protocols.io https://dx.doi.org/10.17504/protocols.io.eh5bb86 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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