Protocols from Sullivan Lab (updates and discussions best at protocols.io where available)


 

Find here protocols for:

Sample Collection
Virus Purification and Concentration
Virus Enumeration
Virus DNA Extraction
Phage-Host Interactions
Preparation for Sequencing and Proteomics
Electron Microscopy

For a complete list of nearly 150 up-to-date protocols, check out the Sullivan lab at protocols.io!


 

Sample Collection

• Treatments for the Preservation of Seawater Samples
Protocol: treatment_of_seawater_samples_v6.3
Updated: March 2014
Description: This protocol describes the treatment of seawater for collection of samples for flow cytometry, quantitative PCR, viral qPCR, SYBR staining, transmission electron microscopy, culturing viruses, fluorescence in situ hybridization, and single cell sampling.

 


 

Virus Purification and Concentration

• Iron Chloride Precipitation of Viruses from Seawater (protocol at protocols.io)
Protocol: iron_chloride_precipitation_of_viruses_v8.3
Updated: November 2014
Description: This protocol describes a technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research.
Citation: John, S.G., Mendez, C.B., Deng, L., Poulos, B., Kauffman, A.K.M., Kern, S., Brum, J., Polz, M.F., Boyle, E.A., &Sullivan, M.B. (2011). A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environ Microbiol Rep. 3(2), 195-202. doi:10.1111/j.1758-2229.2010.00208.x.  LINK PDF
— Minor correction concerning buffer recipes:  PDF
• Concentrating Viruses with Centrifugal Ultrafiltration Devices
Protocol: Concentrating_Viruses_with_Centrifugal_Ultrafiltration_Devices
Updated: April 2011
Description: This protocol describes how to concentrate viruses in liquid samples using an Amicon or Nanosep centrifugal ultrafiltration device. Amicons are used to concentrate medium volumes of samples (10s to 100s of mls) down to a final volume of ∼4 ml. Nanoseps are used to concentrate smaller volumes of sample (<10 ml) down to a final volume of ∼30 μl.
• DNase I Treatment (protocol at protocols.io)
Protocol: DNase_I_Treatment_v1
Updated: August 2012
Description: This protocol decribes the preparation of and treatment with DNase I to degrade DNA in solutions containing iron chloride, EDTA–Mg ascorbate buffer, and cesium chloride. DNase I treatment, CsCl purification, and sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012.
• Cesium Chloride Virus Purification and Dialysis (protocol at protocols.io)
Protocol:  CsCl_Virus_Purification_Dialysis_v3
Updated: March 2013
Description: This protocol describes the use of Cesium Chloride to purify viruses with an optional dialysis step to remove the CsCl. 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. Environ Microbiol. 15(5), 1428–1440. doi:10.1111/j.1462-2920.2012.02836.x. LINK
• Purification of Viruses using Sucrose Cushion
Protocol: Sucrose_Cushion_Virus_Purification_v1
Updated: August 2012
Description: This protocol decribes the use of a sucrose cushion to purify viruses. 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. Environ Microbiol. 15(5), 1428–1440. doi:10.1111/j.1462-2920.2012.02836.x.  LINK
• Phage Tangential Flow Filtration
Protocol: Phage_Tangential_Flow_Filtration_Rohwer_2005
Updated: 2005
Description: This guide shows the basic information for setting up a Tangential Flow Filtration system. It was created by Dr. Forest Rohwer at San Diego State University.

 


 

Virus Enumeration

• Wet-Mount Method for Enumeration of Aquatic Viruses
Protocol: Wet-Mount_Virus_Enumeration_Protocol
Updated: June 2014
Description: This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method.
Citation: Cunningham, B., Brum, J., Schwenck, S., Sullivan, M.B., & John, S. 2015. An inexpensive, accurate and precise wet-mount method for enumerating aquatic viruses. Applied Environmental Microbiology. 81:2995-3000.

 


 

Viral DNA Extraction

• DNA Extraction of Viruses Using Wizard Columns
Protocol: Wizard_Column_Viral_DNA_Extraction_v1
Updated: October 2012
Description: This protocol decribes the extraction of DNA from viral particles using Wizard Columns from Promega.

 


 

Phage-Host Interactions

• phageFISH
Protocol: phageFISH_v2
Updated: October 2015
Description: This protocol combines phage gene detection with rRNA detection for the identification of host cells and detection of free phage particles.
Citation: Allers, E.*, Moraru, C.*, Duhaime, M.B., Beneze, E., Solonenko, N., Barrero-Canosa, J., Amann, R., & Sullivan, M.B. (2013). Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses. Environ Microbiol. 15(8), 2306–2318. doi:10.1111/1462-2920.12100. (* =co-first authors) ( =co-corresponding authors)   PDFLINK
• Viral Tagging
Protocol: Viral_Tagging_v6.1
Updated: August 2014
Description: This protocol describes viral tagging — a high–throughput method to investigate virus-host interactions by using fluorescently labeled viruses to tag host cells which can be analyzed and sorted using flow cytometry.
Citation: Deng, L., Gregory, A., Yilmaz, S., Poulos, B.T., Hugenholtz, P., & Sullivan, M.B. (2012). Contrasting life strategies of viruses that infect photo- and heterotrophic bacteria, as revealed by viral tagging. mBio. 3(6), e00373-12. doi:10.1128/mBio.00373-12.   PDFLINK
Deng, L.*, Ignacio-Espinoza, J.C.*, Gregory, A., Poulos, B.T., Weitz, J.S., Hugenholtz, P., & Sullivan, M.B. (2014). Viral tagging reveals discrete populations in Synechococcus viral genome sequence space. Nature. doi:10.1038/nature13459. (*=co-first authors)   LINK
• Isotopic Labeling of Cyanobacteria and DNA Analysis
Protocol: isotopic_labeling_of_cyanobacteria_and_dna_analysis_v2
Updated: June 2013
Description: Protocol for labeling bacteria with N15. The bacterial DNA was made “heavy” so that community viral and host bacterial DNA could be distinguished from each other after sorting by using density gradient centrifugation.

 


 

Preparation for Sequencing

• Low Input Library Preparation for Illumina sequencing
Protocol: genoscope_low_input_illumina
Updated: November 2012
Description: Protocol for preparation of short single and paired-end libraries from genomic dsDNA starting from low DNA quantity (up to 10 ng) for Illumina sequenincg. Developed by Adriana Alberti at Genoscope.
• Processing Samples for Illumina HiSeq
Protocol: emory_hiseq_sample_prep_guide_v3
Updated: December 2011
Description: Protocol for Illumina SPRIworks library contruction, clustering, and sequencing. Written by Jamie K. Davis at Emory.
• DNA Preparation and Linker Amplification for Pyrosequencing
Protocol: Linker_Amplification_v8
Updated: July 2013
Description: Here, we adapt the linker amplified shotgun library (LASL) approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a “reconditioning PCR” step to increase yield and minimize late-cycle PCR artifacts. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent.
Citation: Duhaime, M.B., Deng, L., Poulos, B.T., & Sullivan, M.B. (2012). Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: A rigorous assessment and optimization of the linker amplification method.Environ Microbiol. 14(9), 2526-2537. doi:10.1111/j.1462-2920.2012.02791.x.   PDFLINK
• Barcoded Oligos for Linker Amplified DNA
Protocol: TMPL_LAs
Updated: July 2013
Description: Sequence for barcoded oligos for linker amplified libraries. Numbers 1 to 29 were developed at the Tucson Marine Phage Lab dervied from MID 1 to 14 recommended by Roche.
Citation: Duhaime, M.B., Deng, L., Poulos, B.T., & Sullivan, M.B. (2012). Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: A rigorous assessment and optimization of the linker amplification method.Environ Microbiol. 14(9), 2526-2537. doi:10.1111/j.1462-2920.2012.02791.x.   PDFLINK

 


 

Preparation for Proteomics

• FASP (Filter-Aided Sample Prep) Optimized Preparation for 2d-LC-MS/MS
Protocol: FASP_Protocol_Viruses
Description: Collaborator, Nathan VerBerkmoes (Oak Ridge National Labs), worked with Tucson Marine Phage Lab to develop more sensitive proteomics assays for viral isolates which we are now also using for environmental viral concentrates. A new sample prep method (FASP) was optimized to maximize the signal from our commonly low-protein containing samples, and then run using 2d-LC-MS-MS to maximize detection across the large dynamic range in isolate and environmental samples.
• Multidimensional chromatography tandem mass spectrometry (2d-LC-MS/MS) Method
Protocol: General_2D_LCMSMS_Method
Description: This method was optimized for bacteriophage for shotgun proteomics. Used with the above FASP Protocol to maximize the detection across the large dynamic range in isolate and environmental viral samples.

 


 

Transmission Electron Microscopy (TEM)

• Adsorbing Viruses onto TEM Grids
Protocol: Adsorbing_Viruses_onto_TEM_Grids
Updated: October 2011
Description: This protocol describes how to adsorb viruses onto TEM (transmission electron microscopy) grids. The sample is allowed to sit on a hydrophilic grid and viruses adsorb onto the surface of the grid. This technique is generally used for viral lysates with high concentrations of viruses. For natural samples, use the protocol “Quantitatively Depositing Viruses onto TEM Grids using an Airfuge”.
• Quantitatively Depositing Viruses onto TEM Grids
Protocol: Quantitatively_Depositing_Viruses_onto_a_TEM_Grid_using_an_Airfuge
Updated: April 2011
Description:This protocol describes how to quantitatively deposit viruses onto TEM grids using an Airfuge (an air-driven ultracentrifuge made by Beckman). The purpose of using this protocol is to prepare aquatic samples to obtain viral assemblage characteristics (e.g. morphotypes, capsid widths, tail lengths) using transmission electron microscopy (TEM).
Citation: Brum, J. R., & Steward, G. F. (2010). Morphological characterization of viruses in the stratified water column of alkaline, hypersaline Mono Lake. Microbial Ecol. 60(3), 636-643.   LINK
• Positive and Negative Staining of Viruses on TEM Grids
Protocol: Positive_and_Negative_Stainging_of_Viruses_on_TEM_Grids
Updated: April 2011
Description: 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. 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. Negative staining is more difficult and is used to obtain publication quality micrographs of viruses.
• FVIC (Frequency of Visibly Infected Cells)
Protocol: FVIC_Protocol
Updated: October 2011
Description: This protocol describes how to pellet bacterial cells onto grids and stain them so that viruses can be visualized within the cells using transmission electron microscopy. Data obtained from these grids can be used to calculate the frequency of visibly infected cells (FVIC), the frequency of infected cells (FIC), the fraction of mortality due to viral lysis (FMVL), and burst size.
• Analysis of Viral Morphological Characteristics
Protocol: Analysis_of_Viral_Morphological_Characteristics
Updated: August 2013
Description: 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 percentage
• Using ImageJ to Measure Viral Dimensions in Micrographs
Protocol: Using_ImageJ_to_Measure_Viral_Dimensions_in_Micrographs
Updated: April 2011
Description: This protocol describes the use of ImageJ to measure dimensions of viruses in TEM micrographs, but can be applied to the measurement of anything in any image.