Viral Genomics

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Viral Genomics
Allie Evans Colin Lappala Chelsea Layes Sheena
Scroggins
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The Sorcerer II Global Ocean Sampling
Expedition Northwest Atlantic through Eastern
Tropical Pacific Rusch DB, Halpern AL, Sutton
G, Heidelberg KB, Williamson S, et al. PLoS
Biology Vol. 5, No. 3, e77 doi10.1371/journal.pbi
o.0050077
The Sorcerer II Global Ocean Sampling
Expedition Expanding the Universe of Protein
Families Yooseph S, Sutton G, Rusch DB, Halpern
AL, Williamson SJ, et al. PLoS Biology Vol. 5,
No. 3, e16 doi10.1371/journal.pbio.0050016
The Sorcerer II Global Ocean Sampling Expedition
Metagenomic Characterization of Viruses within
Aquatic Microbial Samples Shannon J. Williamson,
Douglas B. Rusch, Shibu Yooseph, Aaron L.
Halpern, Karla B. Heidelberg, John I. Glass,
Cynthia Andrews-Pfannkoch, Douglas Fadrosh,
Christopher S. Miller, Granger Sutton, Marvin
Frazier, J. Craig Venter
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Baltimore Classification of Viruses

• dsDNA
• ssDNA
• dsRNA
• ssRNA
• -ssRNA
• ssRNA-RT
• dsDNA-RT

http//upload.wikimedia.org/wikipedia/en/thumb/0/0
7/Baltimore_Classification.png/720px-Baltimore_Cla
ssification.png
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Bacteriophages

• Viruses that infect bacteria
• Numerically dominant type of phage in oceans.

http//www.scienceclarified.com/images/uesc_02_img
0070.jpg
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Cyanophages

• Prochlorococcus
• Viruses have acquired and retained photosynthesis
  gene

http//web.mit.edu/mbsulli/www/NATL2A-40-group-cro
pped.jpg
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Phage Cycles
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Lateral gene transfer
l
http//upload.wikimedia.org/wikipedia/commons/thum
b/4/42/Transduction_(genetics).svg/800px-Transduct
ion_(genetics).svg.png
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Metagenomics

• Contribution of viral genomes to microbial
  environmental processes studied through
  metagenomic techniques.
• Metagenomics enables us to study microorganisms
  by examining DNA that is extracted directly from
  communities of environmental microorganisms

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http//camera.calit2.net/metagenomics/what-is-meta
genomics.php
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Metagenomic Challenges

• Inefficiencies in sampling
• DNA extraction methods
• Construction of libraries
• Inadequacies in data analysis and visualization
  tools

• Low abundance species overlooked
• Lack of reference genomes
• Sequencing complex environments cost prohibitive
• Standardizing metadata

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Methods
First

• Cruise the world
• Collect 90-200 L of seawater
• from each of 37 different stations
• Record pH, salinity, temperature,
• etc. of water

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Methods

• Pass water through 2.0, 0.8, 0.1
• µm filters, TFF to 50Kda for viral
• concentrate
• Store at -20C until shipment from
• next port

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Sequencing Preparation

• Extract DNA
• Nebulize DNA
• Average of 1.0-2.2 kb fragments
• Gel electrophoresis extraction
• purify and determine lengths
• Subclone into E. coli
• Colonies selected for inserts
• Shotgun sequence inserts

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Sequencing

• End sequence each insert
• Average of 822 bp sequenced per end

www.pasteur.fr/recherche/genopole/PF8/equipement_e
n.htmlnopole/PF8/equipement_en.html
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Metagenomic Assembly

• Same procedure as in humans, Drosophila, dogs,
  etc.

Unitigs using 98 or 94 homology for overlap
Scaffolding
Consensus sequence
Venter et al. (2001)
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Metagenomic Assembly

• New uses for shotgun sequencing and assembly
• Multiple organisms at once
• Likely novel organisms

Problems?

• Mate-pair data relied on more heavily, since
  overlap coverage is
• low or unknown
• Need verification of assembly somehow

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Metagenomic Assembly

• Created multiple distinct assemblies
• 98 homology unitigs
• 94 homology unitigs
• non-preassembled end-pairs at various
  stringencies for multiple sequence alignments
• Multiple assemblies allowed cross-referencing,
• quality assurance.

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Taxonomic Assignment

• Protein-ORF based strategy
• 5.6 million sequences from GOS
• All ORFs in same sequence scaffold compared to
  
• NCBI protein database using BLAST
• Votes tallied from each ORF into pools for
  scaffold
• Archea, Bacteria, Eukaryota, Viral
• 5.0 million sequence assigned using this
  method

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Quantitative PCR

• How many copies of studied proteins exist
• from station to station?
• versus one another?

http//www.invitrogen.com/content.cfm?pageid10037
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Quantitative PCR

• Level of fluorescence checked after each PCR
  cycle
• Initial amount can be inferred using standard
  curve
• Multiple dilutions allow comparison
• - Outcome reported only if
• -- Ten-fold above no-template negative control
• AND
• -- 10-2 dilution results in 3-30 more than 10-3
  dilution

http//www.invitrogen.com/content.cfm?pageid10037
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Clustering and Phylogeny

• Proteins clustered and compared to NCBI
• Sequence alignments, not just domains
• Gene families bolstered with new genes
• Phylogeny trees generated
• Multiple sequence alignments CLUSTALW
• Used only long, fairly homologous samples
• PHYLIP used to build trees
• Based on difference matrix

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Results

• 37 marine surface water samples collected
• 7.7 million sequencing reads were produced
• Identified 154,662 viral peptide sequences

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Identification of Viral Sequences

• Data from microbial fraction of water samples was
  examined
• Looked for viral sequences by comparison to the
  NCBI non-redundant protein database
• 154,662 viral peptide sequences were identified
• Approximately 3 of predicted proteins were
  identified as viral sequences
• Number of viral sequences thought to be largely
  underestimated

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Classification through Protein Clustering

• Of 154,662 viral peptide sequences, 117,123 or
  76 fell within 380 protein clusters containing
  at least 20 proteins
• Remaining sequences fell within clusters
  containing less than 20 proteins
• Average cluster size contained 258 peptide
  sequences

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Neighbor Functional Linkage Analysis

• Used to verify that they were on viral instead of
  pro-viral regions of bacterial genomes
• Proportion of viral same-scaffold ORFs range from
  32 to 92 for the metabolic gene families
  studied
• Occurrence of viral neighbors on same scaffolds
  as host-derived viral genes supports hypothesis
  that sources of the sequences are viruses rather
  than bacterial

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Quantitative PCR

• qPCR used on DNA collected from 5 sampling
  locations
• Yields were initially too low, so samples were
  pooled
• Viral gene families psbD, petE, speD, talC, pstS,
  and phoH were included
• Results indicate that host-derived viral genes
  are viral in nature
• Viral genes encoding environmentally significant
  host-specific functions are prevalent in aquatic
  samples

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Phylogenetic Analyses
Figure 2. Phylogenetic trees of all GOS and
publicly available psbA(A) and psbD(B) sequences.
BS indicates bootstrap values. GOS and
public viral sequences are colored aqua and pink
respectively. GOS and public prokaryotic
sequences are navy blue and lime green
respectively. doi10.1371/journal.pone.0001456.g00
2
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Figure 3. Phylogenetic trees of all GOS and
publicly available pstS(A) and talC(B) sequences.
BS indicates bootstrap values. GOS and public
viral sequences are colored aqua and pink
respectively. GOS and public prokaryotic
sequences are navy blue and lime green
respectively. GOS eukaryotic sequences are
colored yellow. doi10.1371/journal.pone.0001456.g
003
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All viral gene families were positively
correlated with water temperature Some viral
gene families were correlated with salinity,
water depth, and calculated trophic status
indices Different environmental pressures may
influence acquisition of these genes by
viruses Table S7 shows the correlations between
viral gene families and environmental parameters
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Discussion

• Most studies have focused on the filtered viral
  fraction of the data
• This is the first study to focus on the viral
  components in the microbial fraction of the data
• Strong evidence for abundance and distribution of
  environmentally important host-derived viral gene
  families
• Distribution patterns of host-derived viral
  families over environmental gradients
• Evidence of interactions between bacteriophage
  and host organisms

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Detection of Viruses in Mircrobial Data

• Large viruses (0.1 µm0.22 µm) get caught in the
  filters because of their size and geometric shape
• Small free living phages flow through the filter,
  but when viruses physically interacting with the
  microbes will be caught along with the microbes
• When filtrating large volumes, biomass
  accumulates on the filter and viruses get caught
• Most viruses found within the aquatic microbial
  communities studies seemed to be in the lytic
  infection cycle therefore they were actively
  replicating their DNA

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Viruses with Metabolic Genes

• Through lateral gene transfer, metabolic genes
  can be acquired from the host
• Acquisition, retention, and expression of
  metabolic genes may increase fitness
• Key metabolic processes and pathways running
  during infection allows maximum replication
• Previous studies on host-derived metabolic viral
  genes has been on the photosynthesis genes psbA
  and psbD of a cyanophage
• Previous studies did not focus on abundance or
  distribution of these genes in the oceans

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Host-Derived Metabolic Gene Families

• In aquatic viral communities sampled,
  host-derived genes were found widely distributed
  in significant proportions
• Quantitative PCR of the these genes confirmed
  high abundance
• Not known if these genes were expressed at the
  time of sampling
• Unlikely to see these genes in high abundance if
  they
• Were not expressed
• Did not have a fitness advantage

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Suggests that viruses may play a more
substantial role in environmentally relevant
metabolic processes than previously recognized
such as the conversion of light to energy,
photoadaptation, phosphate acquisition, and
carbon metabolism
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Potential Evolutionary Viral-Host Relationships

• The study of the cyanophage found that the
  host-derived genes undergo higher mutation rates
  than their cyanobacterial nucleotide counterpart
• After phage acquisition, the genes could
  diversify
• Mutated viral genes could form gene reservoirs
  for the host
• Through horizontal gene transfer, viruses could
  promote diversity and distribution

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Prochlorococcus P-SSM4-like Phage

• Prochlorococcus is one of the most widespread
  picophytoplankton in the ocean
• P-SSM4-like phage may influence the abundance,
  diversity, and distribution of Prochlorococcus
• Statistically significant relationship between
  the Prochlorococcus and the P-SSM4-like phage

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Metagenomic Viral-Microbial Interactions

• This study of viral-microbial association between
  communities was coincidental
• Horizontal transfer of metabolic genes
• More studies necessary on the viral-microbial
  diversity and genetic complement
• Community relationships
• Evolutionary relationships

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Any Questions?