Environmental Genome Shotgun Sequencing of the Sargasso Sea Venter JC, et al

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Environmental Genome Shotgun Sequencing of the
Sargasso SeaVenter JC, et al

• Kristine Briedis
• Evening Journal Club
• 4 April 2005

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Overview

• Craig Venter is sailing around the world
  collecting ocean water
• Performing whole-genome shotgun sequencing on the
  samples (focusing on microbes)

http//www.calacademy.org/calwild/2005winter/stori
es/venter.html
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Big Picture

• Of 1.7 million species classified so far,
  roughly 6000 are microbes
• True number of microbes is obviously larger than
  6000
• Imagine if our entire understanding of biology
  was based on a visit to the zoo. Thats where
  weve been in microbiology.
• Norman Pace, Univ of Colorado, Boulder
• http//www.wired.com/wired/archive/12.08/venter.ht
  ml?pg3topicventertopic_set

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Sargasso Sea
http//www.smithsonianmag.si.edu/smithsonian/issue
s98/nov98/map_jpg.html
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Voyage
http//www.sorcerer2expedition.org/version1/HTML/m
ain.htm
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Methods

• Performed whole-genome shotgun sequencing of
  surface water samples from Sargasso Sea
• Samples were filtered to isolate microbes
• Created genomic libraries with 2 to 6 kb inserts
• Sequenced plasmid clones
• Resulted in gt1.5 Gbp of microbial DNA sequence

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Shotgun Sequencing
http//www.bioteach.ubc.ca/Bioinformatics/GenomePr
ojects/shotgun201.gif
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Organism Identification

• Focused analysis on scaffolds with at least 3X
  coverage depth
• 333 scaffolds 2226 contigs 30.9 Mbp 25 of
  the data set
• Used oligonucleotide frequencies, depth of
  coverage, and similarity to previously sequenced
  genomes to separate some sequences into organism
  bins
• Identified several populations related to known
  species

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Prochlorococcus gene conservation
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SNPs

• Most deeply covered scaffolds 21 scaffolds with
  gt14X coverage contain 1 SNP per 10,000 bp
• Indicates discrete species are present
• Prochlorococcus scaffolds display blend of
  discrepancies from the consensus sequence
• Example-two scaffolds share gene synteny and
  align to MED4, but contain 15 inserted genes of
  probable phage origin
• Also some regions of MED4 not present in
  scaffolds largest region codes for surface
  polysaccharide biosynthesisperhaps not present
  in Sargasso Sea population

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Gene Finding

• Most sequence is in short (lt10kb) unassociated
  scaffolds and singletons
• Used sequence alignments to determine the most
  likely coding frame
• Ends of alignments refined to include start and
  stop codons
• Identified 1,214,207 genes (700 MB)
• Additional hypothetical genes identified by
  conserved open reading frames69,901 genes
• SwissProt database contains 137,885 sequences

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Gene Function
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Phylotypes
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Photosynthesis in Sargasso Sea

• Thought to be dominated by the cyanobacteria
  Prochlorococcus and Synechococcus
• But, gt90 of cyanobacteria scaffolds appear to be
  Prochlorococcus
• Could be due to the gradient sampled and the
  larger size of Synechococcus

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Bacteriorhodopsin

• Transmembrane protein that is a green
  light-driven proton pump
• Protons pumped out of cell then flow back in
  through ATP Synthase to create ATP
• Some rhodopsins found on scaffolds of organisms
  previously unknown to contain them

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Rhodopsin-like Sequences

• Identified 13 subfamilies of rhodopsin-like genes
• Four families of proteins from cultured organisms
  and nine families from uncultured organisms
• Expression levels of these genes are unknown

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Problems

• Large data dump in NCBI angered some
• Unclear how effective filtering was (some
  apparent eukaryotic DNA found)
• Some questioning of how samples were collected
• Had some trouble getting permits from countries
  to collect ocean water samples

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Our Plans

• 8.3 million sequences to analyze
• Sequences clustered by CD-HIT
• Run Superfamily HMMs on data
• Look for FSFs that are unique to particular
  superkingdoms (as found by Song)
• Look for FSFs that are not present in the three
  superkingdoms