the global marine viriome rob edwards dept. biology, sdsu computational sciences research center,...
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THE GLOBAL MARINE VIRIOME
Rob Edwards
Dept. Biology , SDSUComputational Sciences Research Center, SDSU
Center for Microbial Sciences, San Diego,Fellowship for Interpretation of Genomes, Chicago, ILThe Burnham Inst. for Medical Research, San Diego
IMEC, LLC, San Diego
UBC, Vancouver, June 2006
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
Metagenomics
200 liters water 5-500 g fresh fecal matter
DNA/RNA LASL
Sequence
Epifluorescent Microscopy
Concentrate and purify viruses
Extract nucleic acids
Breitbart et al., multiple papers
454So 2004
Pyrosequencing
www.454.com
5-100ng DNA
whole genomeamplification
2-5 µg DNA
454 Sequence Data(Only from Rohwer Lab, in one year)
• 42 libraries– 22 microbial, 20 phage
• 1,028,563,420 bp total– 33% of the human genome– 95% of all complete and partial bacterial
genomes– 10% of community sequencing of JGI per year
• 9,933,184 sequences– Average 236,511 per library
• Average read length 103.5 bp– Av. read length has not increased in 12 months
Human blood Human stool
Marine Near-shore water Off-shore water Near- and off-shore sediments
Metazoanassociated Corals Fish
Sampling Sites
Terrestrial/Soil Amazon rainforest Konza prairie Joshua Tree desert
Freshwater Aquifer Glacial lake
ExtremeHot springs (84oC; 78oC)Soda lake (pH 13)Solar saltern (>35% salt)Air
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
http://theseed.uchicago.edu/FIG/index.cgi
The SEED database developed by FIG
Current version:
580 Bacteria (342 complete)38 Archaea (26 complete)562 Eukarya (29 complete)1335 Viruses2 Environmental Genomes
predicted or measured co-regulation
genome context(virulence islands, prophages,
conserved gene clusters)
virulence mechanism
cellular localization
enzymatic activity
common phenotype
combinations of criteria
Subsystems are not just for gene clusters
Marine Seed:http://theseed.uchicago.edu/FIG/organisms.cgi?
show=marine
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
Assembly of 454 sequences
mitochondrion ca. 17 kb
assembled fragment ca. 10 kbThanks: Lutz Krause
Community structure
Community structure based on frequency of findingoverlapping fragments from the sequences
2-contig
3-contig
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
Phages In The Worlds Oceans
GOM41 samples
13 sites5 years
SAR1 sample
1 site1 year
BBC85 samples
38 sites8 years
ARC56 samples
16 sites1 year
LI4 sites1 year
Most Marine Phage Sequences are Novel
Thanks: Mya Breitbart
Phages are specific to environments
PhageProteomicTree v. 5(Edwards, Rohwer)
ssDNA
-like
T7-likeT4-like
Marine Single-Stranded DNA Viruses
• 6% of SAR sequences ssDNA phage (Chlamydia-like Microviridae)
• 40% viral particles in SAR are ssDNA phage
• Several full-genome sequences were recovered via de novo assembly of these fragments
• Confirmed by PCR and sequencing
12,297 sequence fragments hitusing TBLASTX over a ~4.5 kb genome
SAR Aligned Against the Chlamydia 4
Individual sequence reads
Chlamydia phi 4genome
Coverage
Concatenated hits
Chl4 ORF calls
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
Phages, Reefs, and Human Disturbance
Phages, Reefs, and Human Disturbance
The Northern Line IslandsExpedition, 2005
Christmas
Kingman
Christmas
Kingman
Palmyra
Washington
Fanning
16S rDNA at each island
16S genes from 454 same as from cloning
Cloned and 454 sequenced16S are indistinguishable
Black stuff
Red
ClonedRed
Christmas to Kingman Bias in No. Phage HostsNegative numbers mean relatively more phage hosts at Kingman
More pathogens at Christmas.More people at Christmas.
More photosynthesis at Kingman.No people at Kingman.
Outline
• Forget DGGE, just sequence it– (Fabulous four-five-four for facile functional findings)
• Functional analysis is a blast• Is community structure
antiestablishment?• Are there viruses in the ocean?• Why people suck• Why we’re screwed
Computational Challenges• Sequence annotations and analysis
– What is there?
– What is it doing?
– How is it doing it?
• Gene predictions in unknowns
– Lutz Krause
• Sequence comparisons
– BLAST
– Other ways to rapidly compare short sequences
– What happens when everyone is using 454 sequencing?
Sequence data from 21 libraries
6 million sequences600 million bp
• Each BLASTX search takes 1,000 CPU hours• 42 libraries = 42,000 CPU hours or 4.8 CPU years• Users want
• repeat runs, • TBLASTX, • more analysis• more data• more, more, more, more
SDSU Forest Rohwer Beltran Rodriguez-Brito Lutz Krause
USF Mya Breitbart
Rohwer Lab Linda Wegley Florent Angly Matt Haynes
Math Guys@SDSU Peter Salamon Joe Mahaffy James Nulton Ben Felts David Bangor Steve Rayhawk Jennifer Mueller
MIT: Ed DeLong
FIG Veronika Vonstein Ross Overbeek Annotators
ANL Rick Stevens Bob Olsen CI Support
Also at SDSU Anca Segall Willow R-S Stanley Maloy
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