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

Cyanoseed:http://cyanoseed.theFIG.info

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