THE GLOBAL MARINE VIRIOME

1. UBC, Vancouver, June 2006 THE GLOBAL MARINE VIRIOME Dept. Biology , SDSU Computational Sciences Research Center, SDSUCenter for Microbial Sciences, San Diego, Fellowship for Interpretation of Genomes, Chicago, IL The Burnham Inst. for Medical Research, San Diego IMEC, LLC, San Diego Rob Edwards

2. 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

3. So 2004 454 Metagenomics 200 liters water 5-500 g fresh fecal matter Concentrate and purify viruses Epifluorescent Microscopy Extract nucleic acids DNA/RNA LASL Sequence Breitbart et al., multiple papers

4. Pyrosequencing whole genome amplification 5-100ng DNA 2-5 µg DNA www.454.com

5. 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

6. Metazoan associated Corals Fish Sampling Sites Freshwater Aquifer Glacial lake Marine Near-shore water Off-shore water Near- and off-shore sediments Human blood Human stool Extreme Hot springs (84oC; 78oC) Soda lake (pH 13) Solar saltern (>35% salt) Terrestrial/Soil Amazon rainforest Konza prairie Joshua Tree desert Air

7. 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

8. The SEED database developed by FIG http://theseed.uchicago.edu/FIG/index.cgi • Current version: • 580 Bacteria (342 complete) • 38 Archaea (26 complete) • 562 Eukarya (29 complete) • 1335 Viruses • 2 Environmental Genomes

9. genome context (virulence islands, prophages, conserved gene clusters) virulence mechanism enzymatic activity cellular localization predicted or measured co-regulation common phenotype combinations of criteria Subsystems are not just for gene clusters

10. Cyanoseed: http://cyanoseed.theFIG.info

11. Marine Seed: http://theseed.uchicago.edu/FIG/organisms.cgi?show=marine

12. 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

13. mitochondrion ca. 17 kb assembled fragment ca. 10 kb Assembly of 454 sequences Thanks: Lutz Krause

14. 2-contig 3-contig Community structure Community structure based on frequency of finding overlapping fragments from the sequences

15. 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

16. ARC 56 samples 16 sites 1 year BBC 85 samples 38 sites 8 years SAR 1 sample 1 site 1 year GOM 41 samples 13 sites 5 years LI 4 sites 1 year Phages In The Worlds Oceans

17. Most Marine Phage Sequences are Novel

18. ssDNA -like T4-like T7-like Phages are specific to environments Phage Proteomic Tree v. 5 (Edwards, Rohwer) Thanks: Mya Breitbart

19. 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

20. SAR Aligned Against the Chlamydia 4 Individual sequence reads Coverage Concatenated hits Chlamydia phi 4 genome Chl4 ORF calls 12,297 sequence fragments hit using TBLASTX over a ~4.5 kb genome

21. 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

22. Phages, Reefs, and Human Disturbance

23. Kingman Christmas Phages, Reefs, and Human Disturbance Kingman Palmyra Washington Fanning Christmas The Northern Line Islands Expedition, 2005

24. 16S rDNA at each island

25. 16S genes from 454 same as from cloning Black stuff Cloned and 454 sequenced 16S are indistinguishable Cloned Red Red

26. More photosynthesis at Kingman. No people at Kingman. More pathogens at Christmas. More people at Christmas. Christmas to Kingman Bias in No. Phage Hosts Negative numbers mean relatively more phage hosts at Kingman

27. 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

28. 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?

29. Sequence data from 21 libraries 600 million bp 6 million sequences • 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

30. SDSU Forest Rohwer Beltran Rodriguez-Brito Lutz Krause USF Mya Breitbart Rohwer Lab Linda Wegley Florent Angly Matt Haynes ANL Rick Stevens Bob Olsen CI Support FIG Veronika Vonstein Ross Overbeek Annotators Also at SDSU Anca Segall Willow R-S Stanley Maloy Math Guys@SDSU Peter Salamon Joe Mahaffy James Nulton Ben Felts David Bangor Steve Rayhawk Jennifer Mueller MIT: Ed DeLong