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

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. 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 454 So 2004

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

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. Human blood Human stool Marine Near-shore water Off-shore water Near- and off-shore sediments Metazoan associated Corals Fish Sampling Sites Terrestrial/Soil Amazon rainforest Konza prairie Joshua Tree desert Freshwater Aquifer Glacial lake Extreme Hot springs (84 o C; 78 o C) Soda lake (pH 13) Solar saltern (>35% salt) 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. 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 Viruses 2 Environmental Genomes

9. 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

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

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

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

17. Most Marine Phage Sequences are Novel

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

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

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. The Northern Line Islands Expedition, 2005 Christmas Kingman Christmas Kingman Palmyra Washington Fanning

24. 16S rDNA at each island

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

26. Christmas to Kingman Bias in No. Phage Hosts Negative numbers mean relatively more phage hosts at Kingman More pathogens at Christmas. More people at Christmas. More photosynthesis at Kingman. No people 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 6 million sequences 600 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

30. 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