Environmental Genome Shotgun Sequencing of the Sargasso Sea

1. Environmental Genome Shotgun Sequencing of the Sargasso Sea J. Craig Venter,1 * Karin Remington,1 John F. Heidelberg,3 Aaron L. Halpern,2 Doug Rusch,2 Jonathan A. Eisen,3 Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3 Derrick E. Fouts,3 Samuel Levy,2 Anthony H. Knap,6 Michael W. Lomas,6 Ken Nealson,5 Owen White,3 Jeremy Peterson,3 Jeff Hoffman,1 Rachel Parsons,6 Holly Baden-Tillson,1 Cynthia Pfannkoch, Yu-Hui Rogers,4 Hamilton O. Smith1 Therese Pham D145 Presentation January 29, 2019

2. Why the Sargasso Sea? • This is specifically the northwest Sargasso Sea at Bermuda Atlantic Time-series Study (BATS) site • One of the best studied regions of the global ocean • Has been intensively studied as part of a 50-year time series study on ocean physics and biogeochemistry • These circumstances creates the opportunity for an interpretation of genomic data from an oceanic environment

3. Environmental and Microbial characteristics of the Sargasso Sea • Nutrient poor, open ocean environment • Bounded in the north and west by the Gulf Stream • This keeps the nutrient poor waters of the Sargasso Sea from the more nutrient rich waters of the U.S. continental shelf • In the winter cold fronts travel across the region eroding the seasonal thermoclines and causing convective mixing resulting in subtropical mode waters 150-300 m in depth, with a more nutrient rich profile at the surface level • Causes phytoplankton blooms with Synechococcus and Prochlorococcus contributing the most numerically to overall photosynthetic biomass

4. Collection of samples • 170- 200 liters of surface water was collected from each of three sites off the Bermuda coast in February 2003 via the RV Waterbird II • Later, additional samples were collected in May 2003 with the SV Socerer II Fig. 1

5. DNA extraction • Genomic DNA was extracted from filtering sea water through a series of 0.1- 3.0 micron filters • The filters were then quartered and incubated in TE buffer containing lysozyme • sodium dodecyl sulfate (SDS) was added and the samples were then put through three freeze/thaw cycles • This lysate was then treated with Proteinase K to purify it of proteins

6. Whole Genome Shotgun Sequencing (WGS) • First DNA was randomly sheared • Then it was blunted with consecutive BAL31 nuclease and T4 DNA polymerase treatments • Followed by size-selected by electrophoresis on 1% low-melting-point agarose • It was then purified by 3 rounds of gel electrophoresis to remove excess adapters • 2-6 kB inserts were inserted into Bst XI-linearized plasmid vector and cloned Contigs

7. WGS cont’d • These prepared plasmid clones were then sequenced on ABI 3730XL DNA sequencers which are 96-capillary sequencers • The process involves incorporating fluorescent ddNTPs • DNA is then separated by size through thin capillaries • prepared plasmid clones were sequenced from both ends to provide paired-end reads • These sequences were then assembled by the Celera Assembler (computational assembly)

8. Figure 5A

9. Figures 5B

10. Results of the sequencing and assembly • There were 1.66 million reads produced from WGS of the Weatherbird II samples (1.36 Gbp) • And an additional 325,561 sequences generated from the Socerer II samples (265 Mbp) • Assembly of the Weatherbird II sequences generated 64,398 scaffolds (826 bp- 2.1 Mbp) and 215,015 unassembled pair-ends, termed mini-scaffolds. There were also 215,038 unassembled singleton reads. • The Sorcerer II provided almost no assembly; only 153,458 mini-scaffolds and 18,692 singleton reads

11. Analysis of mapped scaffolds; photobiology • The sequenced and assembled DNA assemblies were compared to published sequences • Notable species identified include Shewanella, a group of scaffolds that were similar to Burkholderia, and of course Prochlorococcus which had been established as abundantly present • There was, however, a lack of scaffolds relating to Synechococcus within the pooled data but this is likely due to Synechococcus being larger and less distributed within the waters sampled.

12. Prochlorococcus

13. Figure 3

14. Bacteriorhodopsin; a closer look • Bacteriorhodopsin allows for the coupling of light energy harvesting and carbon cycling in the ocean through a non–chlorophyll-based pathway • Environmental culture-independent gene surveys with PCR have revealed about 67 additional closely related proteorhodopsin homologs • More than 650 rhodopsin homologs were identified within the Weatherbird II samples and an additional 132 were identified in the Sorcerer II samples

15. Phylogenetic tree of rhodopsinlike genes

16. Table 1

17. Analysis of mapped scaffolds; plasmids • Megaplasmids are extrachromosomal genetic elements in the size range of 100 kbp and larger. • evidence for supposed six plasmids larger than 100 kbp in length, two plasmids 70 to 80 kbp, and two plasmids under 10 kbp was found • UmuCD DNA is a damage induced DNA polymerase of Escherchia coli and these genes could play a role in ultraviolet (UV) resistance

18. Analysis of mapped scaffolds; Bacteriophages • only double-stranded DNA bacteriophages were observable • 71 scaffolds greater than 10 kb in length containing identifiable clusters of phage genes • Burkholderia- and Shewanella-associated scaffolds accounting for ~33% of these

19. PCR based methods vs. WGS • PCR based methods have two major limitations: • They under sample the total number of genotypes • they access only a very small subsample of the genomes • WGS provides greater depth of knowledge on cell function • However there are also cons • There are gaps due to limitations in sequencing resolution • The size of the genome that needs to be assemble proves a sizable roadblock as well

20. Figure 6

21. Critiques of the study • “Shotgun Sequencing in the Sea: A Blast from the Past?” by Paul G. Falkowski and Colomban de Vargas offerssome critiques ofthestudy • Despite the large-scale sequencing effort put forth by Venter et al. they were still only able to reconstruct only two, almost-complete genomes, even with the help of fully sequenced templates available in the microbial genome database • What about sequencing larger eukaryotic microbes such as diatoms and dinoflagellates • PCR based methods + 18s rDNA analysis provided greater number of new and divergent phylotypes • Many labs at the time were not able to access the same technologies Venter et al. were

22. A good additional figure to consider

23. Additional Reading • “The sequence of the human genome” • Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., ... & Gocayne, J. D. (2001). The sequence of the human genome. science, 291(5507), 1304-1351. • “Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea” • Parsons, R. J., Breitbart, M., Lomas, M. W., & Carlson, C. A. (2012). Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea. The ISME journal, 6(2), 273. • “The Genome Warrior” • Preston, R. (2000, June). The Genome Warrior. The New Yorker, pp.66.

24. Bibliography • Falkowski, P. G., & de Vargas, C. (2004). Shotgun sequencing in the sea: a blast from the past?. Science, 304(5667), 58-60. • Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A., ... & Fouts, D. E. (2004). Environmental genome shotgun sequencing of the Sargasso Sea. science, 304(5667), 66-74.