Environmental Genome Shotgun Sequencing of the Sargasso Sea Venter et. al (2004) Presented by Ken Vittayarukskul Steven S. White.

Context of the Problem Evolutionary history is directly tied to microbial genetics. Little is known Until recently, microbial diversity was measured by PCR amplification and sequencing of only ribosomal genes 20 major phyla identified in Bacteria and Archaea through this approach 2 critical limitations: undersampling of total genes undersampling of rest of genome Larger microbes can’t be cultured ex situ and thus are ignored

Objective of Venter et. al: What they did Sought to obtain a more representative study of the “gene content, genetic diversity, and relative abundance of the microbial population found in the oceans." How did they go about doing this? Sargasso Sea, four sites (RV Weatherbird II, and SV Sorcerer II) Extracted DNA from 0.1 to 3.0 um-filtered seawater Applied whole-genome shot sequencing to the DNA samples for the above purpose

Why the Sargasso Sea? Inhospitable for most forms of life -weak currents and light winds block nutrient-rich water -high salinity Eddies pump nutrient-rich water from ocean floor that sustains rich microbial population from oceanservice.noaa.gov

Whole-Genome Shotgun Sequencing from MMG Genetics and Genomics Wiki

Bias in Sequencing High Volume of Genomes Computational assembly algorithms set based on depth of coverage, in turn based on variation in genome size and relative abundance of specific genomes Less abundant species expected to have genomes of only few sequences Thus, when setting coverage depth to identify unique regions for backbone assembly of all these genomes, more abundant genomes would be labeled as repetitive More abundant genomes are assembled more poorly from Genomenewsnetwork.org Alleviated by manual assembly of large, nonrepetitive contigs. Expected coverage based on these assemblies

Figure 1 Satellite image of ocean chlorophyll around their collection site. Station 3 experienced elevated chlorophyll levels relative to 1&11&13

Figure 2 Scaffold sets representing a conglomerate of Prochlorococcus strains Prochlorococcus MED4 genome (outer ring) Chromosome map depicting conserved gene order Color = Position. Red = Start ; Blue = End ; Black = Non-conserved gene. Mismatching colors/positions likely the result of chromosomal rearrangements.

Figure 3 uncultured marine archaeon Comparing Scaffolds to Crenarchael clone 4B7. Predicted 4B7 proteins & scaffolds show significant homology, and arrayed in positional order. BLASTp matches scored at least 25% similarity. Lines delineate scaffold borders.

Figure 4 Megaplasmids Circular diagrams of 9 complete megaplasmids.  Depths ranging from 4 to 36  Inner circles = reverse coding genes  Genes colored according to category [Table 1]

Table 1 Breakdown of predicted genes by category. 28,023 genes sorted into multiple categories. 1,214,207 genes vs 137,885 sequence currently archived. Additional hypothetical genes ID’d via conserved open reading frames. 69,901 novel genes identified.

Figure 5A Prochlorococcus-related scaffold Sample of multiple sequence alignment Blue = Contigs Green = fragments Yellow = Assembly stages used to create contigs. Collapsed several fragments to form the final contig.

Figure 5B Global structure of previously mentioned scaffold, with respect to assembly.

Figure 6 Depiction of the phylogenetic diversity observed.  Phylogenetic markers: 16S rRNA, EF-G, EF-Tu, HSP70, RecA, RpoB and 16S rRNA. Identified via HMM and BLAST library comparison/search

Figure 7 Detection of Proteorhodopsin/homologs.  Detection labeled according to where samples were gathered. Codes for light-driven proton pump.  Allows for light-cycling WITHOUT chlorophyll.

Shortcomings in Research Methodology Their BAC libraries were created in bacteria from either terrestrial, or nutrient- rich ocean environments. Sampling sites may have biased their estimates of biodiversity Despite their effort, they only compiled two nearly complete genomes. With the help of fully sequenced templates from a microbe database All of this work was done on relatively small organisms Employing this method on more complex organisms may entail work orders of magnitude.

Further reading Falkowski, P. G., Vargas, C. Shotgun Sequencing in the Sea: A Blast from the Past? Science. 304, (2004). Selvaraj S., Dixon J. R., Bansal, V., Ren B. (2013). Whole-genome haplotype reconstruction using proximity ligation and shotgun sequencing. Nature biotechnology. 31, Ruder, K. (2004). Exploring the Sargasso Sea. Retrieved from

End Summary: The Take Away Whole-genome shotgun sequencing methodology used on genomes of microbial populations in Sargasso Sea Gene content, genetic diversity, and relative concentration of species elucidated from billion bp of nonredundant DNA sequences 1800 known microbial species identified, 148 new microbial phylotypes detected 1.2 million new genes identified, including more than 782 new rhodopsin-like photoreceptors “Tip of the iceberg”- data here alone suggest massive microbial diversity