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Presented by: Van Doan and Marie Gomez

Presented by: Van Doan and Marie Gomez. Craig Venter on the Sorcerer II. Introduction/Background. Venter used whole-genome shotgun sequencing to identify microbes in the Sargasso Sea Goal: test usefulness in metagenomics.

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Presented by: Van Doan and Marie Gomez

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  1. Presented by: Van Doan and Marie Gomez Craig Venter on the Sorcerer II

  2. Introduction/Background • Venter used whole-genome shotgun sequencing to identify microbes in the Sargasso Sea • Goal: test usefulness in metagenomics • The Sargasso Sea was initially thought to contain a relatively small number of species because of low nutrient levels

  3. Why Do We Care About Microbes? • Microorganisms are the “unseen majority” that shape our environment • Can reconstruct the evolution and consequences of microbial metabolic pathways • Problem? • Hard to study unless we culture them • 99% of prokaryotes unculturable • Previous approaches • PCR-based rRNA studies with 16S rRNA • “Universal” primer biased

  4. Whole-Genome Shotgun Sequencing • Traditionally used to sequence the genome of one organism at a time • Can capture sequences from many different organisms simultaneously • Analogous to assembling thousands of individual jigsaws from a single box with millions of pieces • New genomic approach to gene and species discovery and overall environmental characterization

  5. The Big Picture • Bypass the need for culturing to isolate microbe of interest • Extract data from microbes as they exist in nature • Shotgun sequencing used to sequence the genome of organisms we didn’t even know existed

  6. Methods

  7. Celera Assembler • Starting material in varying abundance • Dominant organisms overrepresented • Less abundant organisms less sequenced • Only a single-genome assembler • Treated high-depth contigs associated with abundant species as repetitive sequence • So, the most abundant organisms in samples would be poorly assembled • Solution? Manual curation of the resulting scaffolds • Separate well-sampled genomic regions from those barely sampled

  8. Genomes and Large Assemblies • Characterize scaffolds with 3X depth coverage • Assembly pieces sorted into organism “bins” • Depth of coverage • Oligonucleotide frequencies • Similarity to previously sequenced genomes • BLAST searches against GenBankdatabase • Basic Logic Algorithm Search Tool

  9. Prochlorococcus-Related Scaffold 2223290 • Green: environmental fragments • Yellow: stages of assembly of fragments into contigs • Blue: resulting contigs

  10. Comparison of Scaffold Sets to Completed Prochlorococus MED4 Genome • Outer circle: completed genomic sequence • Inner circles: fragments from environmental sequencing • Colors represent the position of genes along the chromosome

  11. Evidence-Based Gene Finding • Functions can be predicted from sequences • Approach: • Develop an evidence-based gene finder that can determine the most likely coding frame based on protein alignments • Approximate the start and stop positions from the bounding coordinates of the alignments Breakdown of predicted genes by category

  12. Phylogenetic Diversity

  13. Additional Sequences in Sargasso Sea Data? • Rhodopsins found outside groups of proteobacteria • 782 new rhodopsin-like photoreceptors Phylogenetic tree using sequences of rhodopsin-like genes

  14. Surprising Findings • Burkholderia • Usually found in only terrestrial samples • Shewanella • Usually found in nutrient-rich environments • Identification of an archaeal scaffold that contained an ammonium monooxygenase • Many genes related to phosphorus uptake

  15. Overall Results • 1800 species of microbes • ~150 new species of bacteria • Over 1 billion base pairs of non-redundant sequence • Over 1.2 million new genes Shewanellaoneidensis Prochlorococcus

  16. Limitations • Low sequence coverage of less abundant organisms • Only able to reconstruct two almost complete genomes despite the large amount of sequences • Heterogeneity of sequences made it hard to separate genomes

  17. Summary/Conclusion • Large-scale sequencing can provide insight into microbial diversity at high resolution • There is a diverse amount of prokaryotic life in the Sargasso Sea (~2000 species) • Discovery of new species, proteins, and metabolic strategies • Shotgun sequencing more powerful than conventional PCR-based rRNA studies at assessing phylogenetic diversity of a sample

  18. Take Home Message • Whole-genome shotgun sequencing of the Sargasso Sea opened up the possibility of sequencing the genome of unknown organisms, allowing us to discover more about the living systems in our environment

  19. Further Reading • The Sargasso Sea was a pilot sampling project (2003) for the Global Ocean Sampling Expedition (GOS) • Rusch, DB, et al. (13 March 2007). "The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific". PLoSBiology • Yooseph, S, et al. (13 March 2007). "The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families". PLoS Biology • Williamson, SJ, et al. (23 January 2008). "The Sorcerer II Global Ocean Sampling Expedition: Metagenomic Characterization of Viruses within Aquatic Microbial Samples". In Hall, Neil. PLoSONE

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