1. Environmental Genome Shotgun Sequencing of the Sargasso Sea
J. Craig Venter1,*, Karin Remington1, John F. Heidelberg3, Aaron L. Halpern2, Doug Rusch2, Jonathan A. Eisen3, Dongying Wu3, Ian Paulsen3, Karen E. Nelson3, William Nelson3, Derrick E. Fouts3, Samuel Levy2, Anthony H. Knap6, Michael W. Lomas6, Ken Nealson5, Owen White3, Jeremy Peterson3, Jeff Hoffman1, Rachel Parsons6, Holly Baden-Tillson1, Cynthia Pfannkoch1, Yu-Hui Rogers4, Hamilton O. Smith1
Bianca Sanchez Mora, Meghana Munagala
D145: Genomics
January 26, 2017

2. Introduction
Interested in using environmental shotgun sequencing to study difficult to study microbes
To find new solutions for energy problems—ocean may have answers
Looking for unique genes to create alternative energy/ clean up environment
To catalogue poorly understood microorganisms into a database for researchers
To stimulate more research

3. Why the Sargasso Sea? Well Studied Defined boundaries Low Nutrient
Cyanobacteria:
Synechococcus
Prochlorococcus
= Chlorophyll rich

4. Whole Genome Shotgun Sequencing
Created by Frederic Sanger
A method used to sequence whole genomes by randomly fragmenting DNA then reassembling the overlapping fragments
Initially used to identify sequence from one organism
Here it is applied to find a representative sequence from a diverse population at once
Thermocline:

5. WGS Procedure

6. Environmental Sequencing
The sequencing of naturally occurring populations and communities
Circumvents the need for culturing
Overcomes selection/culture bias
Aims to capture full measure of diversity through the sequencing of genomes

7. Comparing Techniques PCR-based Methods
Whole-Genome Shotgun Sequencing
Previous method used to PCR to assess microbial diversity
Limitations:
Undersampling of genotypes
Small subsample of genomes
Pros:
Fast
Able to sequence large genomes
Diverse phylogenetic markers
Challenges:
Assembly of millions of DNA fragments

8. Gene Conservation among Prochlorococus
= start of genome
= end of genome
Completed Genomic Sequence
Environmentally Sequenced Fragments
*similarities between sequences: can work environmentally; able to detect variation
*differences between environmental sequence and known sequence; diversity

9. Sargasso Sea v. Crenarchael clone 4B7 Scaffolds
tBLASTx = Translated Bursts
Purpose is to find distant relationships between nucleotide sequences
Crenarchael clone 4B7 = archea
Sim = similarity
4B7 = exact clone of protein *known
Numbers = predicted proteins
Line = alignment = similarity
Q: what are they comparing
Nothing below 25%, mostly similar
Look up TblastX

10. Megaplastids found in Scaffold Set
More diversity in this set than previously
9 megaplastids
Unable to separate other megaplastid because maybe they diverged too far
Or possibly shorter assemblies
Main point*able to obtain plasmids; and large

11. Prochlorococcus-related Scaffold M
Examined the multiple sequence alignments of contigs
Identified 2 distinct classes
Shows a homogenous blend of haplotypes
= contigs
= fragments
= stages of assembly of fragments into resulting contigs

12. Multiple Sequence Alignment Sample
Prochlorococcus scaffolds show differences = meaning there is a diverse population
A comparison of scaffolds show variations

13. Phylogenetic Markers of Sargasso Sea Sequences
Diversity of Sargasso Sea sequences using multiple phylogenetic markers. Relative contribution of organisms from different major phylogenetic groups (phylotypes)

14. Phylogenetic tree of rhodopsinlike genes
proteorhodopsin: a light driven proton pump discovered in BAC

15. Challenges/Improvements
Problems in method of sample collection
Possible bias in diversity estimation
Difficult to distinguish between the DNA sequence of closely related
Manual Curation
Rare organisms less likely to be sequenced—low sequence coverage
The real limitation was cost

16. Conclusion/Significance
A new use for shotgun sequencing for studying environmental genomes
Microbes were added to an online public data base for research use
1800 species of microbes, 150 new bacteria and over 1.2 million new genes were found
Genes found in samples indicate microbial life in the ocean is more abundant and diverse than expected

17. Additional Reading For another perspective: The following expedition
Falkowski, P.G., de Vargas, C., Science 58, 58 (2004); published online 4 March 2004 ( /science ).
The following expedition
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, et al. (2007) The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol 5(3): e77. doi: /journal.pbio

18. References
Gross L (2007) Untapped Bounty: Sampling the Seas to Survey Microbial Biodiversity. PLoS Biol 5(3): e85. doi: /journal.pbio
Falkowski, P.G., de Vargas, C., Science 58, 58 (2004); published online 4 March 2004 ( /science ).
Tring, S.G., Rubin, E.M., Nature 6, ; published online