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Assembling the Marine Metagenome, One Cell at a Time

By Tanja Woyke, Gary Xie, Alex Copeland, José M. González, Cliff Han, Hajnalka Kiss, Jimmy H. Saw, Pavel Senin, Chi Yang, Sourav Chatterji, Jan-Fang Cheng, Jonathan A. Eisen, Michael E. Sieracki and Ramunas Stepanauskas


The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa

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    1. (1991). 16S/23S rRNA sequencing.
    2. (2002). A memory-efficient dynamic programming algorithm for optimal alignment of a sequence to an RNA secondary structure.
    3. (1998). Base-calling of automated sequencer traces using Phred. II. Error probabilities.
    4. (1990). Basic local alignment search tool.
    5. (2005). Closing bacterial genomic sequence gaps with adaptor-PCR.
    6. (2004). Community structure and metabolism through reconstruction of microbial genomes from the environment.
    7. (2005). Comparative metagenomics of microbial communities.
    8. (2008). CompostBin: A DNA composition-based algorithm for binning environmental shotgun reads.
    9. (1998). Consed: A graphical tool for sequence finishing.
    10. (1999). CRITICA: Coding region identification tool invoking comparative analysis.
    11. (2002). Cultivating the uncultured.
    12. (2002). Cultivation of the ubiquitous SAR11 marine bacterioplankton clade.
    13. (2007). Dissecting biological ‘‘dark matter’’ with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.
    14. (2007). DNA-DNA hybridization values and their relationship to whole-genome sequence similarities.
    15. (2003). Drugs from the deep: Marine natural products as drug candidates.
    16. (2004). Environmental genome shotgun sequencing of the Sargasso Sea.
    17. (2002). Fast algorithms for largescale genome alignment and comparison.
    18. (2008). Genome analysis of the proteorhodopsin-containing marine bacterium Polaribacter sp.
    19. (2005). Genome sequencing in microfabricated high-density picolitre reactors.
    20. (2005). Genome streamlining in a cosmopolitan oceanic bacterium.
    21. (2006). Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum.
    22. (2001). Global analysis of the general stress response of Bacillus subtilis.
    23. (1999). Improved microbial gene identification with GLIMMER.
    24. (2004). Improved prediction of signal peptides: SignalP 3.0.
    25. (2007). Improvements of high-throughput culturing yielded novel SAR11 strains and other abundant marine bacteria from the Oregon coast and the Bermuda Atlantic Time Series study site.
    26. (2006). ISfinder: the reference centre for bacterial insertion sequences.
    27. (2007). Light stimulates growth of proteorhodopsin-containing marine Flavobacteria.
    28. (2007). Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time.
    29. (2007). Mechanism of chimera formation during the Multiple Displacement Amplification reaction.
    30. (2007). MEGAN analysis of metagenomic data.
    31. (2006). MetaCyc: a multiorganism database of metabolic pathways and enzymes.
    32. (1999). Molecular evidence for zooplankton-associated nitrogen-fixing anaerobes based on amplification of the nifH gene.
    33. (2007). Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
    34. (2006). Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis.
    35. (2008). Photochemical production of molecular hydrogen in lake water and coastal seawater.
    36. (2001). Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes.
    37. (2005). Prediction of twin-arginine signal peptides.
    38. (2002). Principal component analysis.
    39. (2001). Proteorhodopsin phototrophy in the ocean.
    40. (2005). PSORTb v.2.0: Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis.
    41. (2007). Resourceful heterotrophs make the most of light in the coastal ocean.
    42. (2006). Sequencing genomes from single cells by polymerase cloning.
    43. (2000). Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii.
    44. (2006). Symbiosis insights through metagenomic analysis of a microbial consortium.
    45. (1986). The Analysis of Natural Microbial-Populations by Ribosomal-RNA Sequences.
    46. (2007). The Human Microbiome Project.
    47. (2006). The integrated microbial genomes (IMG) system.
    48. (2008). The Microbial Engines that Drive Earth’s Biogeochemical Cycles.
    49. (2007). The SAR92 clade: an abundant coastal clade of culturable marine bacteria possessing proteorhodopsin.
    50. (2007). The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific.
    51. (1997). tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence.
    52. (2004). Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa.
    53. (2004). Versatile and open software for comparing large genomes.
    54. (2008). Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems.

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