Metagenomics: : DNA Sequencing of Environmental Samples

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1 Metagenomics: : DNA Sequencing of Environmental Samples Susannah Green Tringe and Edward M. Rubin Department of Energy Joint Genome Institute

2 What is Metagenomics? Metagenomics is the study of genomes from whole communities rather than individual species recent advances and decreases in cost have allowed biologists to study genomes of organisms that cannot survive on their own, such as symbionts and pathogens This review focuses on methodological advances that have allowed the sequencing of natural populations, examples of these techniques, and possible future directions for research.

3 Methodological Advances Natural samples contain DNA in several forms free DNA, virus particles, cells, etc. These samples can be suspended in water, bound to the soil or other solid particles, or contained within an aggregate of microorganisms (biofilm( biofilm)

4 Methodological Advances Water suspended (aquatic) samples are easier to concentrate than solid- bound particles may be filtered before concentration choose filtration size by type of particles choose filtration based on either eukaryotic, prokaryotic, or viral particles concentrated by normal or tangential flow filtration

5 Methodological Advances Soil-suspended particles often contain enzyme inhibiting substances DNA isolation is either direct (cells lysed within sample material) or indirect (separated before lysing) lysing technique can change what DNA is extracted i.e. gram-positive bacteria are hard to lyse contaminants can be removed with agarose gel electrophoresis or chromatography methods Once DNA is extracted, it can be cloned

6 Example: 16S rrna 99% of microbes are not easily cultured All these microbes have 16S/18S SSU rrna can be used to determine phylogeny Used large-insert clone sequencing Discovery of proteorhodopsin in oceanic bacteria previously thought to be dependent on organic matter instead of light first time a rhodopsin-like protein had been discovered in the microorganism domain Evidence for horizontal gene transfer (exchange of genetic material between two genomes without a parental relationship)

7 Example:Host-Associated Bacteria Treponema pallidum causes syphilis obtained from the testes of rabbits through lysis and centrifugation analysis aided with virulence aspects of bacteria Whipple disease is caused by Tropheryma whipplei analysis revealed reasons for inability to culture and was corrected with amino acid and tissue culture regime Bacteriomes (specialized organs) from Buchnera amphidicola were isolated using dissection, differential lysis,, and pulse-field electrophoresis

8 Example: Paleogenomics By using ancient DNA (as much as 50,000 years), scientists can determine phylogeny of extinct species through genetics rather than solely on morphology with metagenomics,, it doesn t t matter that the DNA is contaminated by microbes 40,000 year old cave bear was studied only 27 kb of genome was sequenced, but enough to compare it to black and brown bears Possibility of looking at Neaderthals

9 Example: Shotgun Sequencing Acid-mine biofilm small number of species (chemilithotrophs( chemilithotrophs) ) can survive in highly acidic and metal filled environment 76.2 million bp from this community 2 near-complete genomes and 3 partial genomes Leptospirillum is the only species in community that can fix nitrogen possibilities for acid mine drainage controls Sargasso Sea-low nutrient level 7 libraries; 1.6 Gb DNA from this study only 3% of this was accounted for 1.2 million genes found similar genes in database less than 1/3 could be assigned to a cellular role most genes couldn t t be assigned to phylogenetic group encode for phosphorus uptake, proteorhodopsin, and many others Nutrient rich environments agricultural soil and whale falls couldn t t complete genomes because of high number of organisms, but identified gene families of importance environmental gene tag (EGT)

10 Future Directions Second human genome project to study the microorganisms in our bodies Current methods of DNA isolation, library construction, sequencing, ng, and analysis will have to be reworked from a metagenomics standpoint techniques have been used to obtain samples from all represented organisms in the community great for overviews of community and determining dominant factors trouble if trying to obtain complete genome sequence Separation techniques have been developed filtration, fractionation based on GC content, differential centrifugation, density gradients, differential lysis,, pulsed-field electrophoresis, and selective use of restriction enzymes Interest in species that perform specific metabolism use of stable isotope probing to isolate organisms flow cytometry (measures fluorescence) isolates organisms by viability, membrane properties, surface protein expression, or SSU rrna sequence affinity purification-uses uses binding of protein, possibly cell wall proteins, on solid surface to separate Isothermal strand displacement is DNA amplification that doesn t t require a large amount of starting material could be used for whole genome studies Library construction with pyrosequencing (detect pyrophosphate) to sequence rather than cloning to eliminate E. coli bias

11 Future Directions Genome assembly is important has been shown that cross species assemblies are uncommon except for highly conserved and rrna sequences effects of heterogeneity on assembly procedures should be studies could there be correlation with growth rate, competition, community stability, etc. Assigning these sequences to phylogenic trees should be done with caution use related species to help with process Biggest advances will occur with more fully sequenced genomes

12 Conclusions & Comments Metagenomics offers information about populations rather than individuals, which is more representative of the natural world Why is there a limit on age for paleogenomics? How many genes are highly conserved and can these ever truly be used in metagenomic studies?

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