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All the basics you need to know about metagenomics.

Published in: Science
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  1. 1. By: Jitesh Jalthuria MBT (Prev) Roll No.1299
  3. 3. INTRODUCTION Total number of prokaryotic cells on earth 4–6 × 1030 Less than 0.1% are culturable Yet to discover the correct culture conditions for culturing the rest 99.9% Metagenomics presently offers a way to access unculturable microorganisms because it is a culture-independent way to study them. It involves extracting DNA directly from an environmental sample –e.g. seawater, soil, the human gut – and then studying the DNA sample.
  4. 4. Metagenomics “The application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species” - Kevin Chen and Lior Pachter Study of metagenome (genomic content of entire microbial community), genetic material recovered directly from environmental samples. Also referred as Environmental genomics, Ecogenomics, or community genomics. The term "metagenomics" was first used by Jo Handelsmann, Jon Clardy, Robert M. Goodman, and others, and first appeared in publication in 1998.
  5. 5. METAGENOMICS AND SYMBIOSIS Many microorganisms with symbiotic relationships with their hosts are difficult to culture away from the host are prime candidates for metagenomics. E.g. the Aphid and Buchnera, ◦ First example of genomics on an uncultured microorganism. ◦ lost almost 2000 genes since it entered the symbiotic relationship 200–250 million years ago. ◦ It contains only 564 genes and does not conduct many of the life functions. • The deep-sea tube worm, Riftia pachyptila, and a bacterium (Boetius, 2005). o These creatures live in harsh environments near thermal vents 2600m below the ocean surface. o The tube worm provides the bacterium with carbon dioxide, hydrogen sulfide and oxygen, which it accumulates from the seawater. o The bacterium, converts the carbon dioxide to amino acids and sugars needed by the tube worm, using the hydrogen sulfide for energy
  6. 6. Extreme Environments Halophilic environments Glacial Deep sea Desert
  7. 7. METAGENOME OF EXTREME HABITATS Metagenomic analyses of seawater revealed some interesting aspects of ocean-dwelling microorganisms. More than one million genes were sequenced and deposited in the public databases. Groups of bacteria that were not previously known to transduce light energy appear to contain genes for such a function e.g. Rhodopsin. Metagenomic analysis of the biofilm led to the computer-based reconstruction of the genomes of some of the community members. A model for the cycling of carbon, nitrogen and metals in the acid mine drainage environment was developed.
  8. 8. Metagenomics • Scope of diversity: Sargasso Sea – Oligotrophic environment – More diverse than expected • Sequenced 1x109 bases • Found 1.2 million new genes • 794,061 open reading frames with no known function • 69,718 open reading frames for energy transduction – 782 rhodopsin-like photoreceptors • 1412 rRNA genes, 148 previously unknown phylotypes (97% similarity cut off) – α- and γ- Proteobacteria dominant groups Venter, J.C. 2004. Science 304:66
  9. 9. METHODOLOGY • rRNA: –“Evolutionary Chronometer:” Very slow mutation rate. –Universal and functionally similar –16S rRNA sequences used. • Data Collection Methods: –Initially, direct sequencing of RNA and sequencing reverse transcription generated DNA. –Progressed to PCR Data Storage: - Metagenomic Library – 2 Approaches • Function-Driven: Focuses on activity of target protein and clones that express a given trait. • Sequence-Driven: Relies on conserved DNA to design PCR primers and hybridization probes; gives functional information about the organism.
  11. 11. TWO APPROACHES FOR METAGENOMICS In the first approach, known as ‘sequence-driven metagenomics’, DNA from the environment of interest is sequenced and subjected to computational analysis. The metagenomic sequences are compared to sequences deposited in publicly available databases such as GENBANK. The genes are then collected into groups of similar predicted function, and the distribution of various functions and types of proteins that conduct those functions can be assessed. In the second approach, ‘function-driven metagenomics’, the DNA extracted from the environment is also captured and stored in a surrogate host, but instead of sequencing it, scientists screen the captured fragments of DNA, or ‘clones’, for a certain function. The function must be absent in the surrogate host so that acquisition of the function can be attributed to the metagenomic DNA.
  12. 12. LIMITATIONS OF TWO APPROACHES The sequence driven approach ◦ limited existing knowledge: if a metagenomic gene does not look like a gene of known function deposited in the databases, then little can be learned about the gene or its product from sequence alone. The function driven approach ◦ most genes from organisms in wild communities cannot be expressed easily by a given surrogate host Therefore, the two approaches are complementary and should be pursued in parallel.
  13. 13. GENERAL METHODOLOGY • Nucleic acid extraction and enrichment technologies • Genome and gene enrichment • Metagenomic libraries • Transcriptome libraries • Metagenome sequencing
  14. 14. TECHNIQUE • Nucleic Acid Extraction: o Cell Extraction and Direct Lysis - Cell lysis (chemical, enzymatic or mechanical) followed by removal of cell fragments and nucleic acid precipitation and purification. • Genome enrichment: o Sample enrichment enhances the screening of metagenomic libraries for a particular gene of interest, the proportion of which is generally smaller than the total nucleic acid content. o Stable isotope probing (SIP) and 5-Bromo-2-deoxyuridine labeling of DNA or RNA, followed by density-gradient centrifugal separation. o Suppressive subtractive hybridization (SSH) o Phage display o DNA microarray
  15. 15. Gene Targeting: • PCR is used to probe genomes for specific metabolic or biodegradative capabilities •Primer design based on known sequence information •Amplification limited mainly to gene fragments rather than full-length genes, requiring additional procedures to attain the full-length genes •RT-PCR has been used to recover genes from environmental samples since RNA is a more sensitive biomarker than DNA Metagenome sequencing: • Complete metagenome sequencing using large fragments of genomic DNA from uncultured microorganisms. • The objectives have been to sequence and identify the thousands of viral and prokaryotic genomes as well as lower eukaryotic species present in small environmental samples such as a gram of soil or liter of seawater.
  16. 16. Shotgun Sequencing
  17. 17. Metagenomics and Applications Successful products • Antibiotics • Antibiotic resistance pathways • Anti-cancer drugs • Degradation pathways - Lipases, amylases, nucleases, hemolytic • Transport proteins
  18. 18. LIMITATIONS • Too much data? • Most genes are not identifiable • Contamination, chimeric clone sequences • Extraction problems • Requires proteomics or expression studies to demonstrate phenotypic characteristics • Need a standard method for annotating genomes • Requires high throughput instrumentation – not readily available to most institutions
  19. 19. FUTURE OF METAGENOMICS • To identify new enzymes & antibiotics • To assess the effects of age, diet, and pathologic states (e.g., inflammatory bowel diseases, obesity, and cancer) on the distal gut micro biome of humans living in different environments • Study of more exotic habitats • Study antibiotic resistance in soil microbes • Improved bioinformatics will quicken analysis for library profiling • Discoveries such as phylogenic tags (rRNA genes, etc) will give momentum to the growing field • Learning novel pathways will lead to knowledge about the current nonculturable bacteria to then culture these systems.
  20. 20. Thank You 