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Metagenomic
- 3. INTRODUCTION 0 Total number of prokaryotic cells on earth 4–6 × 1030 0 Less than 0.1% are culturable 0 Metagenomics presently offers a way to access unculturable microorganisms because it is a culture-independent way to study them. 0 It involves extracting DNA directly from an environmental sample –e.g. seawater, soil, the human gut – and then studying the DNA sample.
- 5. Etymology 0 The term "metagenomics" was first used by Jo Handelsman, Jon Clardy, Robert M. Goodman, Sean F. Brady, and others, and first appeared in publication in 1998. Metagenome referes to the idea, that a collection of genes sequenced from the environment could be analyzed in way analogous to the study of a single genome .
- 6. Metagenomics 0 Metagenomics ( Environmental Genomics or Community Genomics) is the study of genomes recovered from environmental samples without the need for culturing them . 0 Metagenomics processes data using bioinformatics tools. “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
- 7. Why is it revolutionnary? Classical microbiology 1 colony 1 analysis 1 bacterial identification 20 colonies 20 analyses 20 bacterial identifications Time consuming Laborious expensive • If you want to identify one colony, you need to isolate and send this colony for sequencing • If you want to identify more colonies, you have to repeat operations for each single colony
- 8. Why is it revolutionnary? Bacterial diversity profile Metagenomics Over 5.000 identifications amongst the most important populations 1 analysis • If you want to identify one colony, you need to isolate and send this colony for sequencing • If you want to identify more colonies, you have to repeat operations for each single colony
- 11. Why Do METAGENOMICS? Understanding Metabolism Defining the Minimal Gene Set Genome Engineering Understanding Cell Structure & Function Understanding Host Interactions Understanding Protein-Protein Interactions Understanding Expression (RNA/Protein) Discover DNA Variation, Genotyping Forensics Drug/Vaccine Development John H has a family To support The J. Craig Venter InstituteTIGR: The Institute for Genomic Research
- 12. Sampling and nucleic acids extraction: 0 Sampling & nucleic acids extraction Soil is a particularly complex matrix containing many substances, such as humic acids, which can be co-extracted during DNA isolation. 0 Sephadex G-200 spin columns have proven to be one of the best ways to remove contaminants from soil DNA. Recently, a pulse field electrophoresis procedure using a two-phase agarose gel, with one phase containing polyvinylpyrrolidone (PVPP), was developed for removal of humics.
- 13. There are two types of extraction techniques: 0(1) direct, in situ, extraction where the cells are lysed in the soil sample and then the DNA is recovered; and 0 (2) indirect extraction techniques, where the cells are removed from the soil and then lysed for DNA recovery.
- 14. Construction of a metagenomic library: 0 The classical approach includes the construction of small insert libraries (<10 kb) in a standard sequencing vector and in Escherichia coli as a host . 0 However, small insert libraries do not allow detection of large gene clusters or operons. To circumvent this limitation researchers have been employing large insert libraries, such as: 0 cosmid DNA libraries with insert sizes ranging from 25- 35 kb 0 fosmid with inserts of 40 kb 0 (BAC) libraries with insert up to 200 Kb.
- 16. 0 E. coli is still the preferred host for the cloning and expression of any metagenome-derived genes and only very recently have other hosts such as Streptomyces lividans been employed to identify genes involved in the biosynthesis of novel antibiotics . 0 Metagenomic libraries are also being developed in other Gram-negative hosts by several laboratories, and these will become available soon.
- 17. Analysis of metagenomic libraries 0 The function-driven analysis 0 The sequence-driven analysis
- 18. 0 1)identification of clones that express a desired trait 0 2)characterization of the active clones by sequence and biochemical analysis . 0 3)analysis enables identification of new enzymes, antibiotics or other reagents in libraries from diverse environments. 0 4)all gene required for function in one clone & expression in host cell The function-driven analysis
- 19. The sequence-driven analysis 0 use of conserved DNA sequences to design hybridization probes or PCR primers to screen metagenomic libraries for clones that contain sequences of interest. 0 Sequencing of clones carrying phylogenetic anchors, such as the 16S rRNA gene and the Archaeal DNA repair gene radA has led to functional information about the organisms from which these clones were derived.
- 21. LIMITATIONS OF TWO APPROACHES 0 The sequence driven approach: 0 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. 0 The function driven approach : 0 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.
- 22. Enrichment for specialized DNA from enviromental sample 0 One of the sustained frustrations with analysis of metagenomic libraries is the low frequency of clones of a desired nature. To increase the proportion of active clones in a library, several strategies have been designed to enrich for the sequences of interest before cloning. BrdU enrichment stable-isotope probe enrichment
- 24. METAGENOMICS AND SYMBIOSIS 0 Many microorganisms with symbiotic relationships with their hosts are difficult to culture away from the host are prime candidates for metagenomics. 0 E.g. the Aphid and Buchnera, 0 First example of genomics on an uncultured microorganism. 0 lost almost 2000 genes since it entered the symbiotic relationship 200–250 million years ago. 0 It contains only 564 genes and does not conduct many of the life functions.
- 25. • The deep-sea tube worm, Riftia pachyptila, and a bacterium. 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
- 27. Environmental Shotgun Sequencing (ESS). (A) Sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds.
- 28. Application
- 29. Application of soil metagenomics 0 soil a rich source of novel and useful biomolecules. Some examples of application of soil metagenomics are: 0 Antibiotics and pharmaceuticals: A clone found in a soil metagenomic library produces deoxyviolacein and the broad spectrum antibiotic violacein. + nine aminoglycoside and+ tetracycline antibiotics resistance genes from soil . 0 Oxidoreductases/dehydrogenases: Alcohol oxidoreductases are useful biocatalysts in industrial production of hydroxy acids, amino acids and alcohols. 0 Amidases: Amidases are used in the biosynthesis of β-lactam antibiotics .
- 30. 0 Polysaccharide degrading/modifying enzymes/ amylolytic genes 0 Cellulases have numerous applications and biotechnological potential for various industries including chemicals, fuel, food, brewery and wine, animal feed, textile and laundry,pulp and paper and agriculture. 0 Agarases, the enzymes that can liquify agar, have been identified during the screening a soil metagenomic library. 0 Vitamin biosynthesis: 0 Soil metagenomics has been applied to the search for novel genes encoding the synthesis of vitamins such as biotin 0 Lipolytic genes 0 esterases and lipases.
- 31. LIMITATIONS 0 Most genes are not identifiable 0 Contamination, chimeric clone sequences 0 Extraction problems 0 Requires proteomics 0Need a standard method for annotating genomes 0 Requires high throughput instrumentation – not readily available to most institutions
- 32. 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 microbiome 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)
- 35. conclusion 0 Metagenomics is a young and exciting technique that has broad application in biology and biotechnology. 0 Although many advances in, library construction, vector design, and screening will improve it, 0 the current technology is sufficiently powerful to yield products for solving real world problems, including the discovery of new antibiotics and enzymes. 0 Approaches that enrich for a portion of the microbial community or for a collection of metagenomic clones will enhance the power of metagenomic analysis.
Notes de l'éditeur
- If you want to study microorganisms in food samples with previous techniques, you need to use classical microbiology and other molecular techniques. But these methods takes time, are laborious and expensive. Moreover you have identified only a small part of the microorganisms because - you have only the culturable bacteria - and the number of identification is limited
- With metagenomics, you identify several thousand microorganisms in one single analysis. Moreover you also have the quantification of all microorganisms : the culturable and non culturable. With this technique you have a clear picture of the flora inside your food sample.
- This is a particular problem when the DNA is isolated from soil or other environments that contain high concentrations of contaminants that inhibit cloning. Methods to improve the preparation of large fragments of DNA that are clean enough to clone are being pursued vigorously in many laboratories. Removal of humic acids is essential before the DNA can be processed further. For this purpose, a range of DNA purification techniques has been developed. \Specifically, soil microbial communities are composed of a mixture of archaea, bacteria and protists displaying a diversity of cell wall characteristics and varying in their susceptibility to lysis . various kits are commercially available for DNA isolation from environmental samples, many laboratories have developed their own methods with the aim of optimising extraction and reducing bias caused by unequal lysis of different members of the soil microbial community.
- . DNA isolation and purification is followed by the Construction of DNA libraries in suitable cloning vectors and host strains. E. coli is still the preferred host for the cloning and expression of any metagenome-derived genes and only very recently have other hosts such as Streptomyces lividans been employed to identify genes involved in the biosynthesis of novel antibiotics. Metagenomic libraries are also being developed in other Gram-negative hosts by several laboratories, and these will become available soon Recovery of DNA sequences longer than a few thousand base pairs from environmental samples was very difficult until recent advances in molecular biological techniques allowed the construction of libraries in bacterial artificial chromosomes (BACs), which provided better vectors for molecular cloning. The current metagenomic studies have largely progressed due to the construction of efficient gene cloning vectors like bacterial artificial chromosomes (BACs) or cosmids, which allow cloning and expression of larger and complex DNA segments or genes and the development of methods for generation and analysis of the data
- A ‘functional-anchor approach’ involves identifying all of the clones that express a certain function and sequencing them completely to determine the diversity of genomic environments from which that function originates.
- In what is arguably the most dramatic discovery from metagenomics to date, sequencing of a clone isolated from seawater that was initially identified because it carried a bacterial 16S rRNA gene revealed a gene with high similarity to bacteriorhodopsin genes. This result provided the first indication that rhodopsins are not limited to the Archaea, as previously thought Subsequent heterologous expression of the bacteriorhodopsin gene in E. coli produced a functional biochemical characterization of the protein, completing the full spectrum of studies that link phylogeny to function .
- The limitations of the approach are that it requires expression of the function of interest in the host cell and clustering of all of the genes required for the function. It also depends on the availability of an assay for the function of interest that can be performed efficiently on vast libraries, because the frequency of active clones is quite low. Many approaches are being developed to mitigate these limitations. Improved systems for heterologous gene expression are being developed with shuttle vectors that facilitate screening of the metagenomic DNA in diverse host species and with modifications of Escherichia coli to expand the range of gene expression.
- Many microorganisms are able to degrade waste products, make new drugs for medical applications, produce environmentally friendly plastics, or even make some of the food we eat (figure 1). By isolating the DNA from these organisms, it provides us with the opportunity to optimize these processes and adapt them for use by society. Another valuable application of metagenomics is that it provides the capacity to effectively characterize the genetic diversity present water, soil and rumen source samples regardless of the availability of laboratory culturing techniques. Metagenomics is a new and exciting field of molecular biology that is likely to grow into a standard technique for understanding biological diversity
- Developed for characterization of metagenomic shotgun reads LCA assignment based on BLAST bitscore Support for paired-end reads and comparison of datasets. Latest version can analyze RDP files / QIIME OTU files Analysis of metabolism via SEED, KEGG or COG maps Comparison of multiple metagenomes (> 2) Easy to work with on a desktop / laptop computer: Extra things needed: Java, a BLAST server MEGAN gives a visualization of BLAST results Study diversity Compare samples Contamination filtering Special gene of interest Extraction of sequences based on taxonomic /metabolic information.