Metagenomics is the study of genomes recovered from environmental samples without culturing. It involves extracting DNA from an environmental sample and sequencing the DNA. This allows study of the 99% of microbes that cannot be cultured. Metagenomics has applications in discovering new antibiotics, enzymes, and understanding microbial communities and host interactions. It provides a culture-independent way to access genetic diversity and biotechnological potential from uncultured microorganisms.

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.

4. Extreme Environments
DesertDeep sea
GlacialHalophilic
environments

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.