All the basics you need to know about metagenomics.

1. By: Jitesh
Jalthuria
MBT (Prev)
Roll No.1299

2. SIGNIFICANCE OF MICROORGANISMS

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. 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. 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. Extreme Environments
Halophilic environments Glacial
Deep sea Desert

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. 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. 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.

10. TWO APPROACHES FOR METAGENOMIC STUDY

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. 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. GENERAL METHODOLOGY
• Nucleic acid extraction and enrichment technologies
• Genome and gene enrichment
• Metagenomic libraries
• Transcriptome libraries
• Metagenome sequencing

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. 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. Shotgun
Sequencing

17. Metagenomics and Applications
Successful products
• Antibiotics
• Antibiotic resistance pathways
• Anti-cancer drugs
• Degradation pathways
- Lipases, amylases, nucleases, hemolytic
• Transport proteins

19. 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

20. 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.

21. Thank You 