Metagenomics

newer approach in understanding Microbes

Dr.T.V.Rao MD

4/4/2016

Dr.T.V.Rao MD

Metagenomics is the study of metagenomes, genetic material

recovered directly from environmental samples.
The term "metagenomics" was first used by Jo Handelsman, Jon
Clardy, Robert M. Goodman, and first appeared in publication in
1998.
This relatively new field of genetic research enables studies of
organisms that are not easily cultured in a laboratory as well as
studies of organisms in their natural environment.
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Dr.T.V.Rao MD

The broad field may also be referred to as

environmental genomics, ecogenomics or community
genomics.

The term metagenome referenced the idea that a collection of

enes sequenced from the environment could be analyzed in a
way analogous to the study of a single genome.

The human microbiome project (HMP) will require metagenomic

ample sequencing data as a primary resource for analysis of the
elationship between microbial communities and the individuals
hey inhabit.
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Dr.T.V.Rao MD

Metagenomics
Metagenomics is the study of genetic material
recovered directly from environmental samples. The
broad field may also be referred to as environmental
genomics, ecogenomics or community genomics.
While traditional microbiology and microbial
genome sequencing and genomics rely upon
cultivated clonal cultures, early environmental gene
sequencing cloned specific genes to produce a profile
of diversity in a natural sample.
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Dr.T.V.Rao MD

Introduction
...functional analysis of the collective genomes of soil
microflora, which we term the metagenome of the soil.
- J. Handelsman et al., 1998

PubMed: metagenom*[Title/Abstract]

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Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

How do we get the genomes?

Culturing

Few microorganisms can be easily cultured (<<5%)

Microorganisms needs to be studied in their environment

Single cell genomics

Only routinely performed in specialized labs
Very incomplete genomes (mean 40%, range
10-90%)
https://www.bigelow.org/

Metagenomics
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Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Understanding ecosystems
Metabolites

Meta-bolomics

Proteins

Extraction

mRNA

Meta-proteomics

Meta-transcriptomics

DNA

In Situ methods

Community structure

Microbial functions

Meta-genomics

Microbial needs

P-Removal:
N-Removal:

-Removal:
Foaming:
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Dr.T.V.Rao MD

Ethanol production:

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Cultures missed many facts on Microbes

missing links clarified by PCR Techniques
Such work revealed that the
vast majority of microbial
biodiversity had been missed
by cultivation-based methods.
Recent studies use either
"shotgun" or PCR directed
sequencing to get largely
unbiased samples of all genes
from all the members of the
sampled communities.
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Dr.T.V.Rao MD

What have metagenomics been used for?

Extracting genomes

Garcia Martin et al., 2006 Nat. Biotechnol.

Albertsen et al., 2013 Nat. Biotechnol.

Genome extraction from low

complexity metagenome
Candidatus Accumulibacter
phosphatis
The first genome of a polyphosphate
accumulating organism (PAO) with a
major role en enhanced biological
phosphorus removal
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Genome extraction of low

abundant species (< 0.1%)
from metagenomes
First complete TM7 genome
Access to genomes of the
uncultured majority
Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Metagenomics will reveal the hidden

genome bases
Because of its ability to

reveal the previously hidden

diversity of microscopic life,
metagenomics offers a
powerful lens for viewing
the microbial world that has
the potential to
revolutionize understanding
of the entire living world
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Metagenomics gives clarity on

understanding genome bases

As the price of DNA

sequencing continues
to fall, metagenomics
now allows microbial
ecology to be
investigated at a much
greater scale and
detail than before.

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Introduction

Genome = Parts list of a single genome

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Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

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Introduction

Photo: D. Kunkel; color, E. Latypova

Metagenome = Parts list of the community

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Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

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History of Metagenomics
However, early metagenomic studies revealed
that there are probably large groups of
microorganisms in many environments that
cannot be cultured and thus cannot be
sequenced. These early studies focused on 16S
ribosomal RNA sequences which are relatively
short, often conserved within a species, and
generally different between species
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Learning more of the unknown Metagenomics

made to know the future of science

Many 16S rRNA sequences have been found which do

not belong to any known cultured species, indicating
that there are numerous non-isolated organisms. These
surveys of ribosomal RNA (rRNA) genes taken directly
from the environment revealed that cultivation based
methods find less than 1% of the bacterial and archaeal
species in a sample. Much of the interest in
metagenomics comes from these discoveries that
showed that the vast majority of microorganisms had
previously gone unnoticed.
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Metagenomics approaches the new path to

science

Metagenomics provides
a new way of examining
the microbial world that
not only will transform
modern microbiology
but has the potential to
revolutionize
understanding of the
entire living world

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Approach to Microbiology is community

oriented rather than individual microbes
In metagenomics, the power of
genomic analysis is applied to
entire communities of
microbes, bypassing the need to
isolate and culture individual
bacterial community members.
The new approach and its
attendant technologies will
bring to light the myriad
capabilities of microbial
communities
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Interrelation of Species of Microbes is

better understood with Metagenomics
Metagenomics will
generate knowledge of
microbial interactions so
that they can be
harnessed to improve
human health, food
security, and energy
production
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Metagenomics Studies the interrelated

genomes
Metagenomics combines
the power of genomics,
bioinformatics, and
systems biology.
Operationally, it is novel
in that it involves study
of the genomes of many
organisms
simultaneously.
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A Novel approach to non cultivable

Microbes
It provides new access to the
microbial world; the vast majority of
microbes cannot be grown in the
laboratory and therefore cannot be
studied with the classical methods
of microbiology. Although
community ecology is not new to
microbiology, the ability to bring to
bear the power of genomics in the
study of communities initiates an
unpatrolled opportunity.
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Applications of Metagenomics

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Applications | What metagenomics can

do
Global Impacts. The role of microbes is critical in maintaining

atmospheric balances, as they are the main photosynthetic

agents responsible for the generation and consumption of
greenhouse gases involved at all levels in ecosystems and
trophic chains
Bioremediation. Cleaning up environmental contamination,
such as the waste from water treatment facilities gasoline leaks
on lands or oil spills in the oceans toxic chemicals
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Science advances with

Metagenomics
Metagenomics has the
potential to advance
knowledge in a wide
variety of fields. It can also
be applied to solve
practical challenges in
medicine, engineering,
agriculture, sustainability
and ecology
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Advancement in Medicine
Microbial communities play a key role in preserving human
health, but their composition and the mechanism by which
they do so remains mysterious. Metagenomic sequencing is
being used to characterize the microbial communities from
15-18 body sites from at least 250 individuals. This is part of
the Human Microbiome initiative with primary goals to
determine if there is a core human microbiome, to
understand the changes in the human microbiome that can
be correlated with human health, and to develop new
technological and bioinformatics tools to support these
goals.
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Advancement in Medicine
Another medical study as part of the MetaHit (Metagenomics of
the Human Intestinal Tract) project consisted of 124 individuals
from Denmark and Spain consisting of healthy, overweight, and
irritable bowel disease patients. The study attempted to
categorize the depth and phylogenetic diversity of
gastrointestinal bacteria. Using Illumina GA sequence data and
SOAPdenovo, a de Bruijn graph-based tool specifically designed
for assembly short reads, they were able to generate 6.58
million contigs greater than 500 bp for a total contig length of
10.3 Gb and a N50 length of 2.2 kb
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Experimental approaches to
Metagenomics
Techniques of Metagenomics are generally used to explore
the properties of microorganisms without prior cultivation.
Although still a novel field of research, Metagenomics has
already developed different branches, which contain mainly
the following focuses:
Discovery of new proteins by functional screening
techniques (e.g. screening for an enzyme that is able to
degrade a particular toxic compound),analysis of microbial
diversity in a habitat (e.g. by classical rRNA analysis or
phylum estimation from sequence properties), and gene
discovery by sequence analysis.
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Bioinformatics on Metagenomes

Sequenced metagenomes yield fragmented genomic

data that is comprised from a mixture of anonymous
microorganisms. Among others, bioinformatics can
be used to sort the fragmented nucleotide fragments
into 'bins' of different taxonomic/phylogenetic
levels, assemble contigs from short sequence reads
,predict genes on sequence reads (or assembled
contigs),predict gene function,classify predicted
proteins into families or other groups.
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Metagenomics is employed
Metagenomics is employed as a means of systematically
investigating, classifying, and manipulating the entire genetic
material isolated from environmental samples. This is a multistep process that relies on the efficiency of four main steps .
The procedure consists of
(i) the isolation of genetic material,
(ii) manipulation of the genetic material,
(iii) library construction, and the
(iv) the analysis of genetic material in the metagenomic
library.
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What metagenomics can do

Bioenergy. We are harnessing
microbial power in order to
produce ethanol (from cellulose),
hydrogen, methane, butanol...
Smart Farming. Microbes help
our crops by the suppressive
soil phenomenon(buffer effect
against disease-causing
organisms) soil enrichment and
regeneration
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What metagenomics can do

The World Within. Studying the
human microbiome may lead to
valuable new tools and
guidelines in human and animal
nutrition better understanding
of complex diseases(obesity,
cancer, asthma...) drug
discovery
Preventative medicine
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Example for a metagenomic study

Many metagenome
sequencing projects currently
rely on 'shotgun sequencing',
a method which is based on
cloning and subsequent
sequencing of genomic DNA.
The single steps of such a
metagenomic study are
illustrated
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Sampling
he single steps of such a metagenomic study are illustrated below

The first step of all

metagenomic studies is
the extraction of a
sample from some
environmental habitat.
An environmental
habitat could be
A Rain water forest
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Material from an underwater volcano

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Material from an Human Individual

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Indicates all the Genomes of Microbes

living in the Environment
The original environmental
sample contains all material
from the chosen
environment, including the
microorganisms living in
there. The red, green and
yellow thing symbolize
microbes living in an
environmental sample.
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Cloning and Sequencing

The environmental samples are
further on processed in a
molecular biology laboratory.
Some of the intermediate steps
on the way to obtaining a
sequenced metagenome are
these:
DNA isolation: the genomic DNA
of all microorganisms that are
present in a sample is
simultaneously extracted.
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Shearing
Shearing: the isolated
genomic DNA is broken into
shorter fragments that can
be cloned into plasmids (a
vector for smaller insert
size). Some DNA isolations
methods already contain a
shearing step by
themselves.
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Cloning
Cloning: the DNA fragments are
cloned into a vector. From here on,
all steps are shown for one
fragment, only. The vector contains
an origin of replication which
enables the host organsim to
multiply the plasmid. It also
contains a marker which assists in
the selection of host organisms that
have incorporated the plasmid
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Transformation
Transformation:
constructs consisting
of a plasmid with an
insert are
transformed into a
host organism
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Multiplication
Multiplication: the
host organism
multiplies (and
obtains) the
transformed
material.
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Sequencing
Sequencing: after another step of
DNA isolation which in this case
isolates the multiplied plasmid
material from the host organism,
the single fragments can be
sequenced by various methods.
Usually, Sanger sequencing with
fluorescence is applied.
Ref pictures: Katharina Hoff. Last
modified November 2007
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Benefits Humans and other

species

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Metagenomics gives a more true path

to understand Genomics
The valuable property of metagenomics is that it
provides the capacity to effectively characterize
the genetic diversity present in samples
regardless of the availability of laboratory
culturing techniques. Information from
metagenomic libraries has the ability to enrich
the knowledge and applications of many aspects
of industry, therapeutics, and environmental
sustainability.
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Metagenomics continues to improve human

relation with environment
This information can then be
applied to society in an effort to
create a healthy human
population that lives in balance
with the environment.
Metagenomics is a new and
exciting field of molecular
biology that is likely to grow into
a standard technique for
understanding biological
diversity.
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Future Direction
New enzymes, antibiotics, and other reagents
identified
More exotic habitats can be intently studied
Can only progress as library technology
progresses, including sequencing technology
Improved bioinformatics will quicken analysis
for library profiling.
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Information Resources
gobics.de [Research: Metagenomics]
METAGENOMICS: THE SCIENCE OF BIOLOGICAL DIVERSITYby
KJSHELSWELL

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 Program created by Dr.T.V.Rao MD for

Basic understanding on Metagenomics for
Medical, paramedical and scientific
Community in the Developing world
Email
doctortvrao@gmail.com
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