You are on page 1of 47

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.
4/4/2016

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.
4/4/2016

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.
4/4/2016

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]

4/4/2016

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
4/4/2016

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:
4/4/2016

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.
4/4/2016

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
4/4/2016

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
4/4/2016

Dr.T.V.Rao MD

10

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.

4/4/2016

Dr.T.V.Rao MD

11

Introduction

Genome = Parts list of a single genome


4/4/2016

Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

12

Introduction

Photo: D. Kunkel; color, E. Latypova

Metagenome = Parts list of the community


4/4/2016

Dr.T.V.Rao MD

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

13

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
4/4/2016

Dr.T.V.Rao MD

14

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.
4/4/2016

Dr.T.V.Rao MD

15

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

4/4/2016

Dr.T.V.Rao MD

16

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
4/4/2016

Dr.T.V.Rao MD

17

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
4/4/2016

Dr.T.V.Rao MD

18

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.
4/4/2016

Dr.T.V.Rao MD

19

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.
4/4/2016

Dr.T.V.Rao MD

20

Applications of Metagenomics

4/4/2016

Dr.T.V.Rao MD

21

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
4/4/2016

Dr.T.V.Rao MD

22

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
4/4/2016

Dr.T.V.Rao MD

23

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.
4/4/2016

Dr.T.V.Rao MD

24

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
4/4/2016

Dr.T.V.Rao MD

25

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.
4/4/2016

Dr.T.V.Rao MD

26

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.
4/4/2016

Dr.T.V.Rao MD

27

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.
4/4/2016

Dr.T.V.Rao MD

28

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
4/4/2016

Dr.T.V.Rao MD

29

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
4/4/2016

Dr.T.V.Rao MD

30

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
4/4/2016

Dr.T.V.Rao MD

31

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
4/4/2016

Dr.T.V.Rao MD

32

Material from an underwater volcano

4/4/2016

Dr.T.V.Rao MD

33

Material from an Human Individual

4/4/2016

Dr.T.V.Rao MD

34

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.
4/4/2016

Dr.T.V.Rao MD

35

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.
4/4/2016

Dr.T.V.Rao MD

36

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.
4/4/2016

Dr.T.V.Rao MD

37

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
4/4/2016

Dr.T.V.Rao MD

38

Transformation
Transformation:
constructs consisting
of a plasmid with an
insert are
transformed into a
host organism
4/4/2016

Dr.T.V.Rao MD

39

Multiplication
Multiplication: the
host organism
multiplies (and
obtains) the
transformed
material.
4/4/2016

Dr.T.V.Rao MD

40

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
4/4/2016

Dr.T.V.Rao MD

41

Benefits Humans and other


species

4/4/2016

Dr.T.V.Rao MD

42

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.
4/4/2016

Dr.T.V.Rao MD

43

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.
4/4/2016

Dr.T.V.Rao MD

44

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.
4/4/2016

Dr.T.V.Rao MD

45

Information Resources
gobics.de [Research: Metagenomics]
METAGENOMICS: THE SCIENCE OF BIOLOGICAL DIVERSITYby
KJSHELSWELL

4/4/2016

Dr.T.V.Rao MD

46

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
4/4/2016

Dr.T.V.Rao MD

47