Metagenomics newer approach in understanding Microbes

Metagenomicsnewer approach in understanding Microbes
Dr.T.V.Rao MD
4/4/2016 Dr.T.V.Rao MD 1

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 2

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 3

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 4

Introduction
PubMed: metagenom*[Title/Abstract]
”...functional analysis of the collective genomes of soil
microflora, which we term the metagenome of the soil.”
- J. Handelsman et al., 1998
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
4/4/2016 Dr.T.V.Rao MD 5

Single cell genomics
How do we get the genomes?
Culturing
Few microorganisms can be easily cultured (<<5%)
Microorganisms needs to be studied in their environment
Only routinely performed in specialized labs
Very incomplete genomes (mean 40%, range 10-90%)
Metagenomics
https://www.bigelow.org/
4/4/2016 Dr.T.V.Rao MD 6

Metabolites
Proteins
mRNA
DNA
Meta-bolomics
Meta-proteomics
Meta-transcriptomics
Meta-genomics
In Situ methods
Community structure Microbial functions
Extraction
P-Removal:
N-Removal:
-Removal:
Foaming:
Ethanol production:
Microbial needs
Understanding ecosystems
4/4/2016 Dr.T.V.Rao MD 7

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 8

What have metagenomics been used for?
• 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
Extracting genomes
• Genome extraction of low
abundant species (< 0.1%)
from metagenomes
• First complete TM7 genome
• Access to genomes of the
”uncultured majority”
Garcia Martin et al., 2006 Nat. Biotechnol. Albertsen et al., 2013 Nat. Biotechnol.
4/4/2016 Dr.T.V.Rao MD 9

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 12

Introduction
Metagenome = Parts list of the community
Photo: D. Kunkel; color, E. Latypova
4/4/2016 Dr.T.V.Rao MD 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 multi-
step 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

Metagenomics newer approach in understanding Microbes

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