Metagenomicsnewer approach in understanding Microbes

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Metagenomicnewer approach in understanding icrobe

Dr.T.V.Rao MD

4/4/2016 Dr.T.V.Rao MD


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Metagenomics is the study of metagenomes, genetic materialrecovered directly from environmental samples.

The term "metagenomics" was first used by Jo Handelsman, Jo

Clardy, Robert M. Goodman, and first appeared in publication 1998.

This relatively new field of genetic research enables studies oforganisms 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


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The broad field may also be referred to asenvironmental genomics, ecogenomics or commun

genomics.

he term metagenome referenced the idea that a collection oenes sequenced from the environment could be analyzed inay analogous to the study of a single genome.

he human microbiome project (HMP) will require metagenomple sequencing data as a primary resource for analysis of lationship between microbial communities and the individu

ey inhabit.4/4/2016 Dr.T.V.Rao MD


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Metagenomics

•Metagenomics is the study of genetic materiarecovered directly from environmental samplbroad field may also be referred to as environgenomics, ecogenomics or community genom

While traditional microbiology and microbialgenome sequencing and genomics rely uponcultivated clonal cultures, early environmentasequencing cloned specific genes to produce aof diversity in a natural sample.



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Introduction

PubMed: metagenom*[Title/Abstract]

”...functional analysis of the collective genomes of so

microflora, which we term the metagenome of the so

- J. Handelsman et al., 1998

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNI4/4/2016 Dr.T.V.Rao MD


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Single cell genomic

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNI

How do we get the genomes

CulturingFew microorganisms can be easily cultured (

Microorganisms needs to be studied in their env

Only routinely performed in specialized labs

Very incomplete genomes (mean 40%, range

Metagenomics

https://ww



7/47CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNI

Metabolites

Proteins

mRNA

DNA

Meta-bolomics

Meta-proteomics

Meta-transcriptomics

Meta-genomicsIn Situ methods

Community structure Microbial functions

Extraction

P-Removal:

N-Removal:

-Removal:

Foaming:

Ethanol production:

Microbial n

Understanding ecosystems



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Cultures missed many facts on Micro

missing links clarified by PCR Techniq

•

Such work revealed that thevast majority of microbialbiodiversity had been missedby cultivation-based methods.Recent studies use either"shotgun" or PCR directedsequencing to get largelyunbiased samples of all genesfrom all the members of thesampled communities.



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What have metagenomics been used


• 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 genom

• Access to genomes of the

”uncultured majority”

Garcia Martin et al ., 2006 Nat. Biotechnol. Albertsen et al ., 2013 Nat. Biotechnol.



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Metagenomics will reveal the hidd

genome bases

•

Because of its abilitreveal the previousldiversity of microscometagenomics offerpowerful lens for vie

the microbial worldthe potential torevolutionize underof the entire living w



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Metagenomics gives clarity on

understanding genome bases

•

As the price of DNAsequencing continuesto fall, metagenomicsnow allows microbialecology to beinvestigated at a muchgreater scale anddetail than before.



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Introduction


Genome = Parts list of a single genome



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Introduction


Metagenome = Parts list of the community

Photo: D. Kunkel; color, E. Latypova



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History of Metagenomic

•However, early metagenomic studies revethat there are probably large groups ofmicroorganisms in many environments thacannot be cultured and thus cannot be

sequenced. These early studies focused onribosomal RNA sequences which are relatshort, often conserved within a species, agenerally different between species



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Learning more of the unknown Metagen

made to know the future of science

•

Many 16S rRNA sequences have been found whnot belong to any known cultured species, indicthat there are numerous non-isolated organismsurveys of ribosomal RNA (rRNA) genes taken dfrom the environment revealed that cultivation methods find less than 1% of the bacterial and aspecies in a sample. Much of the interest inmetagenomics comes from these discoveries thashowed that the vast majority of microorganism

previously gone unnoticed.4/4/2016 Dr.T.V.Rao MD


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Metagenomics approaches the new pa

science

•

Metagenomics providesa new way of examiningthe microbial world thatnot only will transformmodern microbiology

but has the potential torevolutionizeunderstanding of theentire living world



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Approach to Microbiology is commun

oriented rather than individual micro

•

In metagenomics, the genomic analysis is apentire communities ofmicrobes, bypassing thisolate and culture indbacterial community m

The new approach andattendant technologiebring to light the myriacapabilities of microbicommunities



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Interrelation of Species of Microb

better understood with Metagenom

•

Metagenomics willgenerate knowledge ofmicrobial interactions sothat they can beharnessed to improvehuman health, foodsecurity, and energyproduction



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Metagenomics Studies the interrel

genomes

•

Metagenomics cothe power of genbioinformatics, ansystems biology.Operationally, it iin that it involvesof the genomes oorganismssimultaneously.



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A Novel approach to non cultivab

Microbes

• It provides new access to themicrobial world; the vast majority ofmicrobes cannot be grown in thelaboratory and therefore cannot bestudied with the classical methodsof microbiology. Although

community ecology is not new tomicrobiology, the ability to bring tobear the power of genomics in thestudy of communities initiates anunpatrolled opportunity.



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Applications of Metageno



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Applications | What metagenomics

do

• ● Global Impacts. The role of microbes is critical in maiatmospheric balances, as they are the main photosynthagents responsible for the generation and consumptiongreenhouse gases involved at all levels in ecosystems atrophic chains

• ● Bioremediation. Cleaning up environmental contamisuch as the waste from water treatment facilities gasolon lands or oil spills in the oceans toxic chemicals



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Science advances with

Metagenomics

•

Metagenomics has thepotential to advanceknowledge in a widevariety of fields. It can alsobe applied to solve

practical challenges inmedicine, engineering,agriculture, sustainabilityand ecology



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Advancement in Medicin

•

Microbial communities play a key role in preservinghealth, but their composition and the mechanism bthey do so remains mysterious. Metagenomic sequbeing used to characterize the microbial communit15-18 body sites from at least 250 individuals. This

the Human Microbiome initiative with primary goadetermine if there is a core human microbiome, tounderstand the changes in the human microbiome be correlated with human health, and to develop ntechnological and bioinformatics tools to support th

goals.4/4/2016 Dr.T.V.Rao MD


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Advancement in Medicin

•

Another medical study as part of the MetaHit (Metagenothe Human Intestinal Tract) project consisted of 124 indifrom Denmark and Spain consisting of healthy, overweigirritable bowel disease patients. The study attempted tocategorize the depth and phylogenetic diversity of

gastrointestinal bacteria. Using Illumina GA sequence daSOAPdenovo, a de Bruijn graph-based tool specifically dfor assembly short reads, they were able to generate 6.5million contigs greater than 500 bp for a total contig leng10.3 Gb and a N50 length of 2.2 kb



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Experimental approaches t

Metagenomics

•

Techniques of Metagenomics are generally used to the properties of microorganisms without prior cultAlthough still a novel field of research, Metagenomalready developed different branches, which contaithe following focuses:

• Discovery of new proteins by functional screeningtechniques (e.g. screening for an enzyme that is abldegrade a particular toxic compound),analysis of mdiversity in a habitat (e.g. by classical rRNA analysisphylum estimation from sequence properties), and

discovery by sequence analysis.4/4/2016 Dr.T.V.Rao MD


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Bioinformatics on Metageno

Sequenced metagenomes yield fragmented genomdata that is comprised from a mixture of anonymoumicroorganisms. Among others, bioinformatics canbe used to sort the fragmented nucleotide fragmeninto 'bins' of different taxonomic/phylogenetic

levels, assemble contigs from short sequence reads,predict genes on sequence reads (or assembledcontigs),predict gene function,classify predictedproteins into families or other groups.



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Metagenomics is employ

• Metagenomics is employed as a means of systematicainvestigating, classifying, and manipulating the entire material isolated from environmental samples. This is step process that relies on the efficiency of four main sThe 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 metagenomlibrary.


What metagenomics can


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• Bioenergy. We are harnessing

microbial power in order toproduce ethanol (from cellulose),hydrogen, methane, butanol...

• Smart Farming. Microbes helpour crops by the “suppressive

soil” phenomenon(buffer effectagainst disease-causingorganisms) soil enrichment andregeneration



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• The World Within. Studying thehuman microbiome may lead tovaluable new tools andguidelines in human and animalnutrition better understanding

of complex diseases(obesity,cancer, asthma...) drugdiscovery

• ● Preventative medicine



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Example for a metagenomic st

• Many metagenomesequencing projects currentlyrely on 'shotgun sequencing',a method which is based oncloning and subsequent

sequencing of genomic DNA.The single steps of such ametagenomic study areillustrated



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Sampling

he single steps of such a metagenomic study are illustrated

•

The first step of allmetagenomic studies isthe extraction of asample from someenvironmental habitat.

An environmentalhabitat could be

• A Rain water forest



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Material from an underwater vol



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Material from an Human Indiv



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Indicates all the Genomes of Mic

living in the Environment

•

The original environmentalsample contains all materialfrom the chosenenvironment, including themicroorganisms living in

there. The red, green andyellow thing symbolizemicrobes living in anenvironmental sample.



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Cloning and Sequencin

• The environmental samples arefurther on processed in amolecular biology laboratory.Some of the intermediate stepson the way to obtaining a

sequenced metagenome arethese:

• DNA isolation: the genomic DNAof all microorganisms that arepresent in a sample is

simultaneously extracted.4/4/2016 Dr.T.V.Rao MD


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Shearing

• Shearing: the isolatedgenomic DNA is broken intoshorter fragments that canbe cloned into plasmids (avector for smaller insert

size). Some DNA isolationsmethods already contain ashearing step bythemselves.



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Cloning

• Cloning: the DNA fragments are

cloned into a vector. From here on,all steps are shown for onefragment, only. The vector containsan origin of replication whichenables the host organsim to

multiply the plasmid. It alsocontains a marker which assists inthe selection of host organisms thathave incorporated the plasmid



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Transformation

•

Transformation:constructs consistingof a plasmid with aninsert aretransformed into ahost organism



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Multiplication

•

Multiplication: thehost organismmultiplies (andobtains) thetransformedmaterial.



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Sequencing

• Sequencing: after another step of

DNA isolation which in this caseisolates the multiplied plasmidmaterial from the host organism,the single fragments can besequenced by various methods.

Usually, Sanger sequencing withfluorescence is applied.

• Ref pictures: Katharina Hoff. Lastmodified November 2007



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Benefits Humans and oth

species



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Metagenomics gives a more true

to understand Genomics

•

The valuable property of metagenomics isprovides the capacity to effectively characthe genetic diversity present in samplesregardless of the availability of laboratory

culturing techniques. Information frommetagenomic libraries has the ability to enthe knowledge and applications of many aof industry, therapeutics, and environmen

sustainability4/4/2016 Dr.T.V.Rao MD

Metagenomics continues to improve


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Metagenomics continues to improve

relation with environment

• This information can then beapplied to society in an effort tocreate a healthy humanpopulation that lives in balancewith the environment.Metagenomics is a new andexciting field of molecularbiology that is likely to grow intoa standard technique forunderstanding biological

diversity4/4/2016 Dr.T.V.Rao MD


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Future Direction

•

New enzymes, antibiotics, and other reagidentified

•More exotic habitats can be intently stud

•Can only progress as library technology

progresses, including sequencing techn• Improved bioinformatics will quicken anafor library profiling.



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Information Resources

• gobics.de [Research: Metagenomics]

• METAGENOMICS: THE SCIENCE OF BIOLOGICAL DIVERSITYbyKJSHELSWELL



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•Program created by Dr.T.V.Rao MD

Basic understanding on Metagenomic

Medical paramedical and scientifi

Community in the Developing worl

•Email

• [email protected]


Metagenomicsnewer approach in understanding Microbes

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