metagenomics newer approach in understanding microbes

05/02/2023 Dr.T.V.Rao MD 1

Metagenomicsnewer approach in understanding Microbes

Dr.T.V.Rao MD

05/02/2023 Dr.T.V.Rao MD 2

INTRODUCTION

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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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 genes 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 sample sequencing data as a primary resource for analysis of the relationship between microbial communities and the individuals they inhabit.

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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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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

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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 labsVery incomplete genomes (mean 40%, range 10-90%)

Metagenomics

https://www.bigelow.org/

05/02/2023 Dr.T.V.Rao MD 7CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Metabolites

Proteins

mRNA

DNA

Meta-bolomics

Meta-proteomics

Meta-transcriptomics

Meta-genomics

Data integration

In Situ methods

Community structure Microbial functions

Extraction

P-Removal:

N-Removal:

-Removal:

Foaming:

Ethanol production:

Microbial needsEcology

Understanding ecosystems

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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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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.

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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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Introduction


Metagenome = Parts list of the community

Photo: D. Kunkel; color, E. Latypova

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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 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.

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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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Samplinghe 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

•[email protected]