Metagenomics

Kanika National Research Centre on Plant Biotechnology,

New Delhi-110012

kumarkanika@rediffmail.com

Prokaryotes largest proportions and untapped reservoir

Only 1% microbes culturable – metagenomics bypasses the need for isolation or cultivation of microbes . It allows

functional and sequence-based analysis of collective microbial communities inhabiting any habitat

extreme and inhospitable environments- solfataric hot springs, hypersaline basins and glacier ice, Antarctic desert, ground water can be studied using metagenomic

determination and comparison the biological diversity and the functional activity of different microbial communities

Pictorial representation of Metagenomic Study

Technological advances has allowed mapping of microbial community metabolism onto environmental processes

Genomes in the context of environment

Screening of metagenomic libraries

function-based

sequence-based

libraries in host E. coli – nearly 40% genes expressed

Streptomyces, Thermus thermophilus, Sulfolobus solfataricus various Proteobacteria as host

Identification of novel biomolecules- Lipases, cellulases, chitinases , polymerases proteases and antibiotics etc.

Function based screening

No sequence information required novel classes of genes with novel functions

approaches–

phenotypic detection

Heterologus complementation

Induced gene expressionSubstrate induced gene expression screening (SIGEX)Product induced gene expression (PIGEX)Metabolite-regulated expression (METREX)

Sequence based screening-

Design of DNA probes or primer from already known sequences

Novel variant of already known genes can be identified- genes encoding dioxygenases, hydrazine oxido reductase, chitinase and glycerol dehydratases

Gene targeted metagenomics- PCR based sequencing combined with NGS sequence information to recover full length versions of selected genes

Assesment of taxonomic and functional diverse microbial communities

16rRNA, recA , radA, hsp 70, elongation factor Tu and G used as marker for phylogenetic analysis but PCR introduced bias low copy number representatives may be missed ,

Pyrosequencing of marker genes provides immense depth but intrinsic error rate may result in overestimation

270 bp

M 1 2 3 4

1 kb 850 bp

250bp

16 S rDNA gene amplification from metagenomic DNA isolated from hot water spring of India

Lane M - 1 Kb DNA ladderLane 1 & 2- PCR amplification(270 bp)Lane 3 & 4 -PCR amplification(850 bp)

BLAST results of 16S rDNA gene sequences obtained from cloned 16s rDNA genes

Organism Source E Value. Max. Identity

Uncultured bacterium clone

Hot Spring in Yunnan Province, China

0.0 96%

Uncultured bacterium Gwan Tsu Ling Hot Spring Taiwan

0.0 89%

Paenibacillus sp. South Korea 0.0 93%

Thermonema rossianum Hot spring Tunisia 5e-133 99%

Hydrogenophilus thermoluteolus

NBRC Japan 2e-137 99%

Thermus sp Japan 3e-135 98%

Uncultured Thermodesulfobacterium

Geothermal spring 3e-123 94%

Direct sequencing of metagenome is increasingly being used as it avoids bias in amplification of marker genes

•Sorcerer II Global ocean Sampling Project

•Human Microbiome Project

Various NGS platforms available allows sequencing of metagenome at faster rate

High through put sequencing facility has led to

Exploration of taxonomic and functional biodiversity and system biology of a microbial community in a particular habitat

reconstruction of main metabolic pathway and improve our understanding of adaptation strategies of a microbial community thriving in extreme environment

Bioinformatics tools

Bining: Taxonomic analysis of metagenomic data, sequences assigned to phylogenetic group according to their taxonomic origin

MEGAN- Metagenome analyzer,

Sort ITEMS- Sequence Ortholog Based similarities based on nucleotide databases in NCBI

CARMA- based on similarities to protein families and domain included in protein family data bases

Web based metagenomic annotation platforms-

mg_RAST server,

IMG/M server,

JCVI Metagenomic Reports (METAREP)-

Allow data analysis via generic interface

Allow comparison to databases GO, Pfam, NCBI, SEED, KEGG

Allow analysis of metagenomic data at various taxonomic and functional levels

Fig. A snapshot of METAPREP a tool for carrying out comparative metagenomics(www.jcvi.org/metarep/)

Fig. Snapshot of MG-RAST used for carrying out metagenomics analysis(www.metagenomics.anl.gov/ )

METATRANSCRIPTOMICS

metabolic and functional capacity of a microbial community no differentiation expressed and non expressed genes, sequencing and characterization of metatranscriptomes

Limitations

processing of environmental RNA samples

recovery of high-quality mRNA from environmental samples

short half-lives of mRNA species (91), and

separation of mRNA from other RNA species,

non coverage of low abundance transcript leading to non detection

Direct cDNA sequencing employing next-generation sequencing technologiesIs pradtised

METAPROTEOMICS

The proteomic analysis of mixed microbial communities to assess the immediate catalytic potential of a microbial community

Detect and identify all proteins produced by a complex environmental microbial community

Has a huge potential to link the genetic diversity and activities of microbial communities with their impact on ecosystem function

Human Microbiome Projectgenerating resources enabling comprehensive characterization of the human microbiota and their role in human health and disease 

we are bearers of genetic information, more than 99 percent of it is microbial. This “second genome,” exerts an influence on our health even greater than the genes we inherit from our parents. Inherited genes are fixed, it is, however, possible to reshape, even cultivate, the second genome.

when the contents of a lean donor’s microbiota were transferred to the guts of male patients with metabolic syndrome, the researchers found striking improvements in the recipients’ sensitivity to insulin, an important marker for metabolic health. Somehow, the gut microbes were influencing the patients’ metabolisms

alarming increase in autoimmune diseases possibly to a disruption in the ancient relationship between our bodies and their “old friends” — the microbial symbionts with whom we coevolved

Applications

Diversity patterns of microorganisms can be used for monitoring and predicting environmental conditions and change

genes/operons for desirable enzyme candidates (cellulases, chitinases, lipases,

antibiotics, other natural products of industrial or medical applications

Examining secretory, regulatory and signal transduction mechanisms associated with samples or genes of interest

Examining potential lateral gene transfer events. Knowledge of genome plasticity may give us an idea of selective pressures for gene capture and evolution within a habitat

Allow identification of novel metabolic pathways. Directed approach towards designing culture media for the growth of previously-

uncultured microbes.

Identification of genes that predominate in a given environment compared to others

Designing low- and high-throughput experiments focused on defining the roles of genes and microorganisms in the establishment of a dynamic microbial community

Metatranscriptomics and metaproteomics have the potential to improve understanding the functional dynamics of microbial communities.

Improve our understanding of ecosystem functions of microbial communities

Down Side of Metagenomics Limitations

Often fragmentary

Often highly divergent

Rarely any known activity

No chromosomal placement

No organism of origin

Ab initio ORF predictions

Huge data

Computationally bioinformatics tools not very easy

Substantial database biases toward model organisms

Microbes are adapted for living in very unfavourable conditions like

very high and temperature, high and low pH, high saline conditions

etc. They can be a useful source of information as well as genes which

can be used for designing crops which are tolerant to changed

climatic conditions.

In light of this background our group is studying the microbes from hot

water springs of India which have not been explored to a great extent .

It will help in identification of novel genes related to thermotolerance

through NGS sequencing of metagenomic DNA. These genes can be

used in crops for improving their tolerance towards high temperature .