A concise presentation on the emerging topic on Metagenomic Analysis of Water Bodies. This presentation will help students and scientists to understand the various techniques used in metagenomic studies of rivers as well as oceans. Brief Introduction to various bioinformatic techniques like Lipidomics, LCMS, HPLC and Next Gen Sequencing, with a focus toward ocean and river samples.

1. Aman Nanavaty
METAGENOMIC
ANALYSIS OF
W
ATER BODIES

2. INDEX
PAGE NUMBER TITLE
1
INTRODUCTION TO
METAGENOMICS
2
ADVANTAGES OF
METAGENOMICS
3
WHY STUDY WATER
BODIES?
4-5 OCEAN MICROFLORA
6-7 FRESHWATER MICROFLORA
8-9
BIOPROSPECTING OF
WATER SAMPLES
10-11
STEPS INVOLVED IN
METAGENOMIC ANALYSIS
OF WATER BODIES
12
MAIN TECHNIQUES USED IN
METAGENOMICS OF WATER
BODIES
13
APPLICATIONS OF WATER
BODY METAGENOMICS
13 REFERENCES

3. INTRODUCTION TO
METAGENOMICS
Metagenomics is the direct genetic analysis of genomes present in
environmental samples such as soil and water. It is the detailed
structural and functional study of nucleotide sequences obtained/
collected from bulk environmental samples. The genomes elucidated
are usually microbial, such as from fungi and bacteria, but may also be
eukaryotic or viral in nature.
It involves sample collection and library preparation followed by
advanced sequencing methods (shotgun or high throughput). Binning
and Mapping are performed before comparative metagenomic
analysis.
Metagenomics allows researchers to sequence all possible genomes
found in an environment in an ef
fi
cient manner. Metagenomic analysis
reveals the role of individual species and association between different
species in an environment.
1

4. ADVANTAGES OF
METAGENOMICS
• Metagenomics has helped make studies on microbial ecology faster, easier and
simple. These microbial ecology studies have been used to assess ecosystems’
health and potential applications
• Ef
fi
cient Virus Discovery for previously unknown viruses
• Pharmaceutical, Agrochemical and Nutraceutical industries often use
metagenomics to manufacture high quality drugs, such as malacidin (antibiotic)
• Discovery of novel CRISPR proteins for expanded CRISPR-Cas toolkits and
genetic engineering techniques
• Diagnosis of infectious diseases such as Encephalitis
• Identi
fi
cation of biosynthetic gene clusters for antimicrobial production
• Targeted screening of hydrolases involved in biofuel production and
comparative analysis between convergent microbial systems
• Metagenomics is used to enhance bioremediation strategies by improving rates
of bioaugmentation and biostimulation
• Gut microbe characterization of Insects, Mammals and Birds for disease studies
and industrial applications
2

5. WHY STUDY WATER
BODIES?
Water bodies such as lakes, rivers, oceans and groundwater are extensively exploited by
humans for various needs such as manufacturing, potable water, recreation, transportation and
agriculture. However, we have not yet understood the full importance of water bodies and their
near-limitless potential. Many innovative drugs for cancer and HIV have been obtained from
marine sources, while microbes from lakes and rivers have been used in bioremediation of
ef
fl
uents.
Algae, Bacteria and Fungi from water bodies hold immense potential for applications in biofuel
production, bioremediation, drug discovery, food industries and nutraceutical manufacturing.
Spirulina (biomass generated from cyanobacteria) has been touted as a potential protein
superfood, consisting of 60% protein. Many commercially useful polysaccharides are extracted
from seaweeds such as Red . Even sessile / stationary marine organisms such as corals and
sponges have been studied as potential sources of bioactive secondary metabolites that can be
used in treating persistent diseases. Bio
fi
lms found in rivers, ponds and ditches have been
shown to produce chemical compounds that can be used as antimicrobials in aquaculture and
industrial processes.
Many life science domains related to water bodies involve metagenomics, such as Biodefense,
Pharmaceutical Biotech, Bioremediation, Food Safety Testing, Biochemical Engineering,
Industrial Enzymology, Ecological Studies and Molecular Paleolontology.
3

6. OCEAN MICROFLORA
Oceans host a wide variety of bacteria, viruses, fungi, diatoms, archaeans and
dino
fl
agellates that thrive in highly inter-connected ecological networks. Together, they
clean up wastes and serve as food for higher organisms. They are so prevalent in oceans
that it is believed 70-90% of marine biomass comes from microbes.
Bacteria abound in marine habitats. They have inhabited all kinds of oceans, undergoing
many adaptations that have enabled them to thrive. Some important examples of
marine bacteria include Pelagibacter ubique, Prochlorococcus and Vibrio vulni
fi
cus. P.
ubique produces a signi
fi
cant portion of Earth’s oxygen while Marinomas artica grows
inside arctic ice at subzero temperatures. Many parasitic and symbiotic microbes are
found in oceans, working in conjunction with
fi
sh and sponges.
In recent years, many marine archaeans have been discovered. Their unique adaptations
allow them to survive in deep sea vents, extremely saline waters and even the guts of
whales. They are experienced extremophiles that are able to inhabit waters with high
salinity, very high or low pH, high temperatures and frigid conditions. Some archaeans
have been found from temperate coastal waters and deep ocean trenches. Marine
archaeans are unusual in that they are very ubiquitous, have strange physiologies and
are sometimes part of symbiotic associations. Examples include Nitrosopumilus
maritimus and Cenarchaeum symbiosum.
Marine Diatoms such as Thalassiosira pseudomona, Climacosphenia spp. and
Leptocylindrus spp. are widespread. There are a few diatoms that inhabit extreme ocean
waters near the poles. They play an important role in many biogeochemical processes
such as the Urea Cycle and Silica Cycle. The ability of marine diatoms to sequester silica
is being studied by nanotechnologists.
Although poorly studied, there is amazing virus diversity in marine waters. Marine
viruses are often larger than their terrestrial counterparts. The largest known viruses
(Mimivirus, ) are found in . Some viruses that cause major losses in commercial shrimp
culture are White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV).
4

7. DIATOMS
MIMIVIRUS BOTRYOCOCCUS
MARINE CULTURE
5

8. FRESHWATER
MICROFLORA
Despite the advancements in genomic techniques over the past decades, micro
fl
ora
residing in water bodies remain poorly studied, including those found in lakes, rivers,
ponds and streams. Freshwater micro
fl
ora often form bio
fi
lms, cooperating with
different species for survival.
Freshwater is home to many species of Algae, Bacteria, Phages and Protozoans that may
or may not harm humans. They play a key role in their ecosystems by acting as a food
source for
fi
sh and insects while also recycling excess nutrients and wastes. When natural
micro
fl
ora are harmed or displaced, freshwater bodies often collapse and result in the
death of most involved species. It is of prime importance that river and lake micro
fl
ora
are studied, analysed and conserved in order to protect the balance of natural
ecosystems.
Freshwater Algae commonly found in rivers and lakes include Chlorella, Nostoc,
Charophyta, Phaeophyta and Vaucheria. Many of these freshwater micro-algae have
cosmopolitan distributions.
Diatoms are important and critical components of freshwater ecosystems. The diatom
Didymosphenia geminata causes severe environmental degradation by forming rock
snot. It is a common problem in Oceania and Europe. Other well-known freshwater
diatoms are Asterionella formosa,
Many diverse bacteria such as Cyanobacteria, Actinobacteria, Betaproteobacteria,
Psuedomonas, Bacilli and Bacteriodetes reside in rivers. They are found as parts of
species-speci
fi
c, bacterial and mixed bio
fi
lms attached to rocks, substrate and driftwood.
There are also quite a few pathogenic bacteria that infect
fi
sh and cause mortality.
Flavobacterium columnare is responsible for a fatal disease called Columnaris in Neon
Tetras and related species.
The Ganga river has an unusually high population of bacteriophages that prevent
spoilage of water. Myoviridae, Retroviridae, Microviridae, Circoviridae and
Polyomaviridae are some common viruses found in the Ganga. Many other types of
viruses that infect
fi
sh, crustaceans, amphibians, birds and humans reside in freshwater
bodies.
6

9. MICROCYSTIS FRESHWATER BACTERIA
Naegleria fowleri, freshwater thermophile
7

10. BIOPROSPECTING OF
WATER SAMPLES
Bioprospecting is de
fi
ned as the exploration of natural sources for biological information
that can be used in developing commercial products which could bene
fi
t humans.
Researchers collect various kinds of data such as Micro-molecules, Macromolecules,
Biochemical Information and Genetic Information from natural sources like lakes, farms,
forests and mountains. Bioprospecting is a tool widely used in many important domains
such as Pharmaceutical Industries, Nutraceutical Manufacturers, Aquaculture,
Nanotechnology Research and Agricultural Sciences.
Researchers often look for useful enzymes, genes, secondary metabolites and microbes
that can be commercially exploited. Many microbes isolated from rivers and oceans are
used in bioremediation of industrial ef
fl
uents. Bioactive Agents for viral infections and
fungal infections are being discovered from ocean micro
fl
ora, such as Phlorotannins,
Chitosan Derivatives and Sulfated Glycans.
The well-known enzyme, Taq polymerase, was obtained from Thermus aquaticus, an
extremophile residing in hot springs. Taq polymerase is used in classical Polymerase
Chain Reaction. A 2013 research study observed that many Flavobacteria and
Pseudomonads reside in Iceland’s glacial rivers. They also extracted many industrially
useful enzymes such as amylase, laccase and cellulase from those bacteria.
Many bene
fi
cial secondary metabolites produced by aquatic micro
fl
ora such as toxins,
antibiotics, pigments and vitamins are utilised by various industries. In recent years,
efforts have been made to commercialise the extraction of biomolecules such as
peptides, alkaloids and polyketides from ocean micro
fl
ora. Algal pigments from river
samples such as Fucoxanthin, Carotenoid and Phycoerythrin are obtained by suitable
Bioprospecting methods.
The scope of Marine Biotech is quite vast and has not been fully explored. Scientists
hope that cures for persistent diseases and biochemical compounds that could be used
in bioremediation of nuclear waste can be discovered from water bodies.
8

11. 9
WIDE RANGE OF BIOMOLECULES OBTAINED
FROM MARINE ORGANISMS
BIOPROSPECTING METHODOLOGY

12. STEPS INVOLVED IN
METAGENOMIC
ANALYSIS OF WATER
10
1. Experimental Design: Consideration of Costs, Expenses, Sequencing Methods,
Analytical Techniques, Labor and Time
2. Sample collection: Collection of sample from suitable source (freshwater/saltwater/
ocean)
3. Isolation and Puri
fi
cation: Isolating and Purifying biomolecule of interest by
Chromatography, Electrophoresis, Solubilisation or Centrifugation.
4. Next-Generation Sequencing: Massively Parallel Sequencing techniques to analyse
DNA/RNA samples, such as Illumina Technology, Ion Torrent Technology and 454
Sequencing
5. Binning: Sorting of DNA into groups that may share same genome or similar
genomes. Compositional Binning identi
fi
es conserved nucleotide and compares gene
product of DNA fragment with database. Eg: TACAO, CARMA, MEGAN and
MetaCluster algorithms
6. Data Analysis: Using advanced computer programs and analytical softwares, obtained
data is graphically/numerically represented and then studied by scientists
7. Metagenome Annotation: Feature prediction of reconstructed assemblies using
Orphelia, RAST, IMG, SILVA and FragGeneScan.
8. Statistical Analysis of Obtained Data: Using statistical softwares to compute
mathematical data from metagenomic data. Popular packages used are Primer-E
package and ShotgunFunctionalizeR package

13. 11
AQUATIC ORGANISMS
BIOMOLECULES
OF INTEREST
DATA
EXTRACTION
SEQUENCING
METAGENOMIC
ANALYSIS OF
WATER SAMPLES

14. MAIN TECHNIQUES USED
IN METAGENOMICS OF
WATER BODIES
12
1. Mass Spectroscopy (MS): Analyses Mass-to-Charge Ratio to determine relative
molecular weights and elucidate biochemical identity.
2. High Performance Liquid Chromatography (HPLC): Specialised Chromatographic
technique in which pumps pass pressurised sample-solvent mixture through
adsorbent column. Widely used in separating complex biomolecules from
environmental samples and detecting concentration levels.
3. Next-Generation Sequencing: High-Throughput Optimised Sequencing methods that
improve ef
fi
ciency, accuracy and scalability. Some common Next-Generation
Sequencing techniques are Illumina Sequencing and Pyrosequencing.
4. 16 S rDNA Analysis: Culture-Free method to identify, elucidate and differentiate
between bacterial species from complex microbiomes. Used to identify genus/
species level of bacteria isolated from dif
fi
cult to study biomes (deserts, dense
rainforests, underground caves).
5. Proteomics: Thorough structural, evolutionary and functional study of Proteomes, the
complete set of proteins of an Organism,
6. Extraction and Puri
fi
cation: Suitable extraction methods are applied for different
biomolecules. For lipid extraction, Green Extraction is used. For protein extraction,
Precipitation and Solubilization is used. Puri
fi
cation is done by HPLC, GFC, IEC and
Free Flow Electrophoresis.
7. Polymerase Chain Reaction (PCR): Uses DNA Polymerase to repeatedly amplify even
minute DNA samples. Very useful technique that relies on enzymatic DNA replication
8. RT-PCR: Similar to PCR, but involves RNA Polymerase ampli
fi
cation of DNA/RNA
sample. Often used in DNA virus testing, pathological studies and nuclear material
ampli
fi
cation
9. Lipidomics: Structural and Functional study of complete set of lipids from cell/
organism (Lipidome).

15. APPLICATIONS OF WATER
BODY METAGENOMICS
Marine microbes that have been dif
fi
cult to cultivate in vitro are studied using advanced
marine metagenomics, also known as Marine Omics. Many novel marine fungi isolated from
the Indian Ocean are being studied using advanced metagenomics.
Water Body Metagenomics is a powerful tool to study microbial diversity, ecology and
evolution. It also
fi
nds applications in various
fi
elds such as bioremediation of wastewater,
biofuel production, drug engineering, nutraceutical manufacture and aquaculture.
Potential bioactive agents for diseases such as Retroviral Infections, Cancer and Respiratory
Infections are being developed by researchers globally.
Many enzymes used in industrial processes such as Hydrolase, Esterase, Lipase and Reductase
are obtained from Marine bacteria. They are commonly used in pharmaceuticals, food
industries and cosmetic industries.
Detection of possible pathogens microbes (protozoans, bacteria, viruses) in Potable Water is
performed using high throughput Metagenomic techniques.
Metagenomic analysis of water samples is used to elucidate evolutionary pathways, ancestry
and in classi
fi
cation of newly discovered microbes.
Some examples of key industrial processes enhanced by marine omics are Spirulina
production, biofuel production from algae, marine phage detection and synthesis of additives
and useful biochemical compounds.
13

16. REFERENCES
1. https://www.genome.gov/genetics-glossary/Metagenomics
2. https://mammoth.bio/2020/04/13/discoveries-from-the-
fi
eld-of-
metagenomics/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416622/
4. https://www.sciencedirect.com/science/article/abs/pii/S0165232X13000360
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039460/
6. https://www.frontiersin.org/articles/10.3389/fmars.2021.629629/full
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351745/
8. https://www.ncbi.nlm.nih.gov/books/NBK54011/
9. https://en.wikipedia.org/wiki/Metagenomics
10. https://www.frontiersin.org/articles/10.3389/fmars.2014.00038/full
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351745/
12. https://www.frontiersin.org/articles/10.3389/fmicb.2020.616086/full
13. https://www.sciencedirect.com/science/article/pii/S1874778715300027#
14

17. END
THE