This document discusses metagenomics and its applications in bioremediation. It begins by defining bioremediation as using biological entities like microorganisms to clean up pollution. It then explains that metagenomics uses genetic material directly extracted from environments to analyze culturable and non-culturable microorganisms. Metagenomics seeks to identify genes involved in bioremediation to better understand microbial diversity and activities in polluted environments to improve bioremediation processes. Bioinformatics plays an important role in analyzing the large amounts of metagenomic data generated.

1. METAGENOMICS &
BIOREMEDIATION

2. BIOREMEDIATION
• Bioremediation is the process of cleaning up the environment with the
help of biological entities.
• Microorganism-based bioremediation is now regarded as a cost-effective
and environmentally benign method for environmental management.
• They alter their existing metabolic pathways with the help of genetic
transformation to breakdown or conversion of contaminants.
• Micro - organisms like fungus, yeast, or bacteria have long been thought
to be superior organisms for pollution detoxification.
• These are versatile in terms of nutrition and have the flexibility to cope to
harsh environmental conditions.
• They also have a variety of extracellular and intracellular enzymes that
break down complicated contaminants into simpler molecules including
carbon, water, methane, and sources of energy.

3. METAGENOMICS: Culture-Independent
. Insight
• Metagenomics is a technique for analyzing genetic
material extracted directly from environment.
• Only around 1% of the microbes can be grown using
standard microbiological procedures i.e traditional
culture-based techniques (Handelsman 2004).
• “Metagenomics” is increasingly used for
determining the genetic composition of both
culturable and non-culturable microorganisms from
any source.
• Jo Handelsman et al. introduced the term
metagenomics in 1998.

4. APPLICATION OF METAGENOMIC
IN BIOREMEDIATION
• A pool of genomes taken from a polluted sample is used in the metagenomic
method to identify the genes involved in bioremediation.
• With advancements in vector selection and construction research, it is now
possible to operate effectively on huge genomic segments and then screen vast
clone libraries with functioning metagenomes
• Metagenomics seeks to recognize microbe-related genes in order to better
understand the real diversity of microorganisms, their activities, structures,
dynamics, cooperation, relationships, and evolution in a range of environments,
and so improve bioremediation processes.
• Stable Isotope Probing (SIP) can be used to increase the RNA, DNA, or
phospholipids of dynamic microbial populations. DNA fragments obtained from
environment are cloned in a suitable vector [phage, plasmid, bacterial artificial
chromosome (BAC)] and then rebuilt into a host bacterium to create metagenomic
reference libraries.

5. Continue…
• Pre- and post-contamination, metagenomic data can
reveal taxonomic and enzymatic diversity, allowing for
the identification of potentially active genes and species.
• It will be able to correlate changes in contaminant
composition and concentration to individual genes and
taxa by accumulating metagenomes from a range of
contaminated and uncontaminated similar environment.
• This will provide answers to concerns regarding the
polluted system's microbial ecology, especially how
microorganisms respond to the contaminant's
perturbation.
• metagenomic investigations of bioremediation will also
offer information on how microbial populations respond
to changes in a range of environments.

6. Approaches to Metagenomic Analysis
Sequence-Based Analysis
• Relies on sequence analysis to get a foundation
for function prediction.
• Sequence-based screening consists mostly of
two steps: identifying metagenomic reads with
desirable sequences (gene prediction) and
connecting the desirable sequences to a
database (gene annotation).
• Gene identification, genome assemblages,
elucidating entire metabolic pathways, and
comparing organisms from various communities
Function-Based Analysis
• Functional metagenomics is a strong and
effective approach for researching the functions
of genes.
• Its purpose is to isolate DNA from environment
in order to investigate the functionalities of the
encoded protein.
• DNA fragments are cloned, expressed in a
laboratory host, and tested for enzyme activity in
functional-based study.
• This method is dependent on the expression
profiles of the clones of the metagenomic library.
• This method has a lot of potential for detecting
new gene segments that code for already-
identified or unknown functions.
• Phenotype-based screening is part of the
functional screening method.

7. Workflow of metagenomics research.

8. Metagenomic strategies and tools for
bioremediation
 First generation sequencing (complete genome shotgun
sequencing)
• The first-generation DNA sequencing technologies were Frederick
Sanger's and Allen Maxam's—Walter Gilbert's approaches.
• Sanger sequencing generates DNA fragments of varied lengths using
a denatured DNA template, radioactively tagged primer, DNA
polymerase, and chemically modified nucleotides termed di-
deoxynucleotides. The integrated dNTPs determine the length of
the DNA fragment. On gel electrophoresis, the DNA fragments are
separated depending on their size and may be seen using an X-ray
or UV-light imaging equipment.
• Since it employs chemicals to break nucleotides, Maxam-Gilbert
sequencing is known as the chemical degradation technique.
Chemical treatment causes nucleotide base breakage, resulting in a
collection of marked fragments that may be separated by gel
electrophoresis based on their size.

9.  Next generation sequencing (high throughput sequencing)
(i) Pyrosequencing technique
It is a synthesis-based sequencing method that detects the release of
pyrophosphate when a nucleotide is added to a freshly produced DNA
strand. It is ideal for sequencing small DNA fragments.
(ii) Illumina/Solexa sequencing
the DNA sequence is examined base-by-base, hence very accurate. Cells
are not required, the throughput is maximum, the reads are relatively
short (up to 300 bp), the cost per base is lowest, and the output is
suitable with most applications. Sequencing by synthesis with reversible
terminators is used to determine the nucleotide in the sequences, with
four modified nucleotides, sequencing primers, and DNA polymerases
included such that the primers are hybridised to the sequence.

10. (iii) Sequencing by ligation on beads
It is made up of several rounds of sequencing. The location of the
nucleotide is revealed during sequencing by ligating universal primer to a
fluorescently tagged DNA octamer. The procedure is repeated until every
base has been sequenced twice, increasing the platform's accuracy.
(iv) Ion torrent sequencing
It works in a similar way as pyrosequencing technology. This method
depends on the discharge of a hydrogen when a dNTP is introduced to
DNA polymer instead of fluorescently tagged nucleotides. The
incorporation of nucleotides into DNA strands by polymerase produces
hydrogen ions as a by-product, which lowers the pH, which is detected by
a pH sensor at the microwell's base and converted into a voltage
proportionate to the amount of nucleotides integrated.

11. Third generation sequencing (single molecule
long-read sequencing)
• It does not require PCR amplification for
sample preparation.
(i) Pacific Biosciences
Fluorescent labelling, like other sequencing
methods, is used in this method. In real time, it
identifies nucleotide signals. Each base is
enzymatically incorporated, resulting in a flash of
light that identifies the base and is analysed
repeatedly to form the DNA sequence

12. (ii) Oxford nanopore technology
The DNA/RNA molecule is passed through a nanopore using electrophoresis. It makes use of electrolyte
solution as well as a constant electric field. A termination repair stage shears double-stranded DNA and
forms blunt-ended DNA molecules with this technique. The DNA is then modified with two adaptors (a Y
adapter and a hairpin adaptor) coupled with a unique motor protein that aids in unzipping the double-
stranded DNA at the Y adapter and moving the DNA as a single strand via the nanopore. The activity of the
motor protein as the nucleic acid travels through the nanopore creates a change in ionic current due to
mobile nucleotides filling the pore. The ionic current variation is graphically shown and then clarified for
sequence identification.
(iii) HeliScope
It is another technological platform for single DNA molecule sequencing that uses an exceptionally
sensitive fluorescence detection device. Restriction enzymes fragment DNA strands, which are identified
by the insertion of a poly-A tail. The DNA molecules are hybridized to the flow cell plate, which has billions
of oligo(dT) chains attached to its surface, resulting in an array of primer-annealed single DNA templates.
Labelling is done in "quads," which are made up of four cycles for each of the four nucleotide bases. A
template-dependent extension is created by adding fluorescently labelled bases one at a time. The label is
illuminated by a laser light, which reads the strands that have taken up a specially designated base, which
is then recognized and recorded by a camera. These signals are translated into a nucleotide sequence by a
variety of computer systems. The label is then cleaved, and a fresh base is used in the following cycle.

13. Bioinformatic tools for metagenomic
bioremediation
• Bioinformatics performs a variety of
functions in the field of metagenomic
bioremediation, most notably during the
analysis of metagenomic data.

14. A comparative overview of functions and suitability of
mostly used tools for metagenomic analysis
Suitability SmashCommunity
MG-
RAST
IMG/M RAMMCAP MEGAN MOTHUR
Single Genomes - - + + - -
Single Cell
Genomes
- - - - - -
Shotgun
Metagenomes
+ + + + + -
16S rDNA
Metagenomes
+ + - + + +
Functions
Assembly + - + - - -
Gene Prediction + - + + - -
Functional
Analysis
+ + + + + -
Taxonomy
Assignment
+ + + + + +
Comparative
Analyses
+ + + + + +
Data
Management
+ - + - - -
+Yes,
-No