shortgun by Panjab university microbiology dept.

1. By:-Harkirat kaur(M.sc 1)

2.  Metagenomics (meta: beyond; beyond the simple genome
study) attempts to analyse the complex genomes contained
within a microbial niche.
 Hence, instead of collecting live microorganisms from a
microbial community to be cultured or observed in the
laboratory, the isolation of DNA can provide information
related to the diversity of the microorganisms thriving in
certain area and can reveal information related to their
functions and biological roles.

3.  Shotgun metagenomics also provides a means to
study unculturable microorganisms that are
otherwise difficult or impossible to analyze.
 Unlike capillary sequencing or PCR-based
approaches, next-generation sequencing (NGS)
allows researchers to sequence thousands of
organisms in parallel.
 With the ability to combine many samples in a
single sequencing run and obtain high sequence
coverage per sample, NGS-based metagenomic
sequencing can detect very low abundance
members of the microbial community that may be
missed or are too expensive to identify using other
methods
 16S rRNA Sequencing is another method used for
metagenomics studies.

4. Metagenomics can be divided into two
areas of research:-
1. Environmental single gene surveys : It is
based on single-gene surveys and is
focused on the study of particular genes
that are amplified using polymerase chain
reaction, sequenced and analysed
2.Random shotgun: All genes are involve in
sequencing all of the DNA isolated from a
particular sample with theoretical full
coverage of all genes.

5.  First generation sequencing sanger sequencing
 Second-generation sequencing It is based on the generation of polymerase
colonies or polonies has now replaced Sanger sequencing for small-sized
genomes and environmental genomics.
 Third-generation sequencing A full coverage for shotgun metagenomics will
be achieved only with the advent of third-generation sequencing technology
which is expected to be capable of sequencing a single DNA/RNA strand
without amplification

7.  Take bacterial sample from the environment whose
genome has to be sequenced.
 The bacterium DNA strand to be sequenced directly by
shotgun sequencing method.
 Now decode a genome by shredding it into smaller
fragments of DNA.
 These sequences are known as ‘contigs’.
 These smaller fragments are sequenced using Sanger
method.
 The sequences of these fragments are then ordered.
 Fragments having overlapping nucleobase
sequences assemble to make one complete sequence.

8.  Total metagenomic DNA is extracted from a microbial community
sample, sheared and ligated into an expression vector and is
subsequently transformed into a suitable library host to create a
metagenomic library.
 The metagenomic libraries can be constructed from large DNA
fragments (25-200 Kb).
 The choice of vector will depend on the size of the insert to be
cloned.
 The bacterial artificial chromosome (BAC) supports DNA fragments
from 100-200Kb, cosmids from 25-35Kb, fosmids from 25-40Kb and
yeast artificial chromosome (YAC) over 40Kb.
 Libraries can be classified into 2 groups according to the size of their
inserts; small ones(less than 15 Kb) are constructed using plasmids
and large inserts are constructed in vectors like fosmids, cosmids
and BAC.

9.  The choice of suitable host is another important point during library
construction.
 E. coli is the most commonly used host strain because its genome is well
defined and easily transformable.
 But E.coli can express only 40% of the genes in the environmental samples.
 In such cases, a significant number of genes present in a metagenomic
library cannot be expressed in a single host. In this case, broad host range
vectors are able to replicate and express in more than one type of host.
 An example of broad range host vector is VecA (artificial chromosome vector
of E. coli).
 Broad spectrum hosts include Pseudomonas, Rhizobium and Streptomyces
which have 15 transcriptional factors while the most common host(E.coli)
has only 7.

10.  The library is then plated on media containing
antibiotics inhibitory to the wild type host to select
for metagenomic fragments conferring antibiotic
resistance.
 Metagenomic fragments present in colonies
growing on antibiotic selection media are then
PCR amplified and sequenced using high
throughput sequencing.
 Finally, reads are assembled and annotated in
order to identify the causative antibiotic resistance
genes.

12. ADVANTAGES
This method is useful in sequencing of
data of huge genome i.e. human genome.
As the breaking is random, we don’t need
any restriction enzyme and specific
enzyme.

13. DISADVANTAGE
As breaking of DNA is random, some
nucleotides are cleaved out or lost.
There is missing data in the sequence.
If overlapping region is not there, means
that part of genome remain unknown.

14.  Next generation sequencing and other high
throughout laboratory techniques are
circumventing laborious testing by active
replacement to traditional microbiological and
molecular test for identifying, typing, and
characterizing pathogens.
 Can sequence all DNA in a given sample(e.eg
bacteria, archaea, eukaryotes, parasites, and
viruses).
 Several NGS sequencing platforms are available;
each with their own advantages and
disadvantages differing in sequencing time, read
length, cost and others.

15. The illumina sequencing workflow is
composed of four basic steps:-
1. Sample preparation
2. Cluster generation
3. Sequencing
4. Data analysis

17. 1.Sample preparation :-The first step in sample preparation is
TAGMENTATION.
 It involves the transposon cleaving and tagging of the double- stranded
DNA with the adaptor.
 Through reduced cycle amplification additional molecule were introduced
such as sequencing primer binding sites, index and region complementary
to flow cell oligo.
2.Cluster formation :- It is process where each fragment is isothermally
amplified.
 A flow cell is a glass slide with lanes. Each lane is a channel composed of
two types of oligos.
 Hybridization is enable by the two types of oligos on the surface.
 This oligo is complementary to the adaptor region.
 A polymerase creates a complement of the hybridized fragment.
 The double stranded molecule is denature and the original strand washed
out.
 The strand is amplified through bridge amplification.

18.  In this process, the strand falls over and the adaptor region hybridize with
second type of oligo on the flow cell.
 Again, polymerases generates the complementary strands forming double
stranded bridge. This bridge denature resulting into two single stranded
copies of the molecule that are tagged with the flow cell.
 The process is then repeated over and over and simultaneously form billions
of clusters.
 In end of bridge amplification, the reverse strand are cleaved & washed
leaving behind the forward strand.
 3.Sequencing :- It begins with the extension of the first sequencing primer to
produce the double stranded molecule.
 In each cycle, four fluorescence tagged nucleotides competes for the addition
to growing one is incorporated based on the sequence of the template.
 The cluster are excited by the light source and a signal is emitted.
 This process is known as SEQUENCE BY SYNTHESIS.

19.  The number of cycles determined the length of the read. The
emission wavelength, along with the signal intensity determines
the base call.
 For a given cluster all identical strand are read simultaneously.
 After the completion of the first read the read product is washed
away.
 In this step the index 1 read primer is introduced and hybridize to
the template.The read is generated similar to the first read.
 After the completion of the index read , the read product is
washed off and the3’ ends of the template are deprotected.
 The template now folds over end binds the second oligo of the
flow cell.
 Index 2 is read in the same manner as index 1.
 Polymerase extend the second flow cell oligo forming a double
stranded bridge.