Next generation sequencing

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SUBMITTED TO
S.PALANIANANTH
Ist MSC Microbiology
Dr. S.Sivasankara
Narayani
SUBMITTED BY
Presented to

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INTRODUCTION:
• Next Generation Sequencing (NGS) is a powerful platform that has enabled
the sequencing of thousands to millions of DNA molecules simultaneously.
• Next-generation sequencing (NGS), also known as high-throughput
sequencing, is the catch-all term used to describe a number of different
modern sequencing technologies.
• The high demand for low-cost sequencing has driven the development of
high-throughput sequencing which produce thousands or millions of
sequences at once.
• They are intended to lower the cost of DNA sequencing beyond what is
possible with standard dye-terminator methods.
• Thus, these recent technologies allow us to sequence DNA and RNA much
more quickly and cheaply than the previously used Sanger sequencing, and
as such have revolutionized the study of genomics and molecular biology.

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Classification:
Classified to different generations, NGS has led to overcome the limitations of conventional DNA
sequencing methods and has found usage in a wide range of molecular biology applications.
The generations it is classified into include:
First Generation
• Sanger Sequencing
Second Generation Sequencing
• Pyrosequencing
• Sequencing by Reversible Terminator Chemistry
• Sequencing by Ligation
Third Generation Sequencing
• Single Molecule Fluorescent Sequencing
• Single Molecule Real Time Sequencing
• Semiconductor Sequencing
• Nanopore Sequencing
Fourth Generation Sequencing
• Aims conducting genomic analysis directly in the cell.

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Sanger Sequencing:
• Sanger Sequencing utilizes a high fidelity DNA-dependent polymerase to generate a
complimentary copy to a single stranded DNA template In each reaction a single primer,
complementary to the template, initiates a DNA synthesis reaction from its 3’ end.
Deoxynucleotides or nucleotides, which are the monomers of DNA, are added one after the
other in a template-dependent manner forming phospho-diester bonds between the 3’
hydroxyl of the growing end of the primer and the 5’ tri-phosphate group of the incoming
nucleotide
• Each reaction also contains a mixture of four di-deoxynucleotides, one for each DNA base
(i.e. A, G, T,and C). These di-deoxynucleotides resemble the DNA monomers enough to allow
incorporation into the growing strand, however, they differ from natural deoxynucleotides
in two ways:
• 1) they lack a 3’ hydroxyl group which is required for further DNA extension resulting in
chain termination once incorporated in the DNA molecule, and
• 2) each di-deoxynucleotide has a unique fluorescent dye attached to it allowing for
automatic detection of the DNA sequence

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Sanger Sequencing:
Many copies of different-length DNA fragments are generated in each reaction, terminated at
all of the nucleotide positions of the template molecule by one of the di-deoxynucleotides
The reaction mixtures are loaded on the sequencing machine, either manually onto slab gels
or automatically with capillaries, and are electrophoresed to separate the DNA molecules by
size.
The DNA sequence is read through the fluorescent emission of the di-deoxynucleotide as it
flows through the gel (Modern day Sanger Sequencing instruments use capillary based
automated electrophoresis, which typically analyzes 8–96 sequencing reactions
simultaneously.

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Next generation methods of DNA sequencing have three general steps:
• Library preparation: libraries are created using random fragmentation of
DNA, followed by ligation with custom linkers
• Amplification: the library is amplified using clonal amplification methods
and PCR
• Sequencing: DNA is sequenced using one of several different approaches

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LIBRARY PREPARATION:
• Firstly, DNA is fragmented either enzymatically or by sonication (excitation using ultrasound) to
create smaller strands. Adaptors (short, double-stranded pieces of synthetic DNA) are then ligated to
these fragments with the help of DNA ligase, an enzyme that joins DNA strands. The adaptors enable
the sequence to become bound to a complementary counterpart.
• Adaptors are synthesised so that one end is 'sticky' whilst the other is 'blunt' (non-cohesive) with the
view to joining the blunt end to the blunt ended DNA.
• This could lead to the potential problem of base pairing between molecules and therefore dimer
formation.
• To prevent this, the chemical structure of DNA is utilised, since ligation takes place between the 3′-
OH and 5′-P ends.
• By removing the phosphate from the sticky end of the adaptor and therefore creating a 5′-OH end
instead, the DNA ligase is unable to form a bridge between the two termini

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LIBRARY PREPARATION
In order for sequencing to be
successful, the library fragments need
to be spatially clustered in PCR
colonies or 'polonies' as they are
conventionally known, which consist
of many copies of a particular library
fragment. Since these polonies are
attached in a planar fashion, the
features of the array can be
manipulated enzymatically in
parallel. This method of library
construction is much faster than the
previous labour intensive procedure of
colony picking and E. coli cloning used
to isolate and amplify DNA for Sanger
sequencing, however, this is at the
expense of read length of the
fragments.

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AMPLIFICATION:
• Library amplification is required so that the received signal from the
sequencer is strong enough to be detected accurately. With enzymatic
amplification, phenomena such as 'biasing' and 'duplication' can occur
leading to preferential amplification of certain library fragments. Instead,
there are several types of amplification process which use PCR to create
large numbers of DNA clusters.

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Emulsion PCR:
• Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to the
formation of micro wells
In order for the sequencing process to be successful, each micro well should contain one bead with one
strand of DNA (approximately 15% of micro wells are of this composition). The PCR then denatures the
library fragment leading two separate strands, one of which (the reverse strand) anneals to the bead.
The annealed DNA is amplified by polymerase starting from the bead towards the primer site. The
original reverse strand then denatures and is released from the bead only to re-anneal to the bead to
give two separate strands.

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Emulsion PCR:
• These are both amplified to give two DNA strands attached to the bead. The process is then repeated
over 30-60 cycles leading to clusters of DNA.
• This technique has been criticised for its time consuming nature, since it requires many steps
(forming and breaking the emulsion, PCR amplification, enrichment etc) despite its extensive use in
many of the NGS platforms.
• It is also relatively inefficient since only around two thirds of the emulsion micro reactors will actually
contain one bead. Therefore an extra step is required to separate empty systems leading to more
potential inaccuracies

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Bridge PCR:
• The surface of the flow cell is densely coated with primers that are complementary to the
primers attached to the DNA library fragments (The DNA is then attached to the surface of
the cell at random where it is exposed to reagents for polymerase based extension.
• On addition of nucleotides and enzymes, the free ends of the single strands of DNA attach
themselves to the surface of the cell via complementary primers, creating bridged
structures.
• Enzymes then interact with the bridges to make them double stranded, so that when the
denaturation occurs, two single stranded DNA fragments are attached to the surface in
close proximity. Repetition of this process leads to clonal clusters of localised identical
strands. In order to optimise cluster density, concentrations of reagents must be monitored
very closely to avoid overcrowding.

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Bridge PCR:
SEQUENCING:
• Sequencing: DNA is sequenced using one of several different approaches

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Pyrosequencing:
principle:
• Pyrosequencing is non-electrophoretic, bioluminescence method that measures the release of
inorganic pyrophosphate by proportionally converting it into visible light using a series of enzymatic
reaction.
• 454 sequencing was the first commercially available advanced sequencing technique. It was
introduced in 2005 by the 454 Corporation.
Step-1
• DNA is first denatured into single strands ,joined to adapters at either end of the fragmented DNA
and attached to microscopic beads
Step-2
• The DNA on the beads is amplified by an emulsion PCR. PCR amplified allowing up to 1 million
identical fragments around one bead

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Pyrosequencing:
Step-3
• Each bead is then placed in a well of a Pico Titer tube, which is put into a flow cell where it is
incubated with DNA polymerase, ATP sulfurylase, luciferase, and apyrase long with the substrates
luciferin and adenosine 5’- phosphosulfate (ASP).
Step-4
• The first of four deoxyribonucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase
catalyzes the incorporation of the deoxyribon Nucleotide triphosphate into the DNA strand, if it is
complementary to the base in the template strand. Each incorporation event is accompanied by
release of pyrophosphate(PPi) in a quantity equimolar to the amount of incorporated nucleotide

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Pyrosequencing:
Step-5
• ATP sulfuryla sequantitatively converts PPito ATP in the presence of adenosine5
phosphosulfate(APS). This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin
that generates visible light in amounts that are proportion alto the amount of ATP. The light produced
in the luciferase- catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as
a peak in a PyrogramT”4. The height of each peak (light signal) is proportion alto the number of
nucleotides incorporated
Step-6 Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincorporated dNTPs.
This switches off the light and regenerates the reaction solution. The next dNTP is then added.

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Pyrosequencing:
Step-7
• Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio
triphosphate (dATPalfaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP)
since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process
continues, the complementary DNA strand is built up and the nucleotide sequence is determined from
the signal peaks in the Pyrogram.

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Ion torrent semiconductor sequencing:
• Ion torrent sequencing uses a "sequencing by synthesis" approach, in which a new DNA strand,
complementary to the target strand, is synthesized one base at a time. A semiconductor chip detects
the hydrogen ions produced during DNA polymerization
• Following polony formation using emulsion PCR, the DNA library fragment is flooded sequentially
with each nucleoside triphosphate (dNTP), as in pyrosequencing. The dNTP is then incorporated into
the new strand if complementary to the nucleotide on the target strand. Each time a nucleotide is
successfully added, a hydrogen ion is released, and it detected by the sequencer's pH sensor. As in the
pyrosequencing method, if more than one of the same nucleotide is added, the change in pH/signal
intensity is correspondingly larger.
• Ion torrent sequencing is the first commercial
technique not to use fluorescence and camera
scanning; it is therefore faster and cheaper than
many of the other methods. Unfortunately, it can
be difficult to enumerate the number of identical
bases added consecutively. For example, it may be
difficult to differentiate the pH change for a
homorepeat of length 9 to one of length 10,
making it difficult to decode repetitive sequences.

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Illumina/Solexa sequencing
Principle :
Illumina sequencing was initially developed by Solexa Inc. and introduced in 2006; shortly after its
introduction, Solexa was purchased by Illumina Inc. This technique uses a flow cell surface that allows
for the detection of over 1 million individual reads. This flow surface has eight channels on it, so eight
separate sequencing experiments can be run per cell. The process uses the four nucleotides labeled with
four different fluorescent mole clues and involves the following steps.
STEP 1:
Prepare Genomic DNA Sample
• A strand cannot be sequenced if it is tolarge or if it is double stranded. Therefore the DNA is
fragmented into between 300 and 800 bps long through–
Sonication Nebulization Enzyme digestion
• Adapters are needed to be ligated no the ends of the fragments in order to get the sequence to anneal
to where the DNA sequence can be determined by the sequencing machine. Denature dsDNA into
ssDNA by heating to 95°

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Step-2-
Attach DNA to Surface In bridge amplification, the adapter constructs added to the DNA sequence of
interest have flow cell binding sites, P5 and P7, which allow the P5 and P7 regions of the single-stranded
library fragments to anneal to their complimentary oligo's on the flow cell surface. This means if one of
the DNA fragments P5 anneals to the flow cell, it will anneal by attaching to a P7 oligosthatis attached
to the flow cell, and vise versa. Several samples can be loaded the 8 lane flow cell for simultaneous
analysis
STEP 3:
Bridge Amplification In this step the sequence makes a kind of bridge shape when it is being copied.
Unlabeled nucleotides and polymerase enzyme are added to initiate the solid phase bridge amplification
FLOW CELL

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Step-4-Fragments Become Double Stranded The reagents needed to
sequence are added such as primers to start the sequencing,
nucleotides to form the new sequence, polymerase to actually
sequence the nucleotides together, and buffer to pH at an optimal
level for the enzymatic reaction. The flow cell oligo's act as primers
and a strand complimentary to the library fragment is synthesized.
step 5 Denature the Double-Stranded Molecules
The original strand is washed a way, leaving behind fragment copies
that are covalently bonded to the flow cell surface in a mixture of
orientations

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step-6-Complete Amplification The steps through of the addition of
the sequencing reagents, creation of double stranded DNA, and the
denaturation are repeated multiple times to create thousands of
identical copies of the same sequence in each cluster. In other word
search cluster is made of up thousands of the identical sequence and
each cluster is a different fragment of the original larger sequence of
interest.
Step-7-Determine First Base The P5 region is cleaved, resulting in
clusters containing only fragments which are attached by the P7
region. This ensures that all copies are sequenced in the same
direction. The sequencing primer anneals to the P5 end of the
fragment, and begins the sequencing by synthesis process. The
reagents needed to sequence are added such as primers to start the
sequencing, fluorescently labeled nucleotides to form and detect the
base added to each cluster ,polymerase to actually add the
nucleotides to the forming strand, and buffer to keep the pH at an
optimal level for the enzymatic reaction

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Step-8-Image First Base The unincorporated bases, which are the bases that were not attached by a
polymerase to one of newly forming sequences in the clusters, are washed away. Then the machine can
clearly detect the fluorescent signals that are left and record the base that was added to each of the
clusters. After that the block on the newly added base’s 3 prime part of the sugar is removed so that
another base will be able to added to it by polymerase. The fluorescent is also removed so tha tonly the
new base’s fluorescent is detected in the new cycle.

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step-9-Determine Second Base
• Add sequencing reagents
• Primers
• Polymerase
• Fluorescently labeled nucleotides
• Buffer
• Second base incorporated
Step-10-Image Second Chemistry Cycle
Again the image is detected by first washing a way the un incorporated bases.
Then the flow cell is prepared for the next cycle by removing the fluorescent and blocking the newly
added base so that polymerase can add another base to it.
Step-11-Sequencing Over Multiple Chemistry Cycles
The sequencing cycles are repeated to determine the sequence of bases in a fragment ,one base at a time.

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Step-12-Align Data • After the sequencing is finished they are aligned
• Each was once one larger sequence that had been fragmented • Needs To be realigned to find the
original sequence of the larger sequence . Bioinformatics tools are used to do this using a reference
sequence. A reference sequences digital nucleic acid sequence data base, assembled by scientists as are
presentative example of a species 'set of genes. SNPs are also call by the alignment tools as well. SNP
stands for Single Nucleotide Polymorphism . Using this variations in the new sequence can be found
population in which a Single Nucleotide — A, T, C or G — in the genome differs between members of a
biological species or paired chromosomes

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Advantages of NGS-
1. Construction of a sequencing library for
clonal amplification to generate sequencing
features.
2. No invivo cloning, transformation, colony
picking
3. Array-based sequencing
4. Higher degree of parallelism than
capillary-based sequencing

Next generation sequencing

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