TEJASVI NAVADHITAMASTU

“Let our (the teacher and the taught) learning be radiant”

Let our efforts at learning be luminous and filled with joy, and endowed with the force of purpose

Dr. Prabhakar Singh

Department of Biochemistry
Faculty of Science, V.B.S. Purvanchal University, Jaunpur
PIN-222 003, (U.P.), INDIA.
Mobile No.: +91-9454695363
E-Mail: [email protected]
 VEER BAHADUR SINGH PURVANCHAL UNIVERSITY JAUNPUR-222003

E –content
Course: M.Sc.
Subject: Biochemistry; Biotechnology, Microbiology, Environmental Science
Topic: Instrumentation and Analytical Techniques
Subtopic: DNA Sequencing
Prepared by: Dr. Prabhakar Singh
Department : Biochemistry
Faculty : Science
Email: [email protected]
Contact: +91-9454695363

Note-This E-content has been prepared as a reading material for students without any
commercial interest. Original contributors are acknowledged.
DNA Sequencing
Maxam-Gilbert sequencing
Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on
chemical modification of DNA and subsequent cleavage at specific bases.
Also known as chemical sequencing, this method allowed purified samples of double-
stranded DNA to be used without further cloning. This method's use of radioactive labeling
and its technical complexity discouraged extensive use after refinements in the Sanger
methods had been made.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and
purification of the DNA fragment to be sequenced.

Chemical treatment then generates breaks at a small proportion of one or two of the four
nucleotide bases in each of four reactions (G, A+G, C, C+T).

The concentration of the modifying chemicals is controlled to introduce on average one

modification per DNA molecule. Thus a series of labeled fragments is generated, from the
radiolabeled end to the first "cut" site in each molecule.

The fragments in the four reactions are electrophoresed side by side in denaturing
acrylamide gels for size separation.

To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a
series of dark bands each corresponding to a radiolabeled DNA fragment, from which the
sequence may be inferred
Principle

The first DNA sequencing technique, using chemical reagents, was developed by
Maxam and Gilbert (1977). This method is briefly described as-

A strand of source DNA is labeled at one end with 32P. The two strands of DNA are then
separated. The labeled DNA is distributed into four samples (in separate tubes).

Each sample is subjected to treatment with a chemical that specifically destroys one
(G, C) or two bases (A + G, T + C) in the DNA. Thus, the DNA strands are partially
digested in four samples at sites G, A + G, T + C and C. This results in the formation of a
series of labeled fragments of varying lengths.

The actual length of the fragment depends on the site at which the base is destroyed
from the labeled end. Thus for instance, if there are C residues at positions 4, 7, and
10 away from the labeled end, then the treatment of DNA that specifically destroys C
will give labeled pieces of length 3, 6 and 9 bases.

The labeled DNA fragments obtained in the four tubes are subjected to
electrophoresis side by side and they are detected by autoradiograph.

The sequence of the bases in the DNA can be constructed from the bands on the
electrophoresis.
 Maxam and Gilbert method
for DNA sequencing

Fragments separated
by electrophoresis
SANGER SEQUENCING/ CHAIN-TERMINATOR/
DIDEOXY SEQUENCING

Sanger sequencing is a method of DNAsequencing based on the selective

incorporation of chain-terminating dideoxynucleotides by DNA
polymerase during in vitro DNA replication.

Developed by Frederick Sanger and colleagues in 1977, it was the most

widely used sequencing method for approximately 25 years.

This method was superseded in 1977 by two different methods, that of

Maxam and Gilbert (1977) and the chain-termination or dideoxy method
(Sanger et al.1977b). For a while the Maxam and Gilbert method, which
makes use of chemical reagents to bring about base-specific cleavage of
DNA, was the favored procedure.
 Principle
The chain-terminator or dideoxy procedure for DNA sequencing capitalizes on two
properties of DNA polymerases: (i) their ability to synthesize faithfully a complementary
copy of a single-stranded DNA template; and (ii) their ability to use 2′, 3′-
dideoxynucleotides as substrates.

Once the analog is incorporated at the growing point of the DNA chain,
the 3′end lacks a hydroxyl group and no longer is a substrate for chain elongation.

Thus, the growing DNA chain is terminated,

i.e. dideoxynucleotides act as chain
terminators. In practice, the Klenow
fragment of DNA polymerase is used
because this lacks the 5′→3′exonuclease
activity associated with the intact enzyme.

Initiation of DNA synthesis requires a

primer and usually this is a chemically
synthesized oligonucleotide which is
annealed close to the sequence being
analyzed.
DNA synthesis is carried out in the
presence of the four deoxynucleoside
triphosphates, one or more of which is
labeled with 32P, and in four separate
incubation mixes containing a low
concentration of one each of the four
dideoxynucleoside triphosphate
analogs.

Therefore, in each reaction there is a

population of partially synthesized
radioactive DNA molecules, each
having a common 5′end, but each
varying in length to a base-specific
3′end.
After a suitable incubation
period, the DNA in each
mixture is denatured and
electrophoresed in a
sequencing gel
DNA sequencing with
dideoxynucleoside
triphosphates as chain
terminators.

In this figure asterisks indicate

the presence of 32P and the
prefix “d” indicates the
presence of a
dideoxynucleotide.

At the top of the figure the

DNA to be sequenced is
enclosed within the box.

Note that unless the primer is

also labeled with a
radioisotope the smallest band
with the sequence
CGTAAGGdC will not be
detected by autoradiography
as no labeled bases were
incorporated.
Enlarged autoradiograph of a sequencing gel obtained with the chain-terminator
DNA sequencing method.
PYROSEQUENCING
Pyrosequencing is a DNA sequencing method that involves determining which one
in the four bases is going to incorporate at each step in the DNA Template during
DNA synthesis by DNA polymerase.

DNA polymerase moves along a single stranded template, each of the four
nucleoside triphosphates is fed sequentially and then removed.

If one of the four bases is incorporated then pyrophosphate is released and this is
detected in an enzyme cascade that emits light.

There are two variants of the pyrosequencing technique

1- In solid-phase pyrosequencing (Ronaghi et al.1996): The DNA to be

sequenced is immobilized and a washing step is used to remove the excess
substrate after each nucleotide addition.
2- In liquid-phase sequencing (Ronaghi et al. 1998b): A nucleotide
degrading enzyme (apyrase) is introduced to make a four-enzyme system.
Addition of this enzyme has eliminated the need for a solid support and
intermediate washing thereby enabling the pyrosequencing reaction to be
performed in a single tube.
(a) SOLID-PHASE PYROSEQUENCING: The four different nucleotides are added stepwise
to the immobilized primed DNA template and the incorporation event is followed
using the enzymes ATP sulfurylase and luciferase. After each nucleotide addition, a
washing step is performed to allow iterative addition.
(b) LIQUID-PHASE PYROSEQUENCING:
The primed DNA template and four enzymes involved in liquid-phase pyrosequencing are
placed in the well of a microtiter plate. The four different nucleotides are added stepwise
and incorporation is followed using the enzymes ATP sulfurylase and luciferase. The
nucleotides are continuously degraded by an enzyme allowing the addition of the
subsequent nucleotide. dXTP indicates one of the four nucleotides. (Redrawn with
permission from Ronaghi 2001.)
PRINCIPLE OF PYROSEQUENCING
1. Pyrosequencing is a method of DNA sequencing (determining the order
of nucleotides in DNA) based on the "sequencing by synthesis" principle. it
relies on the detection of pyrophosphate release on nucleotide incorporation,
rather than chain termination with dideoxynucleotides.
2. The desired DNA sequence is able to be determined by light emitted upon
incorporation of the next complementary nucleotide by the fact that only one
out of four of the possible A/T/C/G nucleotides are added and available at a
time so that only one letter can be incorporated on the single stranded
template.
3. Light emitted by the enzyme cascade is directly proportional to the amount of
pyrophosphate released, The intensity of the light determines if there are
continuous more than one of these “Nucleotide" in a row.

The general principle of pyrosequencing. A polymerase catalyzes incorporation of

nucleotides into a nucleic acid chain. As each nucleotide is incorporated a pyrophosphate
(PPi) molecule is released and incorporated into ATP by ATP sulfurylase. On addition of
luciferin and the enzyme luciferase, this ATP is degraded to AMP with the production of light.
TEMPLATE PREPARATION
Template preparation for pyrosequencing is very easy. After generation of the
template by the PCR, unincorporated nucleotides and PCR primers are
removed.

Two methods have been developed for this purification step-

1. In the first, biotinylated PCR product is captured on magnetic beads,

washed, and denatured with alkali (Ronaghi et al.1998a).

2. In the second method, akaline phosphatase or apyrase and exonuclease I are

added to the PCR product to destroy the nucleotides and primers,
respectively. The sequencing primer is then added and the mixture rapidly
heated and cooled. This inactivates the enzymes, denatures the DNA and
enables the primers to anneal to the templates (Nordstrom et al. 2000)

The acceptable read length of pyrosequencing currently is about 200 nucleotides, i.e.
much lessthan is achieved with Sanger sequencing. However, many modifications are
being made to the reaction conditions to extend the read length. For example, the
addition of ssDNA-binding protein to the reaction mixture increases read length,
facilitates sequencing of difficult templates, and provides flexibility in primer design.
PYROGRAM
Pyrogram of the raw data obtained from liquid-phase pyrosequencing.
Proportional signals are obtained for one, two, three, and four base
incorporations. Nucleotide addition, according to the order of nucleotides, is
indicated below the pyrogram and the obtained sequence is indicated above the
pyrogram. (Redrawn with permission from Ronaghi 2001.)
Sanger Sequencing Vs Pyrosequencing

Currently, a limitation of the method is that the lengths of individual reads of

DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the
800-1000 obtainable with chain termination methods (e.g. Sanger sequencing).

This can make the process of genome assembly more difficult, particularly for
sequences containing a large amount of repetitive DNA.

As of 2007, pyrosequencing is most commonly used for resequencing or

sequencing of genomes for which the sequence of a close relative is already
available.

The templates for pyrosequencing can be made both by solid phase template
preparation (streptavidin-coated magnetic beads) and enzymatic template
preparation (apyrase+exonuclease).

So Pyrosequencing can be differentiated into two types, namely Solid Phase

Pyrosequencing and Liquid Phase Pyrosequencing.
Next Generation Sequencing (NGS)
454 Pyrosequencing or Roche454

A parallelized version of pyrosequencing was developed by 454 Life

Sciences, which has since been acquired by Roche Diagnostics in
2007. The 454 DNA sequencer was the first next-generation sequencer
to become commercially successful.

454 utilizes the detection of pyrophosphate released by the DNA

polymerase reaction when adding a nucleotide to the template strain.

The method amplifies DNA inside water droplets in an oil solution

(emulsion PCR), with each droplet containing a single DNA template
attached to a single primer-coated bead that then forms a clonal colony.

The sequencing machine contains many picoliter-volume wells each

containing a single bead and sequencing enzymes.

Pyrosequencing uses luciferase to generate light for detection of the

individual nucleotides added to the nascent DNA, and the combined
data are used to generate sequence reads.
Roche currently manufactures two systems based on their pyrosequencing
technology: the GS FLX+ and the GS Junior System.[11] The GS FLX+ System
promises read lengths of approximately 1000 base pairs while the GS Junior System
promises 400 base pair reads.[12][13]
In 2009, Roche launched the GS Junior, a bench top version of the 454 sequencer
with read length up to 400bp, and simplified library preparation and data processing.
Advantages of 454 systems is their running speed, Manpower can be reduced with
automation of library preparation and semi-automation of emulsion PCR.
Disadvantage: Price of reagents is relatively more expensive compared with other
next-generation sequencers.
Homopolymer Error: 454 system is prone to errors when estimating the number of
bases in a long string of identical nucleotides. This is referred to as a homopolymer
error and occurs when there are 6 or more identical bases in row.

NOTE: In 2013 Roche announced that they would be shutting down development of
454 technology and phasing out 454 machines completely in 2016 but Roche
produces software tools which optimised the analysis of 454 sequencing data.
GS Run Processor converts raw images generated by a sequencing run into
intensity values. The process consists of two main steps: image processing and signal
processing. The software also applies normalization, signal correction, base-calling
and quality scores for individual reads.
 ION TORRENT /
ION SEMICONDUCTOR SEQUENCING
The technology was licensed from DNA Electronics Ltd, developed by Ion Torrent
Systems Inc. and was released in February 2010. Ion Torrent have marketed their
machine as a rapid, compact and economical sequencer that can be utilized in a large
number of laboratories as a bench top machine.

Roche's 454 Life Sciences is partnering with DNA Electronics on the development of a
long-read, high-density semiconductor sequencing platform using this technology.

This technology differs from

other sequencing-by-
synthesis technologies in
that no modified nucleotides
or optics are used. Ion
semiconductor sequencing
may also be referred to as
Ion Torrent sequencing, pH-
mediated sequencing,
silicon sequencing, or
semiconductor sequencing.
Principle
 Ion semiconductor sequencing is a method of DNA sequencing based on the
detection of hydrogen ions that are released during the polymerization of DNA.
This is a method of "sequencing by synthesis", during which
a complementary strand is built based on the sequence of a template strand.
 the incorporation of a deoxyribonucleoside triphosphate (dNTP) into a growing
DNA strand involves the formation of a covalent bond and the release
of pyrophosphate and a positively charged hydrogen ion A dNTP will only be
incorporated if it is complementary to the leading unpaired template nucleotide.
Ion semiconductor sequencing exploits these facts by determining if a
hydrogen ion is released upon providing a single species of dNTP to the
reaction.
 A microwell containing a template DNA strand to be sequenced is flooded with
a single species of deoxyribonucleotide triphosphate (dNTP). If the introduced
dNTP is complementary to the leading template nucleotide, it is incorporated
into the growing complementary strand. This causes the release of a hydrogen
ion that triggers an ISFET ion sensor, which indicates that a reaction has
occurred. If homopolymer repeats are present in the template sequence,
multiple dNTP molecules will be incorporated in a single cycle. This leads to a
corresponding number of released hydrogens and a proportionally higher
electronic signal.
The release of hydrogen ions indicate if zero, one or more nucleotides were
incorporated.
Released hydrogens ions are detected by an ion sensor. Multiple incorporations lead to
a corresponding number of released hydrogens and intensity of signal.
Microwells on a semiconductor chip that each contain many copies of one
single-stranded template DNA molecule to be sequenced and DNA
polymerase are sequentially flooded with unmodified A, C, G or T dNTP.
If an introduced dNTP is complementary to the next unpaired nucleotide on the
template strand it is incorporated into the growing complementary strand by the
DNA polymerase
If the introduced dNTP is
not complementary there
is no incorporation and
no biochemical reaction.
The hydrogen ion that is
released in the reaction
changes the pH of the
solution, which is
detected by an ISFET.
The unattached dNTP
molecules are washed
out before the next cycle
when a different dNTP
species is introduced.
Signal detection
 Beneath the layer of microwells is an ion sensitive layer, below which is an ISFET
ion sensor. All layers are contained within a CMOS semiconductor chip, similar to
that used in the electronics industry.

 Each chip contains an array of microwells with corresponding ISFET

detectors.Each released hydrogen ion then triggers the ISFET ion sensor. The
series of electrical pulses transmitted from the chip to a computer is translated
into a DNA sequence, with no intermediate signal conversion required.

 Because nucleotide incorporation events are measured directly by electronics,

the use of labeled nucleotides and optical measurements are avoided. Signal
processing and DNA assembly can then be carried out in software.

Sequencing characteristics
The per base accuracy achieved in house by Ion Torrent on the Ion Torrent Ion
semiconductor sequencer as of February 2011 was 99.6% based on 50 base reads,
with 100 Mb per run. The read-length as of February 2011 was 100 base pairs. The
accuracy for homopolymer repeats of 5 repeats in length was 98%. Later releases
show a read length of 400 base pairs.
Strengths

 The major benefits of ion semiconductor sequencing are rapid sequencing

speed and low upfront and operating costs. This has been enabled by the
avoidance of modified nucleotides and optical measurements.
 Because the system records natural polymerase-mediated nucleotide
incorporation events, sequencing can occur in real-time. In reality, the
sequencing rate is limited by the cycling of substrate nucleotides through the
system Ion Torrent Systems Inc., the developer of the technology, claims that
each incorporation measurement takes 4 seconds and each run takes about
one hour, during which 100-200 nucleotides are sequenced. If the
semiconductor chips are improved (as predicted by Moore’s law), the number
of reads per chip (and therefore per run) should increase.
 The cost of acquiring a pH-mediated sequencer from Ion Torrent Systems
Inc. at time of launch was priced at around $50,000 USD, excluding sample
preparation equipment and a server for data analysis. The cost per run is also
significantly lower than that of alternative automated sequencing methods, at
roughly $1,000
Limitations
 If homopolymer repeats of the same nucleotide (e.g. TTTTT) are present
on the template strand (strand to be sequenced) then multiple introduced
nucleotides are incorporated and more hydrogen ions are released in a
single cycle. This results in a greater pH change and a proportionally
greater electronic signal.This is a limitation of the system in that it is difficult
to enumerate long repeats. This limitation is shared by other techniques
that detect single nucleotide additions such as pyrosequencing.vSignals
generated from a high repeat number are difficult to differentiate from
repeats of a similar but different number; e.g., homorepeats of length 7 are
difficult to differentiate from those of length 8.

 Another limitation of this system is the short read length compared to other
sequencing methods such as Sanger sequencing or pyrosequencing.
Longer read lengths are beneficial for de novo genome assembly. Ion
Torrent semiconductor sequencers produce an average read length of
approximately 400 nucleotides per read.

 The throughput is currently lower than that of other high-throughput

sequencing technologies, although the developers hope to change this by
increasing the density of the chip
Application

 The developers of Ion Torrent semiconductor sequencing have

marketed it as a rapid, compact and economical sequencer that
can be utilized in a large number of laboratories as a bench top
machine.The company hopes that their system will take
sequencing outside of specialized centers and into the reach of
hospitals and smaller laboratories.[17] A January 2011 New York
Times article, "Taking DNA Sequencing to the Masses",
underlines these ambitions.[17]

 Due to the ability of alternative sequencing methods to achieve a

greater read length (and therefore being more suited to whole
genome analysis) this technology may be best suited to small
scale applications such as microbial genome sequencing,
microbial transcriptome sequencing, targeted
sequencing, amplicon sequencing, or for quality testing of
sequencing libraries
Illumina- Next-generation Sequencing
 llumina dye sequencing is a technique used to determine the series of base
pairs in DNA, also known as DNA sequencing.

 The reversible terminated chemistry concept was invented by Bruno Canard

and Simon Sarfati at the Pasteur Institute in Paris.

 It was developed by Shankar Balasubramanian and David Klenerman of

Cambridge University, who subsequently founded Solexa, a company later
acquired by Illumina.

 This sequencing method is based on reversible dye-terminators that enable

the identification of single bases as they are introduced into DNA strands.

 It can also be used for whole-genome and region

sequencing, transcriptome analysis, metagenomics,
small RNA discovery, methylation profiling, and genome-wide protein-nucleic
acid interaction analysis
Mechanism
1. Illumina sequencing technology works in three basic steps: amplify, sequence, and
analyze. The process begins with purified DNA. The DNA gets chopped up into
smaller pieces and given adapters, indices, and other kinds of molecular
modifications that act as reference points during amplification, sequencing, and
analysis.
2. The modified DNA is loaded onto a specialized chip where amplification and
sequencing will take place. Along the bottom of the chip are hundreds of thousands
of oligonucleotides (short, synthetic pieces of DNA). They are anchored to the chip
and able to grab DNA fragments that have complementary sequences. Once the
fragments have attached, a phase called cluster generation begins. This step makes
about a thousand copies of each fragment of DNA.
3. Next, primers and modified nucleotides enter the chip. These nucleotides have
reversible 3' blockers that force the polymerase to add on only one nucleotide at a
time as well as fluorescent tags. After each round of synthesis, a camera takes a
picture of the chip.
4. A computer determines what base was added by the wavelength of the fluorescent
tag and records it for every spot on the chip. After each round, non-incorporated
molecules are washed away.
5. A chemical deblocking step is then used in the removal of the 3’ terminal blocking
group and the dye in a single step. The process continues until the full DNA molecule
is sequenced.With this technology, thousands of places throughout the genome are
sequenced at once via massive parallel sequencing.
The DNA attaches to the flow cell via complementary sequences. The strand bends over and
attaches to a second oligo forming a bridge. A polymerase synthesizes the reverse strand. The
two strands release and straighten. Each forms a new bridge (bridge amplification). The result is a
cluster of DNA forward and reverse strands clones.
PROCEDURE
1. Tagmentation
The first step after DNA purification is tagmentation. Transposases randomly cut
the DNA into short segments ("tags"). Adapters are added on either side of the
cut points (ligation). Strands that fail to have adapters ligated are washed away

Double stranded DNA is cleaved by transposomes. The cut ends are repaired and adapters,
indices, primer binding sites, and terminal sites are added to each strand of the DNA.
2. Reduced cycle amplification

The next step is called reduced cycle amplification. During this step,
sequences for primer binding, indices, and terminal sequences are added.
Indices are usually six base pairs long and are used during DNA sequence
analysis to identify samples. Indices allow for up to 96 different samples to
be run together. During analysis, the computer will group all reads with the
same index together.
The terminal sequences are used for attaching the DNA strand to the flow
cell. Illumina uses a "sequence by synthesis" approach.
This process takes place inside of an acrylamide-coated glass flow
cell. The flow cell has oligonucleotides (short nucleotide sequences)
coating the bottom of the cell, and they serve to hold the DNA strands in
place during sequencing.
The oligos match the two kinds of terminal sequences added to the DNA
during reduced cycle amplification. As the DNA enters the flow cell, one of
the adapters attaches to a complementary oligo.
2. Bridge Amplification
Once attached, cluster generation can begin. The goal is to create hundreds of
identical strands of DNA. Some will be the forward strand; the rest, the reverse.
Clusters are generated through bridge amplification.

Polymerases move along a strand of DNA, creating its complementary strand. The
original strand is washed away, leaving only the reverse strand. At the top of the
reverse strand there is an adapter sequence. The DNA strand bends and attaches
to the oligo that is complementary to the top adapter sequence. Polymerases attach
to the reverse strand, and its complementary strand (which is identical to the
original) is made.

The now double stranded DNA is denatured so that each strand can separately
attach to an oligonucleotide sequence anchored to the flow cell. One will be the
reverse strand; the other, the forward. This process is called bridge amplification,
and it happens for thousands of clusters all over the flow cell at once

Millions of oligos line

the bottom of each
flow cell lane.
3. Clonal amplification

Over and over again, DNA strands will bend and attach to oligos.
Polymerases will synthesize a new strand to create a double stranded
segment, and that will be denatured so that all of the DNA strands in one area
are from a single source (clonal amplification).

Clonal amplification is important for quality control purposes. If a strand is

found to have an odd sequence, then scientists can check the reverse strand
to make sure that it has the complement of the same oddity. The forward and
reverse strands act as checks to guard against artifacts. Because Illumina
sequencing uses polymerases, base substitution errors have been
observed, especially at the 3' end. Paired end reads combined with cluster
generation can confirm an error took place. T

he reverse and forward strands should be complementary to each other, all

reverse reads should match each other, and all forward reads should match
each other. If a read is not similar enough to its counterparts (with which it
should be a clone), an error may have occurred. A minimum threshold of 97%
similarity has been used in some labs' analyses
4. Sequence by synthesis

At the end of clonal amplification, all of the reverse strands are washed off the flow
cell, leaving only forward strands. Primers attach to the forward strands and a
polymerase adds fluorescently tagged nucleotides to the DNA strand. Only one base
is added per round. A reversible terminator is on every nucleotide to prevent multiple
additions in one round. Using the four-colour chemistry, each of the four bases has a
unique emission, and after each round, the machine records which base was added.
Starting with the launch of the NextSeq and later the MiniSeq, Illumina introduced a
new two-colour sequencing chemistry. Nucleotides are distinguished by either one of
two colours (red or green), no colour ("black") or binding both colours (appearing
orange as a mixture between red and green).
Once the DNA strand has been read, the strand that was just added is washed away.
Then, the index 1 primer attaches, polymerizes the index 1 sequence, and is washed
away. The strand forms a bridge again, and the 3' end of the DNA strand attaches to
an oligo on the flow cell. The index 2 primer attaches, polymerizes the sequence, and
is washed away.
A polymerase sequences the complementary strand on top of the arched strand. They
separate, and the 3' end of each strand is blocked. The forward strand is washed
away, and the process of sequence by synthesis repeats for the reverse strand.
Data analysis

The sequencing occurs for millions of clusters at once, and each cluster has ~1,000
identical copies of a DNA insert. The sequence data is analyzed by finding fragments
with overlapping areas, called contigs, and lining them up. If a reference sequence is
known, the contigs are then compared to it for variant identification.
This piecemeal process allows scientists to see the complete sequence even though
an unfragmented sequence was never run; however, because Illumina read lengths
are not very long (HiSeq sequencing can produce read lengths around 90 bp long, it
can be a struggle to resolve short tandem repeat areas.

Also, if the sequence is de novo and so a reference

doesn't exist, repeated areas can cause a lot of difficulty in
sequence assembly. Additional difficulties include base
substitutions (especially at the 3' end of reads) by
inaccurate polymerases, chimeric sequences, and PCR-
bias, all of which can contribute to generating an incorrect
sequence.

Tagged nucleotides are added in order to the DNA strand. Each

of the four nucleotides have an identifying label that can be
excited to emit a characteristic wavelength. A computer records
all of the emissions, and from this data, base calls are made.
3 rd Generation Sequencing
Single-molecule real-time(SMRT) /
PacBio sequencing
Single-molecule real-time sequencing (SMRT) is a parallelized single
molecule DNA sequencing method. Single-molecule real-time sequencing
utilizes a zero-mode waveguide (ZMW).
A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a
single molecule of DNA as a template. The ZMW is a structure that creates
an illuminated observation volume that is small enough to observe only a
single nucleotide of DNA being incorporated by DNA polymerase.

Each of the four DNA bases is attached to one of four different fluorescent
dyes. When a nucleotide is incorporated by the DNA polymerase, the
fluorescent tag is cleaved off and diffuses out of the observation area of the
ZMW where its fluorescence is no longer observable.

A detector detects the fluorescent signal of the nucleotide incorporation, and

the base call is made according to the corresponding fluorescence of the dye.
The DNA sequencing is done on a chip that contains many ZMWs. Inside each ZMW, a single
active DNA polymerase with a single molecule of single stranded DNA template is immobilized
to the bottom through which light can penetrate and create a visualization chamber that allows
monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a
phospho-linked nucleotide incorporated by the DNA polymerase is detected as the DNA
synthesis proceeds which results in the DNA sequencing in real time.
1. ZERO-MODE WAVEGUIDE
The zero-mode waveguide (ZMW) is a nano photonic confinement structure that
consists of a circular hole in an aluminum cladding film deposited on a clear silica
substrate.
The ZMW holes are ~70 nm in diameter and ~100 nm in depth. Due to the behavior
of light when it travels through a small aperture, the optical field decays exponentially
inside the chamber.
The observation volume within an illuminated ZMW is ~20 zeptoliters (20 X
10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single
nucleotide can be readily detected

2. PHOSPHOLINKED NUCLEOTIDE
For each of the nucleotide bases, there is a corresponding fluorescent dye molecule
that enables the detector to identify the base being incorporated by the DNA
polymerase as it performs the DNA synthesis.

The fluorescent dye molecule is attached to the phosphate chain of the nucleotide.
When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is
cleaved off with the phosphate chain as a part of a natural DNA synthesis process
during which a phosphodiester bond is created to elongate the DNA chain. The
cleaved fluorescent dye molecule then diffuses out of the detection volume so that the
fluorescent signal is no longer detected
History
Pacific Biosciences [PacBio] commercialized SMRT sequencing in 2011,after
releasing a beta version of its RS instrument in late 2010.
On 19 Sep 2018, Pacific Biosciences [PacBio] released the Sequel 6.0
chemistry, synchronizing the chemistry version with the software version.
Performance is contrasted for large-insert libraries with high molecular weight
DNA versus shorter-insert libraries below ~15,000 bases in length. For larger
templates average read lengths are up to 30,000 bases. For shorter-insert
libraries, average read length are up to 100,000 bases while reading the same
molecule in a circle. The latter shorter-insert libraries then yield up to 50 billion
bases from a single SMRT Cell

8M Chip
In April 2019 the company released a new SMRT Cell with eight million
ZMW's, increasing the expected throughput per SMRT Cell by a factor of
eight. Early access customers in March 2019 reported throughput over 58
customer run cells of 250 GB of raw yield per cell with templates about 15 kb in
length, and 67.4 GB yield per cell with templates in higher weight molecules.
System performance is now reported in either high-molecular-weight continuous
long reads or in pre-corrected HiFi (aka CCS) reads. For high-molecular-weight
reads roughly half of all reads are longer than 50 kb in length
Application
1. Single-molecule real-time sequencing may be applicable for a broad range of genomics
research.
2. For de novo genome sequencing, read lengths from the single-molecule real-time
sequencing are comparable to or greater than that from the Sanger sequencing method
based on dideoxynucleotide chain termination.
3. The same DNA molecule can be resequenced independently by creating the circular
DNA template and utilizing a strand displacing enzyme that separates the newly
synthesized DNA strand from the template. In August 2012, scientists from the Broad
Institute published an evaluation of SMRT sequencing for SNP calling.
4. The dynamics of polymerase can indicate whether a base is methylated. Scientists
demonstrated the use of single-molecule real-time sequencing for detecting methylation
and other base modifications. In 2012 a team of scientists used SMRT sequencing to
generate the full methylomes of six bacteria. In November 2012, scientists published a
report on genome-wide methylation of an outbreak strain of E. coli
5. Long reads make it possible to sequence full gene isoforms, including the 5' and 3'
ends. This type of sequencing is useful to capture isoforms and splice variants.
6. SMRT sequencing has several applications in reproductive medical genetics research
when investigating families with suspected parental gonadal mosaicism. Long reads
enable haplotype phasing in patients to investigate parent-of-origin of mutations. Deep
sequencing enables determination of allele frequencies in sperm cells, of relevance for
estimation of recurrence risk for future affected offspring.
OXFORD NANOPORE

Oxford Nanopore Technologies has begun shipping early versions of

its nanopore sequencing MinION sequencer to selected labs.

The device is four inches long and gets power from a USB port. MinION
decodes DNA directly as the molecule is drawn at the rate of 450
bases/second through a nanopore suspended in a membrane.

Changes in electric current indicate which base is present. It is 60 to 85

percent accurate, compared with 99.9 percent in conventional machines.
Even inaccurate results may prove useful because it produces long read
lengths.

GridION is a slightly larger sequencer that processes up to five MinION flow

cells at once.

PromethION is another (unreleased) product that will use as many as

100,000 pores in parallel, more suitable for high volume sequencing
In July 2015, a group published on nanopore sequencing of an
influenza genome, noting “A complete influenza virus genome was
obtained that shared greater than 99% identity with sequence data
obtained from the Illumina Miseq and traditional Sanger-
sequencing.
The laboratory infrastructure and computing resources used to
perform this experiment on the MinION nanopore sequencer would
be available in most molecular laboratories around the world.
Using this system, the concept of portability, and thus sequencing
influenza viruses in the clinic or field is now tenable.“ In a paper and
accompanying editorial published in October 2015, a group of
MinION users wrote, “At the time of this writing, around a dozen
reports have emerged recounting utility of the MinION for de novo
sequencing of viral, bacterial, and eukaryotic genomes.”.
Other Products of Oxford Nanopore

 MinION: this portable protein nanopore sequencing USB device has been
commercially available since May 2015

 GridION X5: this desktop device has been commercially available since March
2017. The device processes up to five MinION Flow Cells and enables generation
of up to 100 Gb of data per run.

 PromethION: this desktop, high throughput device will be available through an

access programme that opened for registration in July 2015. The device contains
channels for 144,000 nanopores (in comparison to MinION’s 512).

 VolTRAX: this device, currently in development, is designed for automated sample

preparation so that users do not need a laboratory or lab skills to run the
device. Registration for the early access programme was opened in October 2016.

 Metrichor: this spinout company from Oxford Nanopore was set up to provide
end to end solutions for biological analyses, using nanopore sensing technologies.

 SmidgION: a mobile phone sequencer announced in May 2016, currently in

development
PICODROP OR NANOQUANTA ANALYZER
The Picodrop Microliter UV/Vis Spectrophotometer is the only system that allows you to use
the best format for your assay without compromise. Use our patented disposable UVpette
Tips or the unique drop-and-read TrayCell for tip-free applications

Use the In-tip detection method for a range between 5-1200ng/ul of dsDNA or for an
extended range switch the holder to use the TrayCell and benefit from the extended
concentration range up to 8500ng/ul dsDNA.

Range of interchangeable cuvette accessories including compatibility with

TrayCell, standard cuvettes and dip probes
No cleaning required when using tips and just a simple wipe when using the Cell

Sample volumes down to 1uL

No dilutions necessary
Kinetic measurements in both tip or cuvette
100% Sample recovery
Zero cross-contamination
3 Second detection time
Scanning wavelength range from 220nm to 950nm
AUTOMATED DNA SEQUENCING

 DNA sequencing in the recent years is carried out by an automated

DNA sequencer. In this technique, flourescent tags are attached to
chainterminating nucleotides (dideoxynucleotides).

 This tag gets incorporated into the DNA molecules, while

terminating new strand synthesis. Four different fluorescent dyes
are used to identify chain-terminating reactions in a sequencing gel.

 The DNA bands are separated by electrophoresis and detected by

their fluorescence. Recently, four dyes that exhibit strong
absorption in laser are in use for automated sequencing.
Synthesis of new DNA fragments in the presence of dideoxynucleotides
(*the size of the new DNA is variable, depending on the chain termination).
Sequence of the newly synthesized DNA fragment (complementary to original strand).
ADVANTAGES OF AUTOMATED SEQUENCING :

 It is a rapid and accurate technique.

 Automated DNA sequencer can accurately sequence up to

100,000 nucleotides per day.

 The cost works out to be not more than $0.2 per

nucleotide.

 Automated DNA sequencing has been successfully used in

the human genome project
METHOD READ LENGTH ADVANTAGES DISADVANTAGES
Single-molecule real- 30,000 bp (N50); Fast. Detects 4mC, 5mC, Moderate throughput.
time sequencing (Pacific maximum read length 6mA. Equipment can be very
Biosciences) >100,000 bases expensive.
Ion semiconductor (Ion up to 600 bp Less expensive equipment. Homopolymer errors.
Torrent sequencing) Fast.
Pyrosequencing (454) 700 bp Long read size. Fast. Runs are expensive.
Homopolymer errors.
Sequencing by MiniSeq, NextSeq: 75–300 bp; Potential for high sequence Equipment can be very
synthesis (Illumina) MiSeq: 50–600 bp; HiSeq 2500: yield, depending upon expensive. Requires high
50–500 bp; HiSeq 3/4000: 50– sequencer model and concentrations of DNA.
300 bp; HiSeq X: 300 bp desired application.
Combinatorial probe BGISEQ-50: 35-50bp; MGISEQ
anchor synthesis (cPAS- 200: 50-200bp; BGISEQ-500,
BGI/MGI) MGISEQ-2000: 50-300bp
Sequencing by ligation 50+35 or 50+50 bp Low cost per base. Slower than other
(SOLiD sequencing) methods. Has issues
sequencing palindromic
sequences.
Nanopore Sequencing Read length (up to 2,272,580 Longest individual reads. Lower throughput than
bp reported Accessible user community. other machines, Single read
Portable (Palm sized). accuracy in 90s.
GenapSys Sequencing Around 150 bp single-end Low-cost of instrument
($10,000)
Chain termination 400 to 900 bp Useful for many applications. More expensive and
(Sanger sequencing) impractical for larger
sequencing projects. This
method also requires the
time-consuming step of
plasmid cloning or PCR.
 References

1. Voet D, Voet J. .G “Biochemistry”, 4th Edition, John Wiley & Sons, Inc.,2010.
ISBN: 978-0-470-57095-1
2. David L. Nelson; Michael M. Cox “Lehninger Principles of Biochemistry”
Seventh Edition, Macmillan 2017, ISBN:9781464187957
3. Wilson, K., Walker, J. (eds.); Cambridge University Press, Cambridge, 2000, 784
pp., ISBN 0‐521‐65873‐X (paperback)
4. Primrose S B, Twyman R. Principles of Gene Manipulation and Genomics, 7th
Edition. John Wiley & Sons, Inc, 2006. ISBN: 978-1-405-13544-3

All the original contributors of the concept and findings published are
gratefully acknowledged while preparing the e-content for the
students of Biochemistry and allied sciences