This document discusses DNA sequencing methods and their history and applications. It covers first generation sequencing methods like Sanger sequencing and Maxam-Gilbert sequencing. It also covers next generation sequencing (NGS) methods like 454 pyrosequencing, Illumina sequencing, and Ion Torrent semiconductor sequencing. NGS allows high-throughput, massively parallel sequencing of DNA fragments. Template preparation for NGS involves fragmenting DNA, attaching fragments to beads, and emulsion PCR. The document provides details on the chemistry and detection methods used for different sequencing platforms.

1. DNA Sequencing Methods
and its Applications
SIDDARAJ B
PGS 17 AGR 7577

2. Determining the precise order of nucleotides within a DNA
molecule.
All the information required for the growth and development of
an organism is encoded in the DNA of its genome.
So,DNA sequencing is fundamental to genome analysis and
understanding the biological processes in general.
DNA SEQUENCING
The process of determining the order of bases adenine (A),
thymine (T), cytosine (C), and guanine (G) along a DNA strand.

4. By exploring this property of nucleic acids i.e. central dogma of molecular biology one can
correlate the linear structure of proteins and sequences of bases in DNA through reverse
engineering based on the principle of complementary base pairing between purines and
pyrimidines.
In this way each protein is responsible for particular trait in organism through its
involvement in particular metabolic pathway ,so decoding of genetic language is an
essential step in understanding about the diversity among different organisms and
difference between individuals of same species.
Final product of gene
expression which may be
involved in structural role or
enzymatic activity
“Central dogma of
molecular biology”

5. HISTORY OF DNA SEQUENCING:
 Early sequencing was performed with tRNA through a technique
developed by Richard Holley, who published the first structure of a
tRNA in 1964.
 The sequencing of DNA molecules began in the 1970s with
development of the Maxam-Gilbert method, and later the
Sanger method.
 1972 – Earliest nucleotide sequencing – RNA sequencing of
Bacteriophage MS2 by WALTER FIESERR.
 Originally developed by Frederick Sanger in 1975, most DNA
sequencing that occurs in medical and research laboratories today is
performed using sequencers employing variations of the Sanger
method.
 1977 - DNA sequencing FREDERICK SANGER by Chain
termination method.

6.  Chemical degradation method by ALLAN MAXAM and WALTER
GILBERT.
 1977 - First DNA genome to be sequenced of Bacteriophage ΦX174.
 1986 - LOREY and SMITH gave Semi automated sequencing.
 1987 – Applied biosystems marketed fully automated sequencing
machines.
 1995 – CRAIG VENTER, HAMILTON SMITH and collegues
published first complete genome sequence of Haemophilus influenzae.
 2003 – Human genome project.

7. Fundamental reasons for knowing the sequence
of DNA molecule:
To characterize the newly cloned DNA.
For predictions about its functions.
To facilitate manipulation of the molecule.
To confirm the identity of a clone or a mutation.
To check the fidelity of newly created mutation and ligation
junction.
Screening tool to identify polymorphisms and mutation in genes of
particular interest.

8. DNA Sequencing methods:
I. First generation sequencing
II. Next generation sequencing(NGS)
III. Third generation sequencing
IV. Methods in pipeline for development

9. I. First generation sequencing
A. Sanger’s Dideoxy or chain termination method:
 Developed by Frederick Sanger.
 Common method.
 Involves controlled synthesis of DNA to generate fragments
terminating at specific point.
Principle:
 Replacement of dNTPs with 2’, 3’ dideoxy NTPs in the DNA chain
terminates DNA synthesis.
 This is because these ddNTPs are nucleotide analogues that lacks the
3’ OH group that is necessary for phosphodiester bond formation and
chain elongation.

10. Method:
 The DNA sample is divided into four separate sequencing reactions, containing all
four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA
polymerase. To each reaction is added only one of the
four dideoxynucleotides(ddATP, ddGTP, ddCTP, or ddTTP), while the other added
nucleotides are ordinary ones.
 Following rounds of template DNA extension from the bound primer, the resulting
DNA fragments are heat denatured and separated by size using gel electrophoresis.
 The DNA bands may then be visualized by autoradiography or UV light and the DNA
sequence can be directly read off the X-ray film or gel image.
 A dark band in a lane indicates a DNA fragment that is the result of chain
termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or
ddTTP). The relative positions of the different bands among the four lanes, from
bottom to top, are then used to read the DNA sequence.

12. B. Chemical degradation method or Maxam and
Gilbert’s method
 Developed by Allan Maxam and Walter Gilbert in 1976–1977.
 Maxam–Gilbert sequencing rapidly became more popular, since
purified DNA could be used directly, while the initial Sanger method
required that each read start be cloned for production of single-
stranded DNA.
Principle:
 This method is based on nucleobase-specific partial chemical
modification of DNA and subsequent cleavage of the DNA backbone
at sites adjacent to the modified nucleotides.

13. Method:
 Maxam–Gilbert sequencing requires radioactive labeling at one 5′
end of the DNA fragment to be sequenced (typically by
a kinase reaction using gamma-32P ATP) and purification of the DNA.
 Chemical treatment 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).
5′ G A C G T G C A A C G A A 3′
5′ G A C G T G C A A C G A A 3′
Base Modification using Dimethyl sulphate
 Purine
• Adenine
• Guanine

14. Only DMS------- G
DMS+ Formic acid-------G+A
Cleavage of Sugar Phosphate backbone using Piperidine.
Base modification using Hydrazine
 Pyrimidine
• Cytosine
• Thymine
Hydrazine----- C+T
Hydrazine + NaCl--------C
Cleavage of Sugar Phosphate backbone using Piperidine.

16. II. Next generation sequencing(NGS)
 The next-generation DNA sequencing methods, also called massively
parallel sequencing (MPS) technologies, are faster and cheaper and require
much less template preparation than the Sanger–Coulson method.
 NGS technologies allow simultaneous sequencing of hundreds of thousands
to hundreds of millions of different DNA fragments (Schendure and Ji
2008).
 At present, there are four NGS methods, namely,
(1) 454 sequencing
(2) Solexa method
(3) ion semiconductor sequencing,
(4) Life/APG – SOLiD system

18. Template Preparation
 The template for sequencing is single-stranded DNA (ssDNA), which can be
prepared from genomic DNA, BAC clones, PCR products, and cDNA.
Genomic DNA and BAC clones are randomly sheared by sonication,
nebulization (mechanical shearing), or enzymatic digestion by DNase I to
produce fragments of suitable size, while PCR products and cDNA may not
need fragmentation.
 The fragments are now made single-stranded, and one single strand is
attached to a single capture bead.These beads, along with the amplification
reagents and the enzymes, are then enclosed in droplets of water-in-oil
mixture. The emulsion around each bead forms a micro-reactor isolated
from all other such beads.
 PCR amplification produces millions of copies of the single fragment
attached to each bead, and all these copies become attached to the same
capture bead. These beads form the in vitro library used for sequencing.

20. 454 Pyrosequencing (454 Life Sciences)
 Pyrosequencing is based on the 'sequencing by synthesis’ principle, where a
complementary strand is synthesized in the presence of polymerase enzyme.
 In contrast to using dideoxynucleotides to terminate chain amplification (as
in Sanger sequencing), pyrosequencing instead detects the release of
pyrophosphate when nucleotides are added to the DNA chain.
 It initially uses the emulsion PCR technique to construct the colonies
required for sequencing and removes the complementary strand.
 Next, a ssDNA sequencing primer hybridizes to the end of the strand
(primer-binding region), then the four different dNTPs are then sequentially
made to flow in and out of the wells over the colonies.

21.  When the correct dNTP is enzymatically incorporated into the strand, it
causes release of pyrophosphate.
 In the presence of ATP sulfurylase and adenosine, the pyrophosphate is
converted into ATP.
 This ATP molecule is used for luciferase-catalysed conversion of luciferin to
oxyluciferin, which produces light that can be detected with a camera.
 The relative intensity of light is proportional to the amount of base added
(i.e. a peak of twice the intensity indicates two identical bases have been
added in succession).
 Pyrosequencing, developed by 454 Life Sciences, was one of the early
successes of Next-generation sequencing; indeed,454 Life Sciences
produced the first commercially available Next-generation sequencer.
 However, the method was eclipsed by other technologies and, in 2013, new
owners Roche announced the closure of 454 Life Sciences and the
discontinuation of the 454 pyrosequencing platform.

23. Ion torrent semiconductor sequencing(Ion Torrent Systems
Inc.)
 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 colony 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.

24.  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
homo repeat of length 9 to one of length 10, making it difficult to
decode repetitive sequences.

26. Illumina (Solexa) sequencing/Reversible terminator sequencing
 Reversible terminator sequencing differs from the traditional Sanger
method in that, instead of terminating the primer extension irreversibly
using dideoxynucleotide, modified nucleotides are used in reversible
termination.
 While many other techniques use emulsion PCR to amplify the DNA
library fragments, reversible termination uses bridge PCR, improving the
efficiency of this stage of the process.
3′-O-blocked reversible terminators
 The mechanism uses a sequencing by synthesis approach,elongating the
primer in a stepwise manner.
 Firstly, the sequencing primers and templates are fixed to a solid support.
 The support is exposed to each of the four DNA bases,which have a
different fluorophore attached (to the nitrogenous base) in addition to a
3’-O-azidomethyl group.

27.  Only the correct base anneals to the target and is subsequently ligated to
the primer.
 The solid support is then imaged and nucleotides that have not been
incorporated are washed away and the fluorescent branch is cleaved
using TCEP (tris(2-carboxyethyl)phosphine).
 TCEP also removes the 3’-O-azidomethyl group, regenerating 3’- OH,
and the cycle can be repeated.

28.  The reversible termination group of 3′-unblocked reversible
terminators is linked to both the base and the fluorescence
group, which now acts as part of the termination group as well
as a reporter.
 The main disadvantage of this technique lies with the
poor read length.
 This technique was pioneered by Illumina, with their HiSeq
and MiSeq platforms.
 It also has a high data output of 600 Gb per run which takes
around 8 days to complete.

30. Life/APG – SOLiD system( Polony method )
 The Applied Biosystems, USA, commercialized the Polony method
in 2005 as SOLiD 3.0 platform (Schendure et al. 2005). SOLiD
stands for“sequencing by oligonucleotide ligation detection”
since this method achieves DNA sequencing by detecting
oligonucleotide ligation.
 The DNA sample is fragmented (fragment size 600 bp to 6 kb) and
processed in a manner similar to that for paired-end sequencing.
The beads along with the attached DNA molecules are
immobilized in a single layer in an acrylamide matrix on a glass
slide.
 An anchor primer is then hybridized to the adaptor sequence
attached to the template DNA.
 Sequencing is done by using a set of 16 oligonucleotides for
hybridization with the template DNA and ligation to the 5’ end of
the anchor primer/elongating chain.

31.  Each oligonucleotide is 8 bases long and is labeled with fluorophore
at the 5’ end, and each member of a set of 16 oligos has a unique
combination of two nucleotides at its 3’ end.
 At a given time, four specific oligonucleotides of the set, each
labeled with a different fluorophore, are added and allowed to pair at
their 3’ ends with the template DNA.
 The 3’ ends of the oligonucleotides paired with the template DNA
are ligated to the 5’ ends of the anchor primer molecules, the color of
fluorescence is recorded, and the unpaired 5’ ends of the
oligonucleotides are removed.
 A new set of four oligonucleotides is now added and the steps
of the first cycle are repeated. After five cycles of oligonucleotide
hybridization and ligation, the DNA is melted and the newly
synthesized DNA strands are removed.

32.  A new anchor primer is now added that is one base shorter than the
adaptor. Therefore, hybridization will begin one base upstream of
the site it began in the first cycle and into the adaptor sequence.
Again five cycles of hybridization and ligation are carried out, and
fluorescence from each cycle is recorded.
 The data from the two repeats of ligation reactions are compared
and analyzed to obtain the base sequence of the template strand.
 The SOLiD 3.0 platform gives sequence reads of ~50 bases and
generates over 20 Gb of total sequence per run, and each run takes
about 6–7 days. In 2011, SOLiD 5500 and SOLiD 5500 XL
systems were introduced; these systems give sequence data of up
to 300 Gb per run at 99.9 % accuracy (Edwards 2013).
 The average error rates are lower when a good quality reference
genome sequence is available and is used for error correction.

35. III. Third generation sequencing
 The third-generation sequencing methods do not use PCR
amplification for template preparation because they sequence single
DNA molecules (Schadt et al. 2010). For this reason, they are
often called single-molecule sequencing (SMS) methods.
 The technologies being developed for TGS are quite diverse and
include captured DNA polymerase, nanopores, electronic detection,
fluorescence energy transfer, and transmission electron microscopy.
1. Helicos Genetic Analysis System
2.Single-Molecule Real-Time Technology
3.The Nanopore Sequencing Technologies
 Several other highly innovative third-generation sequencing
technologies are in different stages of development which are based
on electron microscopy and electronics techniques.

36. 1. Helicos Genetic Analysis System
 In this method, 100–200-bp-long template fragments are subjected to
tailing to generate over 50-nucleotide-long poly(dA) tails at their
3’ ends, followed by blocking of the 3’ ends with a suitable treatment.
These fragments are now hybridized with primers [50-nt-long poly
(dT)] immobilized on a proprietary substrate within a glass
microfluidics cell having 25 channels.
 The dNTPs used for DNA synthesis are labeled with a bright
fluorophore, e.g., Cy3 and Cy5, so that the dNTPs incorporated into
single growing chains are readily detected. The four labeled dNTPs
(blocked with virtual terminators) are added sequentially, one at a
time. When molecules of a given dNTP are added, they will be
incorporated at the 3’ ends of those primers/ growing chains that are
associated with the template molecules having the base
complementary to the given dNTP at the proper site.

37.  fluorescence from the incorporated nucleotide is recorded separately
for each template molecule. The fluorophores of the incorporated
nucleotides and the terminators are removed, and the next dNTP along
with DNA polymerase is added. In this way, base sequence of each
template molecule is determined.
 The length of each read is ~35 bases, and up to one billion reads (and
35 Gb sequence data) can be obtained in one run. Since a virtual
terminator is used, a dNTP can be incorporated only at a
single site in a template during each reaction cycle even when its
complementary base occurs at two or more consecutive sites in the
template.
 Helicos BioSciences Corporation, USA, has commercialized this
process as Helicos Sequencer, HeliScope™. This system generates 1
Gb usable sequence data per day (~100 times greater than the first-
generation sequencers).

39. 2.Single-Molecule Real-Time Technology
 The single-molecule real-time (SMRT) technology was developed by
Pacific Biosciences, USA, and was commercialized as PACBIO RS.
This is the most revolutionary approach as it is based on single
molecules of DNA polymerase immobilized (by biotin–streptavidin
interaction) in zepoliter (10 21 L) wells of nanometers in diameter and
depth. Each well provides a detection volume of only 20 zepoliters.
High concentrations of the four dNTPs labeled with different
fluorophores are used for rapid DNA replication.
 Each DNA polymerase molecule will use a single DNA fragment as
template to add the fluorophore-labeled dNTPs to the primer/
growing chain.

40.  A highly focused detection system continuously records the
fluorescence from the nucleotides added to the growing chain in each
well. Since the fluorophore is attached to the phosphate moiety, it is
automatically removed as the next nucleotide is added, and it diffuses
out of the vicinity of DNA polymerase molecule.
 DNA polymerase can sequence the DNA fragment more than once,
producing multiple coverage of the same molecule (Schadt
et al. 2010; Deschamps and Campbell 2010).
 The sequencing platform generates 20 Gb sequence data per 30 min.
The average read length is ~1,000 bp, while the maximum read length
is over 10,000 bp.
 It can be used for detection of DNA methylation pattern by using
suitable software and for direct RNA sequencing without the need for
cDNA preparation.

42. 3.The Nanopore Sequencing Technologies
 In the case of most nanopore sequencing technologies, the DNA
molecule and its component bases are passed through an extremely
narrow hole (a nanopore), and the component bases are detected by
the changes in an electrical current or optical signal caused by
them (Schadt et al. 2010).
 Genetically engineered proteins or a suitable chemical compound
may be used to construct the nanopores. The Oxford Nanopore
Technologies, UK, uses BASE technology that creates the nanopore
by an engineered protein (α-hemolysin).
 Around 2,000–8,000 nanopores are placed in a lipid bilayer built on a
special application-specific integrated circuit chip. At the extracellular
face of the nanopore, an exonuclease is attached, while a synthetic
cyclodextrin-based sensor is linked at its inside surface; the
cyclodextrin acts as the binding site for DNA bases .

43.  The DNA sample to be analyzed is restriction digested, the digest is
placed onto the chip, and one DNA fragment associates with each
nanopore. An enzyme separates the two strands of the DNA duplex,
and the exonuclease digests one strand, one base at a time, and passes
these bases through the nanopore.
 Each base sequentially binds to the cyclodextrin located on the inside
of the nanopore. This binding creates a disturbance in the electric
current passing through the nanopore, which generates characteristic
signal for each DNA base. This signal is sensed by an electronic
device and is converted into base sequence data.
 Oxford Nanopore Technologies is preparing to launch two models,
namely, MinION and GridION, for sales. MinION USB stick DNA
sequencer is the size of a USB drive, is projected to cost less than US
$ 1,000, works with a PC, has a lifetime of 6 h from activation, and
would generate up to 150 Mb sequence data.

46. Comparison among different methods of DNA sequencing :

47. Online tools or softwares for DNA sequencing

49. IV. Methods in pipeline for development.
 Tunnelling currents DNA sequencing
 Sequencing by hybridization
 Sequencing with mass spectrometry
 Microfluidic Sanger sequencing
 Microscopy-based techniques
 RNAP sequencing
 In vitro virus high-throughput sequencing

50. DNA sequencing applications in Medicine and Biology:
 Molecular biology
Sequencing is used in molecular biology to study genomes and the
proteins they encode. Information obtained using sequencing allows
researchers to identify changes in genes, associations with diseases
and phenotypes, and identify potential drug targets.
 Evolutionary biology
Since DNA is an informative macromolecule in terms of transmission
from one generation to another, DNA sequencing is used
in evolutionary biology to study how different organisms are related
and how they evolved.

51.  Metagenomics
The field of metagenomics involves identification of organisms
present in a body of water, sewage, dirt, debris filtered from the air,
or swab samples from organisms. Knowing which organisms are
present in a particular environment is critical to research in
ecology, epidemiology, microbiology, and other fields. Sequencing
enables researchers to determine which types of microbes may be
present in a microbiome.
 Forensics
DNA sequencing may be used along with DNA profiling methods
for forensic identification and paternity testing.DNA testing has
evolved tremendously in the last few decades to ultimately link a
DNA print to what is under investigation. The DNA patterns in
fingerprint, saliva, hair follicles, etc. uniquely separate each living
organism from one another.

54. HGP-1990-2005
 The Human Genome Project (HGP) was an international scientific
research project with the goal of determining the sequence of
nucleotide base pairs that make up human DNA, and of identifying and
mapping all of the genes of the human genome from both a physical
and a functional standpoint.
 It remains the world's largest collaborative biological project. After the
idea was picked up in 1984 by the US government when the planning
started, the project formally launched in 1990 and was declared
complete in 2003.
 Funding came from the US government through the National Institutes
of Health (NIH) as well as numerous other groups from around the
world.

55.  The project was not able to sequence all the DNA found in human cells.
It sequenced only "euchromatic" regions of the genome, which make up
92% of the human genome.
 The sequencing of the human genome holds benefits for many fields,
from molecular medicine to human evolution.
 The Human Genome Project, through its sequencing of the DNA, can help
us understand diseases including: genotyping of specific viruses to direct
appropriate treatment; identification of mutations linked to different forms
of cancer; the design of medication and more accurate prediction of their
effects; advancement in forensic applied sciences.
 It also provided greatest information in biology and molecular biology which
revealed that only approximately 2 % genome i.e., Exons (expressed
sequences) of human beings is functional and remaining is JUNK DNA what
we call it as Introns ( intervening sequences).

56. The first
printout of
the human
genome to be
presented as
a series of
books,
displayed at
the Wellcome
Collection,
London

57. Cystic fibrosis syndrome-An Example of point mutation
 Cystic fibrosis (CF) is a genetic disorder that affects mostly
the lungs, but also the pancreas , liver , kidneys,
and intestine.Long-term issues include difficulty in
breathing and coughing up mucus as a result of
frequent lung infections. Other signs and symptoms may
include sinus infections, poor growth, fatty stool, clubbing
of the fingers and toes, and infertility in most
males.Different people may have different degrees of
symptoms.
 CF is caused by a mutation in the gene cystic fibrosis
transmembrane conductance regulator (CFTR).
 The most common mutation, ΔF508, is a deletion (Δ
signifying deletion) of three nucleotides that results in a
loss of the amino acid phenylalanine (F) at the 508th
position in the protein.
7q31.2.

59. Applications in crop improvement:
• Particular gene of interest conferring a specific trait can be identified.
• DNA sequencing data provides information to identify SNP’s which are
utilized very effectively in plant breeding programme.
• It also helps to identify some of the promoter and regulator sites
associated with particular gene, those can be utilized in construction of
plasmid vector for transformation in rDNA technology .
• Some of the important mutations which are beneficial can be studied
and utilized for breeding programme by comparing with reference
genome data which is already sequenced.
• DNA sequencing helps to know the consensus sequences in genome.

60. Ethical impacts of genome sequencing of human being
 Information about an individual's ethnic background and
parentage could become cause for discrimination.
 Because a person's DNA reveals so much information about
their physical state, it is sensitive information that must be
carefully guarded.
 In case of paternity issues and parental disputages in forensics
and criminal investigations it may pull the person under
criticism and his carrier and societal status may be affected.

61. 1.Marker assisted plant Breeding: Principles and practices-
B.D.Singh,A.K.Singh
2. Genetic Engineering by Smitha Rasthogi and
N. Pathak.
3. Mardis, E. R. A decade’s perspective on DNA sequencing
technology. Nature (2011) 470:198 – 203.
4. Metzker, M.L. Sequencing technologies – the next generation.
Nature Review Genetics(2010) 11:31 – 46.
5. https://en.wikipedia.org/wiki/DNA_sequencing
6. https://en.wikipedia.org/wiki/Human_Genome_Project
7. https://en.wikipedia.org/wiki/Cystic_fibrosis
8. https://www.slideshare.net/