DNA SEQUENCING is the process of determining the precise order of
nucleotides within a
DNA molecule. It includes any method or
technology that is used to determine the order of the four
bases—adenine , guanine , cytosine , and thymine —in a strand of
DNA. The advent of rapid
DNA sequencing methods has greatly
accelerated biological and medical research and discovery.
DNA sequences has become indispensable for basic
biological research, and in numerous applied fields such as medical
diagnosis , biotechnology , forensic biology , virology and biological
systematics . The rapid speed of sequencing attained with modern DNA
sequencing technology has been instrumental in the sequencing of
DNA sequences, or genomes of numerous types and species of
life, including the human genome and other complete
DNA sequences of
many animal, plant, and microbial species. An example of the
results of automated chain-termination
DNA sequences were obtained in the early 1970s by academic
researchers using laborious methods based on two-dimensional
chromatography . Following the development of fluorescence -based
sequencing methods with a
DNA sequencer ,
DNA sequencing has become
easier and orders of magnitude faster.
* 1 Applications
* 1.4 Medicine
* 2 The four canonical bases
* 3 History
* 4 Early
DNA sequencing methods
* 4.1 Sequencing of full genomes
* 4.2 High-throughput sequencing (HTP) methods
* 5 Basic methods
* 5.2 Chain-termination methods
* 6 Advanced methods and de novo sequencing
* 6.2 Bridge PCR
* 7 High-throughput methods
Massively parallel signature sequencing (MPSS)
* 7.3 454 pyrosequencing
* 7.4 Illumina (Solexa) sequencing
* 7.5 SOLiD sequencing
* 7.6 Ion Torrent semiconductor sequencing
DNA nanoball sequencing
* 7.8 Heliscope single molecule sequencing
* 7.9 Single molecule real time (SMRT) sequencing
* 7.10 Nanopore
* 8 Methods in development
* 8.1 Tunnelling currents
Sequencing by hybridization
* 8.3 Sequencing with mass spectrometry
* 8.4 Microfluidic
* 8.5 Microscopy-based techniques
* 8.6 RNAP sequencing
In vitro virus high-throughput sequencing
* 9 Sample preparation
* 10 Development initiatives
* 11 Computational challenges
* 11.1 Read trimming
* 12 Ethical issues
* 13 See also
* 14 References
* 15 External links
DNA sequencing may be used to determine the sequence of individual
genes , larger genetic regions (i.e. clusters of genes or operons ),
full chromosomes or entire genomes , of any organism.
is also the most efficient way to sequence
RNA or proteins (via their
open reading frames ). In fact,
DNA sequencing has become a key
technology in many areas of biology and other sciences such as
medicine, forensics , or anthropology .
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.
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
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 , for example.
Medical technicians may sequence genes (or, theoretically, full
genomes) from patients to determine if there is risk of genetic
diseases. This is a form of genetic testing , though some genetic
tests may not involve
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. Testing
DNA is a technique in which can detect
specific genomes in a
DNA strand to produce a unique and
individualized pattern. Every living organism ever created has a one
of a kind
DNA pattern, which can be determined through
It's extremely rare that two people have the exact same
DNA testing is highly successful.
THE FOUR CANONICAL BASES
The canonical structure of
DNA has four bases: thymine (T), adenine
(A), cytosine (C), and guanine (G).
DNA sequencing is the
determination of the physical order of these bases in a molecule of
DNA. However, there are many other bases that may be present in a
molecule. In some viruses (specifically, bacteriophage ), cytosine may
be replaced by hydroxy methyl or hydroxy methyl glucose cytosine. In
mammalian DNA, variant bases with methyl groups or phosphosulfate may
be found. Depending on the sequencing technique, a particular
modification, e.g., the 5mC (
5 methyl cytosine ) common in humans, may
or may not be detected.
Deoxyribonucleic acid (
DNA ) was first discovered and isolated by
Friedrich Miescher in 1869, but it remained understudied for many
decades because proteins, rather than DNA, were thought to hold the
genetic blueprint to life. This situation changed after 1944 as a
result of some experiments by
Oswald Avery , Colin MacLeod , and
Maclyn McCarty demonstrating that purified
DNA could change one strain
of bacteria into another. This was the first time that
DNA was shown
capable of transforming the properties of cells.
James Watson and
Francis Crick put forward their
double-helix model of DNA, based on crystallized X-ray structures
being studied by
Rosalind Franklin . According to the model,
composed of two strands of nucleotides coiled around each other,
linked together by hydrogen bonds and running in opposite directions.
Each strand is composed of four complementary nucleotides – adenine
(A), cytosine (C), guanine (G) and thymine (T) – with an A on one
strand always paired with T on the other, and C always paired with G.
They proposed such a structure allowed each strand to be used to
reconstruct the other, an idea central to the passing on of hereditary
information between generations.
Frederick Sanger , a pioneer of
sequencing. Sanger is one of the few scientists who was awarded two
Nobel prizes, one for the sequencing of proteins , and the other for
the sequencing of DNA.
The foundation for sequencing proteins was first laid by the work of
Fred Sanger who by 1955 had completed the sequence of all the amino
acids in insulin , a small protein secreted by the pancreas. This
provided the first conclusive evidence that proteins were chemical
entities with a specific molecular pattern rather than a random
mixture of material suspended in fluid. Sanger's success in sequencing
insulin greatly electrified x-ray crystallographers, including Watson
and Crick who by now were trying to understand how
DNA directed the
formation of proteins within a cell. Soon after attending a series of
lectures given by
Fred Sanger in October 1954, Crick began to develop
a theory which argued that the arrangement of nucleotides in DNA
determined the sequence of amino acids in proteins which in turn
helped determine the function of a protein. He published this theory
RNA sequencing was one of the earliest forms of nucleotide
sequencing. The major landmark of
RNA sequencing is the sequence of
the first complete gene and the complete genome of
Bacteriophage MS2 ,
identified and published by
Walter Fiers and his coworkers at the
University of Ghent (
Belgium ), in 1972 and 1976.
RNA sequencing methods require the creation of a cDNA
molecule which must be sequenced.
DNA SEQUENCING METHODS
The first method for determining
DNA sequences involved a
location-specific primer extension strategy established by
Ray Wu at
Cornell University in 1970.
DNA polymerase catalysis and specific
nucleotide labeling, both of which figure prominently in current
sequencing schemes, were used to sequence the cohesive ends of lambda
phage DNA. Between 1970 and 1973, Wu, R Padmanabhan and colleagues
demonstrated that this method can be employed to determine any DNA
sequence using synthetic location-specific primers. Frederick
Sanger then adopted this primer-extension strategy to develop more
DNA sequencing methods at the MRC Centre ,
Cambridge , UK and
published a method for "
DNA sequencing with chain-terminating
inhibitors" in 1977.
Walter Gilbert and
Allan Maxam at Harvard also
developed sequencing methods, including one for "
DNA sequencing by
chemical degradation". In 1973, Gilbert and Maxam reported the
sequence of 24 basepairs using a method known as wandering-spot
analysis. Advancements in sequencing were aided by the concurrent
development of recombinant
DNA technology, allowing
DNA samples to be
isolated from sources other than viruses.
SEQUENCING OF FULL GENOMES
The 5,386 bp genome of bacteriophage φX174 . Each coloured
block represents a gene.
The first full
DNA genome to be sequenced was that of bacteriophage
φX174 in 1977. Medical Research Council scientists deciphered the
DNA sequence of the
Epstein-Barr virus in 1984, finding it
contained 172,282 nucleotides. Completion of the sequence marked a
significant turning point in
DNA sequencing because it was achieved
with no prior genetic profile knowledge of the virus.
A non-radioactive method for transferring the
DNA molecules of
sequencing reaction mixtures onto an immobilizing matrix during
electrophoresis was developed by Pohl and co-workers in the early
1980s. Followed by the commercialization of the
"Direct-Blotting-Electrophoresis-System GATC 1500" by
GATC Biotech ,
which was intensively used in the framework of the EU
genome-sequencing programme, the complete
DNA sequence of the yeast
Saccharomyces cerevisiae chromosome II.
Leroy E. Hood 's laboratory
California Institute of Technology
California Institute of Technology announced the first
DNA sequencing machine in 1986. This was followed by
Applied Biosystems ' marketing of the first fully automated sequencing
machine, the ABI 370, in 1987 and by Dupont's Genesis 2000 which used
a novel fluorescent labeling technique enabling all four
dideoxynucleotides to be identified in a single lane. By 1990, the
National Institutes of Health
National Institutes of Health (NIH) had begun large-scale
sequencing trials on
Mycoplasma capricolum ,
Escherichia coli ,
Caenorhabditis elegans , and
Saccharomyces cerevisiae at a cost of
US$0.75 per base. Meanwhile, sequencing of human c
DNA sequences called
expressed sequence tags began in
Craig Venter 's lab, an attempt to
capture the coding fraction of the human genome . In 1995, Venter,
Hamilton Smith , and colleagues at The Institute for Genomic Research
(TIGR) published the first complete genome of a free-living organism,
Haemophilus influenzae . The circular chromosome
contains 1,830,137 bases and its publication in the journal Science
marked the first published use of whole-genome shotgun sequencing,
eliminating the need for initial mapping efforts.
By 2001, shotgun sequencing methods had been used to produce a draft
sequence of the human genome.
HIGH-THROUGHPUT SEQUENCING (HTP) METHODS
Several new methods for
DNA sequencing were developed in the mid to
late 1990s and were implemented in commercial
DNA sequencers by the
year 2000. Together these were called the "next-generation" or
"second-generation" sequencing methods.
On October 26, 1990,
Roger Tsien , Pepi Ross, Margaret Fahnestock and
Allan J Johnston filed a patent describing stepwise ("base-by-base")
sequencing with removable 3' blockers on
DNA arrays (blots and single
DNA molecules). In 1996,
Pål Nyrén and his student Mostafa Ronaghi
at the Royal Institute of Technology in
Stockholm published their
method of pyrosequencing .
On April 1, 1997, Pascal Mayer and Laurent Farinelli submitted
patents to the World Intellectual Property Organization describing DNA
colony sequencing. The
DNA sample preparation and random surface-PCR
arraying methods described in this patent, coupled to
Roger Tsien et
al.'s "base-by-base" sequencing method, is now implemented in Illumina
's Hi-Seq genome sequencers.
Lynx Therapeutics published and marketed Massively parallel signature
sequencing (MPSS), in 2000. This method incorporated a parallelized,
adapter/ligation-mediated, bead-based sequencing technology and served
as the first commercially available "next-generation" sequencing
method, though no
DNA sequencers were sold to independent
The large quantities of data produced by
DNA sequencing have also
required development of new methods and programs for sequence
analysis. Phil Green and Brent Ewing of the University of Washington
described their phred quality score for sequencer data analysis in
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
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.
The chain-termination method developed by
Frederick Sanger and
coworkers in 1977 soon became the method of choice, owing to its
relative ease and reliability. When invented, the chain-terminator
method used fewer toxic chemicals and lower amounts of radioactivity
than the Maxam and Gilbert method. Because of its comparative ease,
the Sanger method was soon automated and was the method used in the
first generation of
DNA sequencers .
Sanger sequencing is the method which prevailed from the 1980s until
the mid-2000s. Over that period, great advances were made in the
technique, such as fluorescent labelling, capillary electrophoresis,
and general automation. These developments allowed much more efficient
sequencing, leading to lower costs. The Sanger method, in mass
production form, is the technology which produced the first human
genome in 2001, ushering in the age of genomics . However, later in
the decade, radically different approaches reached the market,
bringing the cost per genome down from $100 million in 2001 to $10,000
ADVANCED METHODS AND DE NOVO SEQUENCING
DNA is fragmented into random pieces and cloned as a
DNA from individual bacterial clones is sequenced
and the sequence is assembled by using overlapping
Large-scale sequencing often aims at sequencing very long
such as whole chromosomes , although large-scale sequencing can also
be used to generate very large numbers of short sequences, such as
found in phage display . For longer targets such as chromosomes,
common approaches consist of cutting (with restriction enzymes ) or
shearing (with mechanical forces) large
DNA fragments into shorter DNA
fragments. The fragmented
DNA may then be cloned into a
DNA vector and
amplified in a bacterial host such as
Escherichia coli . Short DNA
fragments purified from individual bacterial colonies are individually
sequenced and assembled electronically into one long, contiguous
sequence. Studies have shown that adding a size selection step to
DNA fragments of uniform size can improve sequencing
efficiency and accuracy of the genome assembly. In these studies,
automated sizing has proven to be more reproducible and precise than
manual gel sizing.
The term "de novo sequencing" specifically refers to methods used to
determine the sequence of
DNA with no previously known sequence. De
novo translates from Latin as "from the beginning". Gaps in the
assembled sequence may be filled by primer walking . The different
strategies have different tradeoffs in speed and accuracy; shotgun
methods are often used for sequencing large genomes, but its assembly
is complex and difficult, particularly with sequence repeats often
causing gaps in genome assembly.
Most sequencing approaches use an in vitro cloning step to amplify
DNA molecules, because their molecular detection methods
are not sensitive enough for single molecule sequencing. Emulsion PCR
DNA molecules along with primer-coated beads in
aqueous droplets within an oil phase. A polymerase chain reaction
(PCR) then coats each bead with clonal copies of the
followed by immobilization for later sequencing. Emulsion PCR is used
in the methods developed by Marguilis et al. (commercialized by 454
Life Sciences ), Shendure and Porreca et al. (also known as "Polony
sequencing ") and SOLiD sequencing , (developed by
Agencourt , later
Applied Biosystems , now Life Technologies ). Emulsion PCR is also
used in the GemCode and Chromium platforms developed by 10x
Shotgun sequencing is a sequencing method designed for analysis of
DNA sequences longer than 1000 base pairs, up to and including entire
chromosomes. This method requires the target
DNA to be broken into
random fragments. After sequencing individual fragments, the sequences
can be reassembled on the basis of their overlapping regions.
Another method for in vitro clonal amplification is bridge PCR, in
which fragments are amplified upon primers attached to a solid surface
and form "
DNA colonies " or "
DNA clusters". This method is used in
Genome Analyzer sequencers . Single-molecule methods,
such as that developed by
Stephen Quake 's laboratory (later
commercialized by Helicos ) are an exception: they use bright
fluorophores and laser excitation to detect base addition events from
DNA molecules fixed to a surface, eliminating the need for
Multiple, fragmented sequence reads must be assembled together
on the basis of their overlapping areas.
High-throughput (formerly "next-generation") sequencing applies to
genome sequencing, genome resequencing, transcriptome profiling
RNA-Seq ), DNA-protein interactions (
ChIP-sequencing ), and epigenome
characterization. Resequencing is necessary, because the genome of a
single individual of a species will not indicate all of the genome
variations among other individuals of the same species.
The high demand for low-cost sequencing has driven the development of
high-throughput sequencing technologies that parallelize the
sequencing process, producing thousands or millions of sequences
concurrently. High-throughput sequencing technologies are intended
to lower the cost of
DNA sequencing beyond what is possible with
standard dye-terminator methods. In ultra-high-throughput sequencing
as many as 500,000 sequencing-by-synthesis operations may be run in
Comparison of high-throughput sequencing methods
ACCURACY (SINGLE READ NOT CONSENSUS)
READS PER RUN
TIME PER RUN
COST PER 1 MILLION BASES (IN US$)
SINGLE-MOLECULE REAL-TIME SEQUENCING (PACIFIC BIOSCIENCES)
10,000 bp to 15,000 bp avg (14,000 bp N50 ); maximum read length
87% single-read accuracy
50,000 per SMRT cell, or 500–1000 megabases
30 minutes to 4 hours
Longest read length. Fast. Detects 4mC, 5mC, 6mA.
Moderate throughput. Equipment can be very expensive.
ION SEMICONDUCTOR (ION TORRENT SEQUENCING)
up to 400 bp
up to 80 million
Less expensive equipment. Fast.
Long read size. Fast.
Runs are expensive.
SEQUENCING BY SYNTHESIS (ILLUMINA)
MiniSeq, NextSeq: 75-300 bp; MiSeq: 50-600 bp; HiSeq 2500: 50-500
bp; HiSeq 3/4000: 50-300 bp; HiSeq X: 300 bp
MiniSeq/MiSeq: 1-25 Million; NextSeq: 130-00 Million, HiSeq 2500:
300 million - 2 billion, HiSeq 3/4000 2.5 billion, HiSeq X: 3 billion
1 to 11 days, depending upon sequencer and specified read length
$0.05 to $0.15
Potential for high sequence yield, depending upon sequencer model
and desired application.
Equipment can be very expensive. Requires high concentrations of
SEQUENCING BY LIGATION (SOLID SEQUENCING)
50+35 or 50+50 bp
1.2 to 1.4 billion
1 to 2 weeks
Low cost per base.
Slower than other methods. Has issues sequencing palindromic
NANOPORE SEQUENCING )
Dependent on library prep, not the device, so user chooses read
length. (up to 500 kb reported)
~92–97% single read (up to 99.96% consensus)
dependent on read length selected by user
data streamed in real time. Choose 1 min to 48 hrs
$500–999 per Flow Cell, base cost dependent on expt
Very long reads, Portable (Palm sized)
Lower throughput than other machines, Single read accuracy in 90s.
CHAIN TERMINATION (SANGER SEQUENCING)
400 to 900 bp
20 minutes to 3 hours
Long individual reads. Useful for many applications.
More expensive and impractical for larger sequencing projects. This
method also requires the time consuming step of plasmid cloning or
MASSIVELY PARALLEL SIGNATURE SEQUENCING (MPSS)
The first of the high-throughput sequencing technologies, massively
parallel signature sequencing (or MPSS), was developed in the 1990s at
Lynx Therapeutics, a company founded in 1992 by
Sydney Brenner and Sam
Eletr . MPSS was a bead-based method that used a complex approach of
adapter ligation followed by adapter decoding, reading the sequence in
increments of four nucleotides. This method made it susceptible to
sequence-specific bias or loss of specific sequences. Because the
technology was so complex, MPSS was only performed 'in-house' by Lynx
Therapeutics and no
DNA sequencing machines were sold to independent
laboratories. Lynx Therapeutics merged with
Solexa (later acquired by
Illumina ) in 2004, leading to the development of
sequencing-by-synthesis, a simpler approach acquired from Manteia
Predictive Medicine , which rendered MPSS obsolete. However, the
essential properties of the MPSS output were typical of later
high-throughput data types, including hundreds of thousands of short
DNA sequences. In the case of MPSS, these were typically used for
DNA for measurements of gene expression levels.
Polony sequencing method, developed in the laboratory of George
M. Church at Harvard, was among the first high-throughput sequencing
systems and was used to sequence a full
E. coli genome in 2005. It
combined an in vitro paired-tag library with emulsion PCR, an
automated microscope, and ligation-based sequencing chemistry to
E. coli genome at an accuracy of >99.9999% and a cost
approximately 1/9 that of Sanger sequencing. The technology was
Agencourt Biosciences, subsequently spun out into
Agencourt Personal Genomics, and eventually incorporated into the
Applied Biosystems SOLiD platform.
Applied Biosystems was later
acquired by Life Technologies , now part of
Thermo Fisher Scientific .
454 Life Sciences § Technology
A parallelized version of pyrosequencing was developed by 454 Life
Sciences , which has since been acquired by
Roche Diagnostics . The
DNA inside water droplets in an oil solution
(emulsion PCR), with each droplet containing a single
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 . This technology provides intermediate
read length and price per base compared to
Sanger sequencing on one
Solexa and SOLiD on the other.
ILLUMINA (SOLEXA) SEQUENCING
Illumina dye sequencing
Solexa , now part of Illumina , was founded by Shankar
David Klenerman in 1998, and developed a
sequencing method based on reversible dye-terminators technology, and
engineered polymerases. The reversible terminated chemistry concept
was invented by Bruno Canard and Simon Sarfati at the Pasteur
Institute in Paris. It was developed internally at
Solexa by those
named on the relevant patents. In 2004,
Solexa acquired the company
Manteia Predictive Medicine in order to gain a massivelly parallel
sequencing technology invented in 1997 by Pascal Mayer and Laurent
Farinelli. It is based on "
DNA Clusters" or "
DNA colonies", which
involves the clonal amplification of
DNA on a surface. The cluster
technology was co-acquired with Lynx Therapeutics of California.
Solexa Ltd. later merged with Lynx to form
Solexa Inc. An
Illumina HiSeq 2500 sequencer
In this method,
DNA molecules and primers are first attached on a
slide or flow cell and amplified with polymerase so that local clonal
DNA colonies, later coined "
DNA clusters", are formed. To determine
the sequence, four types of reversible terminator bases (RT-bases) are
added and non-incorporated nucleotides are washed away. A camera takes
images of the fluorescently labeled nucleotides. Then the dye, along
with the terminal 3' blocker, is chemically removed from the DNA,
allowing for the next cycle to begin. Unlike pyrosequencing, the DNA
chains are extended one nucleotide at a time and image acquisition can
be performed at a delayed moment, allowing for very large arrays of
DNA colonies to be captured by sequential images taken from a single
camera. An Illumina MiSeq sequencer
Decoupling the enzymatic reaction and the image capture allows for
optimal throughput and theoretically unlimited sequencing capacity.
With an optimal configuration, the ultimately reachable instrument
throughput is thus dictated solely by the analog-to-digital conversion
rate of the camera, multiplied by the number of cameras and divided by
the number of pixels per
DNA colony required for visualizing them
optimally (approximately 10 pixels/colony). In 2012, with cameras
operating at more than 10 MHz A/D conversion rates and available
optics, fluidics and enzymatics, throughput can be multiples of 1
million nucleotides/second, corresponding roughly to 1 human genome
equivalent at 1x coverage per hour per instrument, and 1 human genome
re-sequenced (at approx. 30x) per day per instrument (equipped with a
Library preparation for the SOLiD platform Main article: 2
Applied Biosystems ' (now a Life Technologies brand) SOLiD technology
employs sequencing by ligation . Here, a pool of all possible
oligonucleotides of a fixed length are labeled according to the
sequenced position. Oligonucleotides are annealed and ligated; the
preferential ligation by
DNA ligase for matching sequences results in
a signal informative of the nucleotide at that position. Before
DNA is amplified by emulsion PCR. The resulting beads,
each containing single copies of the same
DNA molecule, are deposited
on a glass slide. The result is sequences of quantities and lengths
comparable to Illumina sequencing. This sequencing by ligation method
has been reported to have some issue sequencing palindromic sequences.
ION TORRENT SEMICONDUCTOR SEQUENCING
Ion semiconductor sequencing
Ion Torrent Systems Inc. (now owned by Life Technologies ) developed
a system based on using standard sequencing chemistry, but with a
novel, semiconductor based detection system. This method of sequencing
is based on the detection of hydrogen ions that are released during
the polymerisation of
DNA , as opposed to the optical methods used in
other sequencing systems. A microwell containing a template
to be sequenced is flooded with a single type of nucleotide . If the
introduced nucleotide 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 a
hypersensitive ion sensor, which indicates that a reaction has
occurred. If homopolymer repeats are present in the template sequence,
multiple nucleotides will be incorporated in a single cycle. This
leads to a corresponding number of released hydrogens and a
proportionally higher electronic signal. Sequencing of the
TAGGCT template with IonTorrent, PacBioRS and GridION
DNA NANOBALL SEQUENCING
DNA nanoball sequencing
DNA nanoball sequencing is a type of high throughput sequencing
technology used to determine the entire genomic sequence of an
organism. The company Complete
Genomics uses this technology to
sequence samples submitted by independent researchers. The method uses
rolling circle replication to amplify small fragments of genomic DNA
DNA nanoballs. Unchained sequencing by ligation is then used to
determine the nucleotide sequence. This method of
allows large numbers of
DNA nanoballs to be sequenced per run and at
low reagent costs compared to other high-throughput sequencing
platforms. However, only short sequences of
DNA are determined from
DNA nanoball which makes mapping the short reads to a reference
genome difficult. This technology has been used for multiple genome
sequencing projects and is scheduled to be used for more.
HELISCOPE SINGLE MOLECULE SEQUENCING
Helicos single molecule fluorescent sequencing
Heliscope sequencing is a method of single-molecule sequencing
Helicos Biosciences . It uses
DNA fragments with added
poly-A tail adapters which are attached to the flow cell surface. The
next steps involve extension-based sequencing with cyclic washes of
the flow cell with fluorescently labeled nucleotides (one nucleotide
type at a time, as with the Sanger method). The reads are performed by
the Heliscope sequencer. The reads are short, averaging 35 bp. In
2009 a human genome was sequenced using the Heliscope, however in 2012
the company went bankrupt.
SINGLE MOLECULE REAL TIME (SMRT) SEQUENCING
Single molecule real time sequencing
SMRT sequencing is based on the sequencing by synthesis approach. The
DNA is synthesized in zero-mode wave-guides (ZMWs) – small well-like
containers with the capturing tools located at the bottom of the well.
The sequencing is performed with use of unmodified polymerase
(attached to the ZMW bottom) and fluorescently labelled nucleotides
flowing freely in the solution. The wells are constructed in a way
that only the fluorescence occurring by the bottom of the well is
detected. The fluorescent label is detached from the nucleotide upon
its incorporation into the
DNA strand, leaving an unmodified DNA
strand. According to
Pacific Biosciences (PacBio), the SMRT technology
developer, this methodology allows detection of nucleotide
modifications (such as cytosine methylation). This happens through the
observation of polymerase kinetics. This approach allows reads of
20,000 nucleotides or more, with average read lengths of 5 kilobases.
Pacific Biosciences announced the launch of a new sequencing
instrument called the Sequel System, with 1 million ZMWs compared to
150,000 ZMWs in the PacBio RS II instrument. SMRT sequencing is
referred to as "third-generation " or "long-read" sequencing.
DNA passing through the nanopore changes its ion current. This
change is dependent on the shape, size and length of the
Each type of the nucleotide blocks the ion flow through the pore for a
different period of time. The method does not require modified
nucleotides and is performed in real time.
Nanopore sequencing is
referred to as "third-generation " or "long-read" sequencing, along
with SMRT sequencing.
Early industrial research into this method was based on a technique
called 'Exonuclease sequencing', where the readout of electrical
signals occurring at nucleotides passing by alpha-hemolysin pores
covalently bound with cyclodextrin . However the subsequently
commercial method, 'strand sequencing' sequencing
DNA bases in an
Two main areas of nanopore sequencing in development are solid state
nanopore sequencing, and protein based nanopore sequencing. Protein
nanopore sequencing utilizes membrane protein complexes such as
Hemolysin , MspA (Mycobacterium Smegmatis Porin A) or CssG, which
show great promise given their ability to distinguish between
individual and groups of nucleotides. In contrast, solid-state
nanopore sequencing utilizes synthetic materials such as silicon
nitride and aluminum oxide and it is preferred for its superior
mechanical ability and thermal and chemical stability. The
fabrication method is essential for this type of sequencing given that
the nanopore array can contain hundreds of pores with diameters
smaller than eight nanometers.
The concept originated from the idea that single stranded
DNA or RNA
molecules can be electrophoretically driven in a strict linear
sequence through a biological pore that can be less than eight
nanometers, and can be detected given that the molecules release an
ionic current while moving through the pore. The pore contains a
detection region capable of recognizing different bases, with each
base generating various time specific signals corresponding to the
sequence of bases as they cross the pore which are then evaluated.
Precise control over the
DNA transport through the pore is crucial for
success. Various enzymes such as exonucleases and polymerases have
been used to moderate this process by positioning them near the
METHODS IN DEVELOPMENT
DNA sequencing methods currently under development include reading
the sequence as a
DNA strand transits through nanopores (a method that
is now commercial but subsequent generations such as solid-state
nanopores are still in development), and microscopy-based
techniques, such as atomic force microscopy or transmission electron
microscopy that are used to identify the positions of individual
nucleotides within long
DNA fragments (>5,000 bp) by nucleotide
labeling with heavier elements (e.g., halogens) for visual detection
and recording. Third generation technologies aim to increase
throughput and decrease the time to result and cost by eliminating the
need for excessive reagents and harnessing the processivity of DNA
Another approach uses measurements of the electrical tunnelling
currents across single-strand
DNA as it moves through a channel.
Depending on its electronic structure, each base affects the
tunnelling current differently, allowing differentiation between
The use of tunnelling currents has the potential to sequence orders
of magnitude faster than ionic current methods and the sequencing of
DNA oligomers and micro-
RNA has already been achieved.
SEQUENCING BY HYBRIDIZATION
Sequencing by hybridization is a non-enzymatic method that uses a DNA
microarray . A single pool of
DNA whose sequence is to be determined
is fluorescently labeled and hybridized to an array containing known
sequences. Strong hybridization signals from a given spot on the array
identifies its sequence in the
DNA being sequenced.
This method of sequencing utilizes binding characteristics of a
library of short single stranded
DNA molecules (oligonucleotides),
DNA probes, to reconstruct a target
Non-specific hybrids are removed by washing and the target
eluted. Hybrids are re-arranged such that the
DNA sequence can be
reconstructed. The benefit of this sequencing type is its ability to
capture a large number of targets with a homogenous coverage. A large
number of chemicals and starting
DNA is usually required. However,
with the advent of solution-based hybridization, much less equipment
and chemicals are necessary.
SEQUENCING WITH MASS SPECTROMETRY
Mass spectrometry may be used to determine
Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry, or MALDI-TOF MS , has specifically been investigated as
an alternative method to gel electrophoresis for visualizing DNA
fragments. With this method,
DNA fragments generated by
chain-termination sequencing reactions are compared by mass rather
than by size. The mass of each nucleotide is different from the others
and this difference is detectable by mass spectrometry.
Single-nucleotide mutations in a fragment can be more easily detected
with MS than by gel electrophoresis alone. MALDI-TOF MS can more
easily detect differences between
RNA fragments, so researchers may
DNA with MS-based methods by converting it to RNA
The higher resolution of
DNA fragments permitted by MS-based methods
is of special interest to researchers in forensic science, as they may
wish to find single-nucleotide polymorphisms in human
DNA samples to
identify individuals. These samples may be highly degraded so forensic
researchers often prefer mitochondrial
DNA for its higher stability
and applications for lineage studies. MS-based sequencing methods have
been used to compare the sequences of human mitochondrial
samples in a
Federal Bureau of Investigation
Federal Bureau of Investigation database and from bones
found in mass graves of World War I soldiers.
Early chain-termination and TOF MS methods demonstrated read lengths
of up to 100 base pairs. Researchers have been unable to exceed this
average read size; like chain-termination sequencing alone, MS-based
DNA sequencing may not be suitable for large de novo sequencing
projects. Even so, a recent study did use the short sequence reads and
mass spectroscopy to compare single-nucleotide polymorphisms in
MICROFLUIDIC SANGER SEQUENCING
Sanger sequencing the entire thermocycling
DNA fragments as well as their separation by
electrophoresis is done on a single glass wafer (approximately 10 cm
in diameter) thus reducing the reagent usage as well as cost. In some
instances researchers have shown that they can increase the throughput
of conventional sequencing through the use of microchips. Research
will still need to be done in order to make this use of technology
Main article: Transmission electron microscopy
This approach directly visualizes the sequence of
DNA molecules using
electron microscopy. The first identification of
DNA base pairs within
DNA molecules by enzymatically incorporating modified bases,
which contain atoms of increased atomic number, direct visualization
and identification of individually labeled bases within a synthetic
DNA molecule and a 7,249 base-pair viral genome has
This method is based on use of
RNA polymerase (RNAP), which is
attached to a polystyrene bead. One end of
DNA to be sequenced is
attached to another bead, with both beads being placed in optical
traps. RNAP motion during transcription brings the beads in closer and
their relative distance changes, which can then be recorded at a
single nucleotide resolution. The sequence is deduced based on the
four readouts with lowered concentrations of each of the four
nucleotide types, similarly to the Sanger method. A comparison is
made between regions and sequence information is deduced by comparing
the known sequence regions to the unknown sequence regions.
IN VITRO VIRUS HIGH-THROUGHPUT SEQUENCING
A method has been developed to analyze full sets of protein
interactions using a combination of 454 pyrosequencing and an in vitro
RNA display method. Specifically, this method covalently links
proteins of interest to the mRNAs encoding them, then detects the mRNA
pieces using reverse transcription PCRs . The m
RNA may then be
amplified and sequenced. The combined method was titled IVV-HiTSeq and
can be performed under cell-free conditions, though its results may
not be representative of in vivo conditions.
The success of a
DNA sequencing protocol is dependent on the sample
preparation. A successful
DNA extraction will yield a sample with
long, non-degraded strands of
DNA which require further preparation
according to the sequencing technology to be used. For Sanger
sequencing, either cloning procedures or PCR are required prior to
sequencing. In the case of next generation sequencing methods, library
preparation is required before processing.
With the advent of next generation sequencing, Illumina and Roche 454
methods have become a common approach to transcriptomic studies
RNA can be extracted from tissues of interest and converted
DNA (cDNA) using reverse transcriptase—a DNA
polymerase that synthesizes a complimentary
DNA based on existing
RNA in a PCR-like manner. Complementary
DNA can be
processed the same way as genomic DNA, allowing the expression levels
of RNAs to be determined for the tissue selected.
Total cost of sequencing a human genome over time as calculated
by the NHGRI .
In October 2006, the
X Prize Foundation established an initiative to
promote the development of full genome sequencing technologies, called
Archon X Prize , intending to award $10 million to "the first Team
that can build a device and use it to sequence 100 human genomes
within 10 days or less, with an accuracy of no more than one error in
every 100,000 bases sequenced, with sequences accurately covering at
least 98% of the genome, and at a recurring cost of no more than
$10,000 (US) per genome."
Each year the National Human
Genome Research Institute , or NHGRI,
promotes grants for new research and developments in genomics . 2010
grants and 2011 candidates include continuing work in microfluidic,
polony and base-heavy sequencing methodologies.
The sequencing technologies described here produce raw data that
needs to be assembled into longer sequences such as complete genomes
(sequence assembly ). There are many computational challenges to
achieve this, such as the evaluation of the raw sequence data which is
done by programs and algorithms such as Phred and
Phrap . Other
challenges have to deal with repetitive sequences that often prevent
complete genome assemblies because they occur in many places of the
genome. As a consequence, many sequences may not be assigned to
particular chromosomes . The production of raw sequence data is only
the beginning of its detailed bioinformatical analysis. Yet new
methods for sequencing and correcting sequencing errors were
Sometimes, the raw reads produced by the sequencer are correct and
precise only in a fraction of their length. Using the entire read may
introduce artifacts in the downstream analyses like genome assembly,
snp calling, or gene expression estimation. Two classes of trimming
programs have been introduced, based on the window-based or the
running-sum classes of algorithms. This is a partial list of the
trimming algorithms currently available, specifying the algorithm
class they belong to:
Read Trimming Algorithms
NAME OF ALGORITHM
TYPE OF ALGORITHM
FASTX quality trimmer
FASTX quality trimmer
THIS SECTION NEEDS EXPANSION. You can help by adding to it . (May
Human genetics have been included within the field of bioethics since
the early 1970s and the growth in the use of
(particularly high-throughput sequencing) has introduced a number of
ethical issues. One key issue is the ownership of an individual's DNA
and the data produced when that
DNA is sequenced. Regarding the DNA
molecule itself, the leading legal case on this topic, Moore v.
Regents of the University of California (1990) ruled that individuals
have no property rights to discarded cells or any profits made using
these cells (for instance, as a patented cell line ). However,
individuals have a right to informed consent regarding removal and use
of cells. Regarding the data produced through
DNA sequencing, Moore
gives the individual no rights to the information derived from their
DNA sequencing becomes more widespread, the storage, security and
sharing of genomic data has also become more important. For
instance, one concern is that insurers may use an individual's genomic
data to modify their quote, depending on the perceived future health
of the individual based on their DNA. In May 2008, the Genetic
Information Nondiscrimination Act (GINA) was signed in the United
States, prohibiting discrimination on the basis of genetic information
with respect to health insurance and employment. In 2012, the US
Presidential Commission for the Study of Bioethical Issues reported
that existing privacy legislation for
DNA sequencing data such as GINA
Health Insurance Portability and Accountability Act
Health Insurance Portability and Accountability Act were
insufficient, noting that whole-genome sequencing data was
particularly sensitive, as it could be used to identify not only the
individual from which the data was created, but also their relatives.
Ethical issues have also been raised by the increasing use of genetic
variation screening, both in newborns, and in adults by companies such
23andMe . It has been asserted that screening for genetic
variations can be harmful, increasing anxiety in individuals who have
been found to have an increased risk of disease. For example, in one
case noted in Time , doctors screening an ill baby for genetic
variants chose not to inform the parents of an unrelated variant
linked to dementia due to the harm it would cause to the parents.
However, a 2011 study in
The New England Journal of Medicine
The New England Journal of Medicine has shown
that individuals undergoing disease risk profiling did not show
increased levels of anxiety.
Cancer genome sequencing
DNA field-effect transistor
DNA sequencing theory
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Nucleic acid sequence
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