DNA sequencing
Overview
 
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...

 bases—adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...

, guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...

, cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...

, and thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...

—in a molecule of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

.

Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...

, forensic biology
Forensic biology
Forensic biology is the application of biology to law enforcement.It includes the subdisciplines of Forensic anthropology, Forensic botany, Forensic entomology, Forensic odontology and various DNA or protein based techniques.- Applications :...

 and biological systematics
Systematics
Biological systematics is the study of the diversification of terrestrial life, both past and present, and the relationships among living things through time. Relationships are visualized as evolutionary trees...

. The advent of DNA sequencing has significantly accelerated biological research and discovery.
Encyclopedia
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...

 bases—adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...

, guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...

, cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...

, and thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...

—in a molecule of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

.

Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...

, forensic biology
Forensic biology
Forensic biology is the application of biology to law enforcement.It includes the subdisciplines of Forensic anthropology, Forensic botany, Forensic entomology, Forensic odontology and various DNA or protein based techniques.- Applications :...

 and biological systematics
Systematics
Biological systematics is the study of the diversification of terrestrial life, both past and present, and the relationships among living things through time. Relationships are visualized as evolutionary trees...

. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome
Human genome
The human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA. 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining...

, in the Human Genome Project
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...

. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.

The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography
Two-dimensional chromatography
Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. This is done by injecting the eluent from the first column onto a second column...

. Following the development of dye
Cyanine
Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...

-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster.

History

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
Bacteriophage MS2
The bacteriophage MS2 is an icosahedral, positive-sense single-stranded RNA virus that infects the bacterium Escherichia coli.-History:...

, identified and published by Walter Fiers
Walter Fiers
Walter Fiers is a Belgian molecular biologist.He obtained a degree of Engineer for Chemistry and Agricultural Industries at the University of Ghent in 1954, and started his research career as an enzymologist in the laboratory of Laurent Vandendriessche in Ghent. In 1956-57, he worked with Heinz...

 and his coworkers at the University of Ghent (Ghent
Ghent
Ghent is a city and a municipality located in the Flemish region of Belgium. It is the capital and biggest city of the East Flanders province. The city started as a settlement at the confluence of the Rivers Scheldt and Lys and in the Middle Ages became one of the largest and richest cities of...

, Belgium
Belgium
Belgium , officially the Kingdom of Belgium, is a federal state in Western Europe. It is a founding member of the European Union and hosts the EU's headquarters, and those of several other major international organisations such as NATO.Belgium is also a member of, or affiliated to, many...

), between 1972 and 1976.

Prior to the development of rapid DNA sequencing methods in the early 1970s by Frederick Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...

 at the University of Cambridge
University of Cambridge
The University of Cambridge is a public research university located in Cambridge, United Kingdom. It is the second-oldest university in both the United Kingdom and the English-speaking world , and the seventh-oldest globally...

, in England and Walter Gilbert
Walter Gilbert
Walter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts, on March 21, 1932...

 and Allan Maxam
Allan Maxam
Allan Maxam is one of the pioneers of molecular genetics. He was one of the contributors to develop a DNA sequencing method at Harvard University, while working as a student in the laboratory of Walter Gilbert....

 at Harvard
Harvard University
Harvard University is a private Ivy League university located in Cambridge, Massachusetts, United States, established in 1636 by the Massachusetts legislature. Harvard is the oldest institution of higher learning in the United States and the first corporation chartered in the country...

, a number of laborious methods were used. For instance, in 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.

The chain-termination method developed by Sanger and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability. It involves separating DNA bases from different DNA fragments
Chemical species
Chemical species are atoms, molecules, molecular fragments, ions, etc., being subjected to a chemical process or to a measurement. Generally, a chemical species can be defined as an ensemble of chemically identical molecular entities that can explore the same set of molecular energy levels on a...

.

Maxam–Gilbert sequencing

In 1976–1977, Allan Maxam
Allan Maxam
Allan Maxam is one of the pioneers of molecular genetics. He was one of the contributors to develop a DNA sequencing method at Harvard University, while working as a student in the laboratory of Walter Gilbert....

 and Walter Gilbert
Walter Gilbert
Walter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts, on March 21, 1932...

 developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases.
Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing, 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. However, with the improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.

The method requires radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA fragment to be sequenced. 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). For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are methylated using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the methylation of thymine for the C-only reaction. The modified DNAs are then cleaved by hot piperidine at the position of the modified base. 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.

Also sometimes known as "chemical sequencing", this method led to the Methylation Interference Assay used to map DNA-binding sites for DNA-binding proteins.

Chain-termination methods

Because the chain-terminator method (or Sanger method after its developer Frederick Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...

) is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method was the use of dideoxynucleotide
Dideoxynucleotides
Dideoxynucleotides, or ddNTPs, are nucleotides lacking a 3'-hydroxyl group on their deoxyribose sugar. Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks hydroxyl groups at both its 2' and 3' carbons...

 triphosphates (ddNTPs) as DNA chain terminators.

The classical chain-termination method requires a single-stranded DNA template, a DNA primer
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...

, a DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation. These ddNTPs will also be radioactively or fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

ly labelled for detection in automated sequencing machines. 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
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

. To each reaction is added only one of the four dideoxynucleotides
Dideoxynucleotides
Dideoxynucleotides, or ddNTPs, are nucleotides lacking a 3'-hydroxyl group on their deoxyribose sugar. Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks hydroxyl groups at both its 2' and 3' carbons...

 (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...

 group required for the formation of a phosphodiester bond
Phosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...

 between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.

The newly synthesized and labelled DNA fragments are heat denatured
DNA denaturation
Nucleic acid thermodynamics is the study of the thermodynamics of nucleic acid molecules, or how temperature affects nucleic acid structure. For multiple copies of DNA molecules, the melting temperature is defined as the temperature at which half of the DNA strands are in the double-helical state...

, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

 on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film
Radiography
Radiography is the use of X-rays to view a non-uniformly composed material such as the human body. By using the physical properties of the ray an image can be developed which displays areas of different density and composition....

 or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. 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 are then used to read (from bottom to top) the DNA sequence.

Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood
Leroy Hood
Leroy Hood is an American biologist. He won the 2011 Fritz J. and Dolores H. Russ Prize “for automating DNA sequencing that revolutionized biomedicine and forensic science” and the 2003 Lemelson-MIT Prize for inventing "four instruments that have unlocked much of the mystery of human biology" by...

 and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.

Dye-terminator sequencing

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

s.

Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis
Capillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...

 (see figure to the left).

This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

Challenges

Common challenges of DNA sequencing include poor quality in the first 15–40 bases of the sequence and deteriorating quality of sequencing traces after 700–900 bases. Base calling software typically gives an estimate of quality to aid in quality trimming.

In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector
Cloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments...

. In contrast, PCR-based cloning and emerging sequencing technologies based on pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...

 often avoid using cloning vectors. Recently, one-step Sanger sequencing (combined amplification and sequencing) methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.

Current methods can directly sequence only relatively short (300–1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. In all cases the use of a primer with a free 3' end is essential.

Automation and sample preparation

Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis
Capillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...

 for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...

 before loading onto the sequencer are performed separately. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks (generally located at the ends of the sequence). The accuracy of such algorithms is below visual examination by a human operator, but sufficient for automated processing of large sequence data sets.

Amplification and clonal selection

Large-scale sequencing often aims at sequencing very long DNA pieces, such as whole chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

s, 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 enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...

s) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, and amplified in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically
Sequence assembly
In bioinformatics, sequence assembly refers to aligning and merging fragments of a much longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 1000 bases,...

 into one long, contiguous sequence.

This method does not require any pre-existing information about the sequence of the DNA and is referred to as de novo sequencing. Gaps in the assembled sequence may be filled by primer walking
Primer walking
Primer walking is a sequencing method of choice for sequencing DNA fragments between 1.3 and 7 kilobases. Such fragments are too long to be sequenced in a single sequence read using the chain termination method. This method works by dividing the long sequence into several consecutive short ones...

. The different strategies have different tradeoffs in speed and accuracy; shotgun methods
Shotgun sequencing
In genetics, shotgun sequencing, also known as shotgun cloning, is a method used for sequencing long DNA strands. It is named by analogy with the rapidly-expanding, quasi-random firing pattern of a shotgun....

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 individual DNA molecules, because their molecular detection methods are not sensitive enough for single molecule sequencing. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. Polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

 (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Emulsion PCR is used in the methods by Marguilis et al. (commercialized by 454 Life Sciences
454 Life Sciences
454 Life Sciences, is a biotechnology company based in Branford, Connecticut. It is a subsidiary of Roche, and specializes in high-throughput DNA sequencing.-History and Major Achievements:...

), Shendure and Porreca et al. (also known as "Polony sequencing
Polony
Polony is a contraction of "polymerase colony," a small colony of DNA.Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase...

") and SOLiD sequencing
ABI Solid Sequencing
SOLiD is a next-generation sequencing technology developed by Life Technologies and has been commercially available since 2008. These next generation technologies generate hundreds of millions to billions of small sequence reads at one time...

, (developed by Agencourt
Agencourt
Agencourt is a commune in the Côte-d'Or department in Bourgogne in eastern France.-Population:-External links:...

, now Applied Biosystems
Applied Biosystems
Applied Biosystems, Inc. started as GeneCo , was the name of a pioneer biotechnology company founded in 1981 in Foster City, California, in the San Francisco Bay Area...

).

Another method for in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

clonal amplification is bridge PCR, where fragments are amplified upon primers attached to a solid surface, used in the Illumina
Illumina (company)
Illumina, Inc. is a company incorporated in April 1998 that develops, manufactures and markets integrated systems for the analysis of genetic variation and biological function. Using its technologies, the company provides a line of products and services that serve the sequencing, genotyping and...

 Genome Analyzer. Single-molecule methods, such as that developed by Stephen Quake's laboratory (later commercialized by Helicos
Helicos Biosciences
Helicos BioSciences Corporation, is a publicly-traded life science company headquartered in Cambridge, Massachusetts focused on genetic analysis technologies for the research, drug discovery and diagnostic markets. The firm's Helicos Genetic Analysis Platform was the first DNA-sequencing...

) is an exception: it uses bright fluorophores and laser excitation to detect base addition events from individual DNA molecules fixed to a surface, eliminating the need for molecular amplification.

High-throughput sequencing

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize
Multiplex (assay)
A multiplex assay is a type of laboratory procedure that simultaneously measures multiple analytes in a single assay. It is distinguished from procedures that measure one or a few analytes at a time...

 the sequencing process, producing thousands or millions of sequences at once. High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.

Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)

The first of the "next-generation" sequencing technologies, MPSS was developed in the 1990s at Lynx Therapeutics, a company founded in 1992 by Sydney Brenner
Sydney Brenner
Sydney Brenner, CH FRS is a South African biologist and a 2002 Nobel prize in Physiology or Medicine laureate, shared with H...

 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 machines were sold; when the merger with Solexa later led to the development of sequencing-by-synthesis, a more simple approach with numerous advantages, MPSS became obsolete. However, the essential properties of the MPSS output were typical of later "next-gen" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Lynx Therapeutics merged with Solexa in 2004, and this company was later purchased by Illumina.

Polony sequencing

Polony sequencing
Polony sequencing
Polony Sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church group in Harvard Medical School...

, developed in the laboratory of George Church
George Church
George Church is an American molecular geneticist. He is currently Professor of Genetics at Harvard Medical School, Professor of Health Sciences and Technology at Harvard and MIT, and a core faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard University.With...

 at Harvard, was among the first next-generation sequencing systems used to sequence a full genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing. The technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and ultimately incorporated into the Applied Biosystems SOLiD platform.

454 pyrosequencing

A parallelized version of pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...

 was developed by 454 Life Sciences
454 Life Sciences
454 Life Sciences, is a biotechnology company based in Branford, Connecticut. It is a subsidiary of Roche, and specializes in high-throughput DNA sequencing.-History and Major Achievements:...

, which has since been acquired by Roche Diagnostics
Roche Diagnostics
Roche Diagnostics Division is a subsidiary of Hoffmann-La Roche which manufactures equipment and reagents for research and medical diagnostic applications. Internally, it is organized into five major business areas: Roche Applied Science, Roche Professional Diagnostics, Roche Diabetes Care, Roche...

. 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 picolitre-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 read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.

Illumina (Solexa) sequencing

Solexa, now part of Illumina
Illumina (company)
Illumina, Inc. is a company incorporated in April 1998 that develops, manufactures and markets integrated systems for the analysis of genetic variation and biological function. Using its technologies, the company provides a line of products and services that serve the sequencing, genotyping and...

, developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). Four types of ddNTPs are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. 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 the next cycle.

SOLiD sequencing

Applied Biosystems
Applied Biosystems
Applied Biosystems, Inc. started as GeneCo , was the name of a pioneer biotechnology company founded in 1981 in Foster City, California, in the San Francisco Bay Area...

' SOLiD technology employs sequencing by ligation
Sequencing by ligation
Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand...

. 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 sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing.

Ion semiconductor sequencing

Ion Torrent Systems Inc. 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 ion
Hydrogen ion
Hydrogen ion is recommended by IUPAC as a general term for all ions of hydrogen and its isotopes.Depending on the charge of the ion, two different classes can be distinguished: positively charged ions and negatively charged ions....

s that are released during the polymerisation
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

 of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...

. If the introduced nucleotide is complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...

 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.

DNA nanoball sequencing

DNA nanoball sequencing
DNA nanoball sequencing
DNA nanoball sequencing is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Fluorescent probes bind to complementary DNA and the...

 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 that researchers submit from several projects. The method uses rolling circle replication
Rolling circle replication
Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids...

 to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent
Reagent
A reagent is a "substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs." Although the terms reactant and reagent are often used interchangeably, a reactant is less specifically a "substance that is consumed in the course of...

 costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome
Reference genome
A reference genome is a digital nucleic acid sequence database, assembled by scientists as a representative example of a species' genetic code. As they are often assembled from the sequencing of DNA from a number of donors, reference genomes do not accurately represent the genetic code of any...

 difficult. This technology has been used for multiple genome sequencing projects and is scheduled to be used for more.

Helioscope(TM) single molecule sequencing

Based on "true single molecule sequencing" technology, Helioscope sequencing uses DNA fragments with added polyA 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 Helioscope sequencer. The reads are short, up to 55 bases per run, but recent improvemend of the methodology allowes more accurate reads of homopolymers (stretches of one type of nucleotides) and RNA sequencing.

Single Molecule SMRT(TM) sequencing

SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesisd in so called 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 at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMTR 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 1000 nucleotides.

Single Molecule real time (RNAP) sequencing

This method is based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA 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 Sangers method).

Nanopore DNA sequencing

This method is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. The method has a potential of development as it does not require modified nucleotides, however single nucleotide resolution is not yet available.

VisiGen Biotechnologies approach

VisiGen Biotechnologies introduced a specially engineered DNA polymerase for use in their sequencing. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides. This approach allows reads performed at the spead at which polymerase incorporates nucleotides into the sequence (several hundred per second). The nucleotide fluorochrome is released after the incorporation into the DNA strand. The expected read lengths in this approach should reach 1000 nucleotides, however this will have to be confirmed.

Future methods

Sequencing by hybridization
Sequencing by Hybridization
Sequencing by Hybridization is a class of methods for determining the order in which nucleotides occur on a strand of DNA. Typically used for looking for small changes relative to a known DNA sequence....

is a non-enzymatic method that uses a DNA microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...

. 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. Mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

 may be used to determine mass differences between DNA fragments produced in chain-termination reactions.

DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores
Nanopore sequencing
Nanopore sequencing is a method under development since 1995 for determining the order in which nucleotides occur on a strand of DNA.A nanopore is simply a small hole, of the order of 1 nanometer in internal diameter...

, and microscopy-based techniques, such as AFM
Atomic force microscope
Atomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit...

 or transmission electron microscopy
Transmission electron microscopy DNA sequencing
Transmission electron microscopy DNA sequencing is an emerging third-generation, single-molecule sequencing technology that uses transmission electron microscopy techniques. DNA is visible under the electron microscope; however, it must be labeled with heavy atoms so that the DNA bases can be...

 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 polymerase.

In microfluidic Sanger sequencing
Microfluidic Sanger Sequencing
The completion of the Human Genome Project has been a cornerstone in the advancement of biological studies. The outcomes of obtaining a complete reference map of the human genome have ushered in the post-genome era of studies...

 the entire thermocycling amplification of 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 effective.

In October 2006, the X Prize Foundation
X Prize Foundation
The X PRIZE Foundation is a non-profit organization that designs and manages public competitions intended to encourage technological development that could benefit mankind....

 established an initiative to promote the development of full genome sequencing
Full genome sequencing
Full genome sequencing , also known as whole genome sequencing , complete genome sequencing, or entire genome sequencing, is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time...

 technologies, called the Archon X Prize
Archon X Prize
The Archon X Prize for Genomics, the second X Prize to be offered by the X Prize Foundation, based in Santa Monica, California, was announced on October 4, 2006. The Archon X Prize in genomics is a joint effort of the X Prize Foundation and the J...

, 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 NHGRI promotes grants for new research and developments in genomics
Genomics
Genomics is a discipline in genetics concerning the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis,...

. 2010 grants and 2011 candidates include continuing work in microfluidic, polony and base-heavy sequencing methodologies

Major landmarks in DNA sequencing

  • 1953
    1953 in science
    The year 1953 in science and technology involved some significant events, listed below.-Biochemistry:* April 25 - Francis Crick and James D...

     Discovery of the structure of the DNA double helix.
  • 1972
    1972 in science
    The year 1972 in science and technology involved some significant events, listed below.-Astronomy and space exploration:* January 5 - President of the United States Richard Nixon orders the development of a space shuttle program....

     Development of recombinant DNA
    Recombinant DNA
    Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...

     technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA.
  • 1977
    1977 in science
    The year 1977 in science and technology involved some significant events, listed below.- Astronomy and space exploration :* March 10 – Rings of Uranus discovered by Kuiper Airborne Observatory measurements of star occultation....

     The first complete DNA genome to be sequenced is that of bacteriophage φX174.
  • 1977
    1977 in science
    The year 1977 in science and technology involved some significant events, listed below.- Astronomy and space exploration :* March 10 – Rings of Uranus discovered by Kuiper Airborne Observatory measurements of star occultation....

     Allan Maxam
    Allan Maxam
    Allan Maxam is one of the pioneers of molecular genetics. He was one of the contributors to develop a DNA sequencing method at Harvard University, while working as a student in the laboratory of Walter Gilbert....

     and Walter Gilbert
    Walter Gilbert
    Walter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts, on March 21, 1932...

     publish "DNA sequencing by chemical degradation". Frederick Sanger
    Frederick Sanger
    Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...

    , independently, publishes "DNA sequencing with chain-terminating inhibitors".
  • 1984
    1984 in science
    The year 1984 in science and technology involved some significant events.-Astronomy and space exploration:* February 7 – Astronauts Bruce McCandless II and Robert L...

     Medical Research Council
    Medical Research Council (UK)
    The Medical Research Council is a publicly-funded agency responsible for co-ordinating and funding medical research in the United Kingdom. It is one of seven Research Councils in the UK and is answerable to, although politically independent from, the Department for Business, Innovation and Skills...

     scientists decipher the complete DNA sequence of the Epstein-Barr virus
    Epstein-Barr virus
    The Epstein–Barr virus , also called human herpesvirus 4 , is a virus of the herpes family and is one of the most common viruses in humans. It is best known as the cause of infectious mononucleosis...

    , 170 kb.
  • 1986
    1986 in science
    The year 1986 in science and technology involved many significant events, some listed below.-Astronomy and space exploration:* January 24 – Voyager 2 space probe makes first encounter with Uranus....

     Leroy E. Hood's laboratory at the California Institute of Technology
    California Institute of Technology
    The California Institute of Technology is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering...

     and Smith announce the first semi-automated DNA sequencing machine.
  • 1987
    1987 in science
    The year 1987 in science and technology involved many significant events, some listed below.-Astronomy:* February 23 – Supernova 1987a is observed, the first "naked-eye" supernova since 1604.* Asteroid 7816 Hanoi is discovered by Masahiro Koishikawa....

     Applied Biosystems markets first automated sequencing machine, the model ABI 370.
  • 1990
    1990 in science
    The year 1990 in science and technology involved some significant events.-Astronomy and space exploration:* April 24 – The Space Shuttle Discovery places the Hubble Space Telescope into orbit.-Computer science:...

     The U.S. National Institutes of Health
    National Institutes of Health
    The National Institutes of Health are an agency of the United States Department of Health and Human Services and are the primary agency of the United States government responsible for biomedical and health-related research. Its science and engineering counterpart is the National Science Foundation...

     (NIH) begins large-scale sequencing trials on Mycoplasma capricolum
    Mycoplasma capricolum
    Mycoplasma capricolum is a species of Mycoplasma bacteria. It is primarily a pathogen of goats, but has also been found in sheep and cows.-External links:* at MicrobeWiki...

    , Escherichia coli
    Escherichia coli
    Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

    , Caenorhabditis elegans
    Caenorhabditis elegans
    Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...

    , and Saccharomyces cerevisiae
    Saccharomyces cerevisiae
    Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

    (at US$0.75/base).
  • 1991
    1991 in science
    The year 1991 in science and technology involved many significant events, some listed below.-Astronomy and space exploration:* May 18 – Helen Sharman becomes the first British person in space, flying with the Soyuz TM-12 mission...

     Sequencing of human expressed sequence tag
    Expressed sequence tag
    An expressed sequence tag or EST is a short sub-sequence of a cDNA sequence. They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination. The identification of ESTs has proceeded rapidly, with approximately 65.9 million ESTs now available in...

    s begins in Craig Venter
    Craig Venter
    John Craig Venter is an American biologist and entrepreneur, most famous for his role in being one of the first to sequence the human genome and for his role in creating the first cell with a synthetic genome in 2010. Venter founded Celera Genomics, The Institute for Genomic Research and the J...

    's lab, an attempt to capture the coding fraction of the human genome.
  • 1995
    1995 in science
    The year 1995 in science and technology involved many significant events, listed below.-Archaeology:* January 18 - In southern France near Vallon-Pont-d'Arc a network of caves are discovered that contain paintings and engravings that are 17,000 to 20,000 years old.* Wes Linster discovers the first...

     Craig Venter
    Craig Venter
    John Craig Venter is an American biologist and entrepreneur, most famous for his role in being one of the first to sequence the human genome and for his role in creating the first cell with a synthetic genome in 2010. Venter founded Celera Genomics, The Institute for Genomic Research and the J...

    , Hamilton Smith
    Hamilton O. Smith
    Hamilton Othanel Smith is an American microbiologist and Nobel laureate.Smith was born on August 23, 1931, and graduated from University Laboratory High School of Urbana, Illinois. He attended the University of Illinois at Urbana-Champaign, but in 1950 transferred to the University of California,...

    , and colleagues at The Institute for Genomic Research
    The Institute for Genomic Research
    The Institute for Genomic Research was a non-profit genomics research institute founded in 1992 by Craig Venter in Rockville, Maryland, United States. It is now a part of the J. Craig Venter Institute.-History:...

     (TIGR) publish the first complete genome of a free-living organism, the bacterium Haemophilus influenzae
    Haemophilus influenzae
    Haemophilus influenzae, formerly called Pfeiffer's bacillus or Bacillus influenzae, Gram-negative, rod-shaped bacterium first described in 1892 by Richard Pfeiffer during an influenza pandemic. A member of the Pasteurellaceae family, it is generally aerobic, but can grow as a facultative anaerobe. H...

    . The circular chromosome contains 1,830,137 bases and its publication in the journal Science marks the first use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts.
  • 1996
    1996 in science
    The year 1996 in science and technology involved many significant events, listed below.-Astronomy and space exploration:* January 30 – Comet Hyakutake is discovered.* February 17 – NEAR Shoemaker spacecraft launched...

     Pål Nyrén
    Pål Nyrén
    Pål Nyrén is a biochemistry professor at the Royal Institute of Technology , Stockholm. He is most famous for developing the pyrosequencing method for DNA sequencing.-Career:*1999 Professor in Biochemistry, KTH, Stockholm...

     and his student Mostafa Ronaghi
    Mostafa Ronaghi
    Mostafa Ronaghi, born 1968. is a molecular biologist, specializing in DNA sequencing methodology . He earned his Ph.D. from the Royal Institute of Technology in Sweden in 1998.He is currently the Chief Technology Officer and Senior Vice President at Illumina...

     at the Royal Institute of Technology in Stockholm publish their method of pyrosequencing
    Pyrosequencing
    Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...

  • 1998
    1998 in science
    The year 1998 in science and technology involved many events, some of which are included below.-Astronomy and space exploration:* January–September – Cosmologists from the Supernova Cosmology Project led by Saul Perlmutter and the High-z Supernova Search Team led by Adam Riess and Brian...

     Phil Green and Brent Ewing of the University of Washington publish "phred
    Phred quality score
    Phred quality scores were originally developed by the program Phred to help in the automation of DNA sequencing in the Human Genome Project. Phred quality scores are assigned to each base call in automated sequencer traces...

    " for sequencer data analysis.
  • 2000
    2000 in science
    The year 2000 in science and technology involved some significant events.-Astronomy and space exploration:* May 4 – A rare conjunction occurs on the New Moon including all seven of the traditional celestial bodies known from ancient times up until 1781 with the discovery of Uranus...

     Lynx Therapeutics publishes and markets "MPSS" - a parallelized, adapter/ligation-mediated, bead-based sequencing technology, launching "next-generation" sequencing.
  • 2001
    2001 in science
    The year 2001 in science and technology involved many events, some of which are included below.-Astronomy and space exploration:* The NEAR Shoemaker spacecraft lands in the "saddle" region of 433 Eros, becoming the first spacecraft to land on an asteroid....

     A draft sequence of the human genome
    Human genome
    The human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA. 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining...

     is published.
  • 2004
    2004 in science
    The year 2004 in science and technology involved some significant events.-Anthropology:*October 27 - Remains of a previously unknown species of human is discovered in Indonesia...

     454 Life Sciences
    454 Life Sciences
    454 Life Sciences, is a biotechnology company based in Branford, Connecticut. It is a subsidiary of Roche, and specializes in high-throughput DNA sequencing.-History and Major Achievements:...

     markets a parallelized version of pyrosequencing. The first version of their machine reduced sequencing costs 6-fold compared to automated Sanger sequencing, and was the second of a new generation of sequencing technologies, after MPSS.

See also

  • Cancer genome sequencing
    Cancer genome sequencing
    Cancer genome sequencing is the resequencing of genomes of several human cancers.It is an effort and a necessity, in the War on Cancer to improve cancer diagnosis, treatment, and prevention through a better understanding of the molecular basis of this disease...

  • Complete Genomics
    Complete Genomics
    Complete Genomics is a life sciences company that has developed and commercialized a DNA sequencing platform for human genome sequencing and analysis. This solution combines the company’s proprietary human genome sequencing technology with its informatics and data management software in an...

  • DNA field-effect transistor
  • DNA sequencing theory
    DNA sequencing theory
    DNA sequencing theory is the broad body of work that attempts to lay analytical foundations for DNA sequencing. The practical aspects revolve around designing and optimizing sequencing projects , predicting project performance, troubleshooting experimental results, characterizing factors such as...

  • Genome project
    Genome project
    Genome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism and to annotate protein-coding genes and other important genome-encoded features...

  • Multiplex ligation-dependent probe amplification
    Multiplex ligation-dependent probe amplification
    Multiplex ligation-dependent probe amplification is a variation of the polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair. Each probe consists of a two oligonucleotides which recognise adjacent target sites on the DNA...

  • Sequence profiling tool
    Sequence profiling tool
    A sequence profiling tool in bioinformatics is a type of software that presents information related to a genetic sequence, gene name, or keyword input. Such tools generally take a query such as a DNA, RNA, or protein sequence or ‘keyword’ and search one or more databases for information related to...

  • Single Molecule Real Time Sequencing
    Single Molecule Real Time Sequencing
    Single molecule real time sequencing is a parallelized single molecule DNA sequencing by synthesis technology developed by Pacific Biosciences. Single molecule real time sequencing utilizes the zero-mode waveguide , developed in the laboratories of Harold G. Craighead and Watt W. Webb at Cornell...

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