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DNA sequencing is the process of determining the
nucleic acid sequence A nucleic acid sequence is a succession of Nucleobase, bases signified by a series of a set of five different letters that indicate the order of nucleotides forming alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequence ...
– the order of
nucleotides Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomer In chemistry, a monomer ( ; ''wikt:mono-, mono-'', "one" + ''wikt:-mer, -mer'', "part") is a molecule that can chemical reaction, react tog ...

nucleotides
in
DNA
DNA
. It includes any method or technology that is used to determine the order of the four bases:
adenine Adenine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a nucleobase (a purine derivative). It is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. Th ...

adenine
,
guanine Guanine () (symbol A symbol is a mark, sign, or word that indicates, signifies, or is understood as representing an idea, object, or relationship. Symbols allow people to go beyond what is known or seen by creating linkages between othe ...

guanine
,
cytosine Cytosine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol C or Cyt) is one of the four Nucleobase, nucleobases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA). It is a pyrimidine derivative, ...

cytosine
, and
thymine Thymine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol T or Thy) 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
. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as
medical diagnosis Medical diagnosis (abbreviated Dx, Dx, or Ds) is the process of determining which disease or condition explains a person's symptoms and medical sign, signs. It is most often referred to as diagnosis with the medicine, medical context being implic ...
,
biotechnology Biotechnology is the integration of Natural science, natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The te ...

biotechnology
, forensic biology,
virology Virology is the Scientific method, scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host (biology), ...
and biological
systematics Biological systematics is the study of the diversification of living forms, both past and present, and the Correlation and dependence, relationships among living things through time. Relationships are visualized as evolutionary trees (synonyms: c ...
. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers, characterize antibody repertoire, and can be used to guide patient treatment. Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or
genomes In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding gene ...
, of numerous types and species of life, including the
human genome The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual Mitochondrial DNA, mitochondria. These are usually treated s ...

human genome
and other complete DNA sequences of many animal, plant, and microbial species. The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy ...

fluorescence
-based sequencing methods with a
DNA sequencer A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then ...
, DNA sequencing has become easier and orders of magnitude faster.


Applications

DNA sequencing may be used to determine the sequence of individual
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian inheritance#History, Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanin ...

gene
s, larger genetic regions (i.e. clusters of genes or operons), full chromosomes, or entire genomes of any organism. DNA sequencing is also the most efficient way to indirectly sequence
RNA Ribonucleic acid (RNA) is a polymer A polymer (; Greek ''wikt:poly-, poly-'', "many" + ''wikt:-mer, -mer'', "part") is a Chemical substance, substance or material consisting of very large molecules called macromolecules, composed of many ...

RNA
or
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metabo ...

protein
s (via their
open reading frame In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible read ...

open reading frame
s). In fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine,
forensics Forensic science, also known as criminalistics, is the application of science to Criminal law, criminal and Civil law (legal system), civil laws, mainly—on the criminal side—during criminal investigation, as governed by the legal standard ...

forensics
, and
anthropology Anthropology is the scientific study of humanity, concerned with human behavior, human biology, cultures, societies, and linguistics Linguistics is the scientific study of human language. It is called a scientific study because i ...
.


Molecular biology

Sequencing is used in
molecular biology Molecular biology is the branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interact ...
to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes and noncoding DNA (including regulatory sequences), associations with diseases and phenotypes, and identify potential drug targets.


Evolutionary biology

Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in
evolutionary biology Evolutionary biology is the subfield of biology that studies the evolution, evolutionary processes (natural selection, common descent, speciation) that produced the Biodiversity, diversity of life on Earth. It is also defined as the study of ...
to study how different organisms are related and how they evolved. In February 2021, scientists reported, for the first time, the sequencing of from , a
mammoth A mammoth is any species of the extinct elephantidae, elephantid genus ''Mammuthus'', one of the many genera that make up the Order (biology), order of trunked mammals called proboscideans. The various species of mammoth were commonly equipped ...

mammoth
in this instance, over a million years old, the oldest DNA sequenced to date.


Metagenomics

The field of
metagenomics Metagenomics is the study of genetics, genetic material recovered directly from Natural environment, environmental or clinical samples by a method called sequencing. The broad field may also be referred to as environmental genomics, ecogenomics ...

metagenomics
involves identification of organisms present in a body of water,
sewage Sewage (or domestic sewage, domestic wastewater, municipal wastewater) is a type of wastewater Wastewater is water generated after the use of Fresh water, freshwater, raw water, drinking water or saline water in a variety of deliberate applic ...
, 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 Ecology () is the study of the relationships between living organisms, including humans, and their biophysical environment, physical environment. Ecology considers organisms at the individual, population, community (ecology), community, ecosy ...
,
epidemiology Epidemiology is the study and analysis of the distribution (who, when, and where), patterns and risk factor, determinants of health and disease conditions in a defined population. It is a cornerstone of public health, and shapes policy decision ...
,
microbiology Microbiology () is the scientific study of microorganisms, those being unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virology, bacteriolog ...

microbiology
, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a
microbiome A microbiome () is the community of microorganisms that can usually be found living together in any given habitat. It was defined more precisely in 1988 by Whipps ''et al.'' as "a characteristic microbial community occupying a reasonably well ...
, for example.


Virology

As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in virology to identify and study the virus. Viral genomes can be based in DNA or RNA. RNA viruses are more time-sensitive for genome sequencing, as they degrade faster in clinical samples. Traditional
Sanger sequencing Sanger sequencing is a method of DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bas ...
and next-generation sequencing are used to sequence viruses in basic and clinical research, as well as for the diagnosis of emerging viral infections,
molecular epidemiology Molecular epidemiology is a branch of epidemiology Epidemiology is the study and analysis of the distribution (who, when, and where), patterns and risk factor, determinants of health and disease conditions in a defined population. It is a corne ...
of viral pathogens, and drug-resistance testing. There are more than 2.3 million unique viral sequences in
GenBank The GenBank sequence database is an Open access (publishing), open access, annotated collection of all publicly available nucleotide sequences and their protein translations. It is produced and maintained by the National Center for Biotechnology I ...
. Recently, NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes. During the 1990 avian influenza outbreak, viral sequencing determined that the influenza sub-type originated through
reassortment Reassortment is the mixing of the genetics, genetic material of a species into new combinations in different individuals. Several different processes contribute to reassortment, including assortment of chromosomes, and chromosomal crossover. It is ...

reassortment
between
quail Quail is a collective name for several genera of mid-sized birds generally placed in the order Galliformes. The collective noun for a group of quail is a flock (birds), flock, covey, or bevy. Old World quail are placed in the family Phasianida ...

quail
and poultry. This led to legislation in
Hong Kong Hong Kong ( (US) or (UK); , ), officially the Hong Kong Special Administrative Region of the People's Republic of China (abbr. Hong Kong SAR or HKSAR), is a List of cities in China, city and Special administrative regions of China, special ...

Hong Kong
that prohibited selling live quail and poultry together at market. Viral sequencing can also be used to estimate when a viral outbreak began by using a
molecular clock The molecular clock is a figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory Prehistory, also known as pre-literary history, is the period of human history between the use of the ...
technique.


Medicine

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 Genetic testing, also known as DNA testing, is used to identify changes in DNA sequence or chromosome structure. Genetic testing can also include measuring the results of genetic changes, such as RNA analysis as an output of gene expression, or t ...
, though some genetic tests may not involve DNA sequencing. DNA sequencing is also being increasingly used to diagnose and treat rare diseases. As more and more genes are identified that cause rare genetic diseases, molecular diagnoses for patients becomes more mainstream. DNA sequencing allows clinicians to identify genetic diseases, improve disease management, provide reproductive counseling, and more effective therapies. Also, DNA sequencing may be useful for determining a specific bacteria, to allow for more precise antibiotics treatments, hereby reducing the risk of creating
antimicrobial resistance Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. P ...

antimicrobial resistance
in bacteria populations.


Forensic investigation

DNA sequencing may be used along with
DNA profiling DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding. DNA profiling is a Forensic DNA ...
methods for
forensic identification Forensic identification is the application of forensic science, or "forensics", and technology to identify specific objects from the trace evidence they leave, often at a crime scene or the scene of an accident. Forensic means "for the courts". Hu ...
and . 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 another. Testing DNA is a technique which can detect specific genomes in a DNA strand to produce a unique and individualized pattern.


The four canonical bases

The canonical structure of DNA has four bases:
thymine Thymine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol T or Thy) 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
(T),
adenine Adenine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a nucleobase (a purine derivative). It is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. Th ...

adenine
(A),
cytosine Cytosine () (nucleoside#List of nucleosides and corresponding nucleobases, symbol C or Cyt) is one of the four Nucleobase, nucleobases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA). It is a pyrimidine derivative, ...

cytosine
(C), and
guanine Guanine () (symbol A symbol is a mark, sign, or word that indicates, signifies, or is understood as representing an idea, object, or relationship. Symbols allow people to go beyond what is known or seen by creating linkages between othe ...

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 A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek language, Greek φαγεῖν ('), meaning "to d ...

bacteriophage
), cytosine may be replaced by hydroxy methyl or hydroxy methyl glucose cytosine. In mammalian DNA, variant bases with
methyl In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom chemical bond, bonded to three hydrogen atoms, having chemical formula . In chemical formula, formulas, the group is often skeletal formula#Pseudoe ...

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.


History


Discovery of DNA structure and function

Deoxyribonucleic acid () was first discovered and isolated by
Friedrich Miescher Johannes Friedrich Miescher (13 August 1844 – 26 August 1895) was a Swiss physician and biologist. He was the first scientist to isolate nucleic acid in 1869. He also identified protamine and made a number of other discoveries. Miescher had is ...

Friedrich Miescher
in 1869, but it remained under-studied 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 Oswald Theodore Avery Jr. (October 21, 1877 – February 20, 1955) was a Canadian-American physician and medical researcher. The major part of his career was spent at the Rockefeller University Hospital, Rockefeller Hospital in New York City. Ave ...
, Colin MacLeod, and
Maclyn McCarty Maclyn McCarty (June 9, 1911 – January 2, 2005) was an American geneticist, a research scientist described in 2005 as "the last surviving member of a Manhattan scientific team that overturned medical dogma in the 1940's and became the first to ...
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. In 1953,
James Watson James Dewey Watson (born April 6, 1928) is an American molecular biology, molecular biologist, geneticist, and zoologist. In 1953, he co-authored with Francis Crick the academic paper proposing the Nucleic acid double helix, double helix struc ...

James Watson
and
Francis Crick Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was an English molecular biology, molecular biologist, biophysics, biophysicist, and neuroscientist. He, James Watson, Rosalind Franklin, and Maurice Wilkins played crucial roles in de ...

Francis Crick
put forward their double-helix model of DNA, based on structures being studied by
Rosalind Franklin Rosalind Elsie Franklin (25 July 192016 April 1958) was a British chemist and X-ray crystallographer whose work was central to the understanding of the molecular structures of DNA (deoxyribonucleic acid), RNA (ribonucleic acid), viruses, coa ...

Rosalind Franklin
. According to the model, DNA is 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 that 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. The foundation for sequencing proteins was first laid by the work of
Frederick Sanger Frederick Sanger (; 13 August 1918 – 19 November 2013) was an English biochemist who received the Nobel Prize in Chemistry twice. He won the 1958 Chemistry Prize for determining the amino acid sequence of insulin and numerous other pr ...

Frederick Sanger
who by 1955 had completed the sequence of all the amino acids in
insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main Anabolism, anabolic hormone of the body. It regulates the m ...

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 spurred on 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 Frederick Sanger in October 1954, Crick began developing 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 in 1958.


RNA sequencing

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 (''Emesvirus zinderi''), commonly called MS2, is an icosahedral, positive-strand RNA virus, positive-sense single-stranded RNA virus that infects the bacterium ''Escherichia coli'' and other members of the Enterobacteriaceae. MS ...
, identified and published by Walter Fiers and his coworkers at the
University of Ghent Ghent University ( nl, Universiteit Gent, abbreviated as UGent) is a public In public relations and communication science, publics are groups of individual people, and the public (a.k.a. the general public) is the totality of such groupings. ...
(
Ghent Ghent ( nl, Gent ; french: Gand ; traditional English: Gaunt) is a city and a Municipalities of Belgium, municipality in the Flemish Region of Belgium. It is the capital and largest city of the East Flanders province, and the third largest i ...

Ghent
,
Belgium Belgium, ; french: Belgique ; german: Belgien officially the Kingdom of Belgium, is a country in Northwestern Europe. The country is bordered by the Netherlands to the north, Germany to the east, Luxembourg to the southeast, France to the ...

Belgium
), in 1972 and 1976. Traditional RNA sequencing methods require the creation of a
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to gene expression, ...
molecule which must be sequenced.


Early DNA sequencing methods

The first method for determining DNA sequences involved a location-specific primer extension strategy established by Ray Wu at
Cornell University Cornell University is a private statutory land-grant research university based in Ithaca, New York. It is a member of the Ivy League. Founded in 1865 by Ezra Cornell and Andrew Dickson White, Cornell was founded with the intention to ...

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 Frederick Sanger (; 13 August 1918 – 19 November 2013) was an English biochemist who received the Nobel Prize in Chemistry twice. He won the 1958 Chemistry Prize for determining the amino acid sequence of insulin and numerous other pr ...

Frederick Sanger
then adopted this primer-extension strategy to develop more rapid DNA sequencing methods at the MRC Centre,
Cambridge Cambridge ( ) is a College town, university city and the county town in Cambridgeshire, England. It is located on the River Cam approximately north of London. As of the 2021 United Kingdom census, the population of Cambridge was 145,700. Cam ...

Cambridge
, UK and published a method for "DNA sequencing with chain-terminating inhibitors" in 1977.
Walter Gilbert Walter Gilbert (born March 21, 1932) is an American biochemist, physicist, molecular biology pioneer, and Nobel laureate. Education and early life Walter Gilbert was born in Boston, Massachusetts, on March 21, 1932, the son of Emma (Cohen), a ch ...
and Allan Maxam at
Harvard Harvard University is a private Ivy League research university in Cambridge, Massachusetts. Founded in 1636 as Harvard College and named for its first benefactor, the History of the Puritans in North America, Puritan clergyman John Harvard ...

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 Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating DNA sequence, sequences that would not othe ...

recombinant DNA
technology, allowing DNA samples to be isolated from sources other than viruses.


Sequencing of full genomes

The first full DNA genome to be sequenced was that of bacteriophage φX174 in 1977. Medical Research Council scientists deciphered the complete 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 Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of Interface and colloid science, dispersed particles relative to a fluid under the influence of a ...

electrophoresis
was developed by Herbert Pohl and co-workers in the early 1980s. Followed by the commercialization of the DNA sequencer "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 ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...

Saccharomyces cerevisiae
'' chromosome II. Leroy E. Hood's laboratory at the
California Institute of Technology The California Institute of Technology (branded as Caltech or CIT)The university itself only spells its short form as "Caltech"; the institution considers other spellings such a"Cal Tech" and "CalTech" incorrect. The institute is also occasional ...
announced the first semi-automated 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 U.S.
National Institutes of Health The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the late 1 ...
(NIH) had begun large-scale sequencing trials on '' Mycoplasma capricolum'', ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative bacteria, Gram-negative, Facultative anaerobic organism, facultative anaer ...

Escherichia coli
'', ''
Caenorhabditis elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a Hybrid word, blend of the Greek ''caeno-'' (recent), ''rh ...

Caenorhabditis elegans
'', and ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...

Saccharomyces cerevisiae
'' at a cost of US$0.75 per base. Meanwhile, sequencing of human
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to gene expression, ...

cDNA
sequences called
expressed sequence tag In genetics, an expressed sequence tag (EST) is a short sub-sequence of a cDNA sequence. ESTs may be used to identify gene Transcription (genetics), transcripts, and were instrumental in gene discovery and in gene-sequence determination. The identif ...
s began in Craig Venter's lab, an attempt to capture the coding fraction of the
human genome The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual Mitochondrial DNA, mitochondria. These are usually treated s ...

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, the bacterium ''
Haemophilus influenzae ''Haemophilus influenzae'' (formerly called Pfeiffer's bacillus or ''Bacillus influenzae'') is a Gram-negative, Motility, non-motile, Coccobacillus, coccobacillary, facultative anaerobic organism, facultatively anaerobic, Capnophile, capnophili ...

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 (HTS) methods

Several new methods for DNA sequencing were developed in the mid to late 1990s and were implemented in commercial DNA sequencers by 2000. Together these were called the "next-generation" or "second-generation" sequencing (NGS) methods, in order to distinguish them from the earlier methods, including
Sanger sequencing Sanger sequencing is a method of DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bas ...
. In contrast to the first generation of sequencing, NGS technology is typically characterized by being highly scalable, allowing the entire genome to be sequenced at once. Usually, this is accomplished by fragmenting the genome into small pieces, randomly sampling for a fragment, and sequencing it using one of a variety of technologies, such as those described below. An entire genome is possible because multiple fragments are sequenced at once (giving it the name "massively parallel" sequencing) in an automated process. NGS technology has tremendously empowered researchers to look for insights into health, anthropologists to investigate human origins, and is catalyzing the "
Personalized Medicine Personalized medicine, also referred to as precision medicine, is a medical model that separates people into different groups—with medical decisions, practices, interventions and/or products being tailored to the individual patient based on thei ...
" movement. However, it has also opened the door to more room for error. There are many software tools to carry out the computational analysis of NGS data, often compiled at online platforms such as CSI NGS Portal, each with its own algorithm. Even the parameters within one software package can change the outcome of the analysis. In addition, the large quantities of data produced by DNA sequencing have also required development of new methods and programs for sequence analysis. Several efforts to develop standards in the NGS field have been attempted to address these challenges, most of which have been small-scale efforts arising from individual labs. Most recently, a large, organized, FDA-funded effort has culminated in the BioCompute standard. On 26 October 1990,
Roger Tsien Roger Yonchien Tsien (pronounced , "''Help:Pronunciation respelling key, CHEN''"'';'' February 1, 1952 – August 24, 2016) was an American biochemist. He was a professor of chemistry and biochemistry at the University of California, San Diego a ...
, 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 Pål Nyrén (born 1955) is a biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three ...
and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm published their method of pyrosequencing. On 1 April 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-polymerase chain reaction (PCR) arraying methods described in this patent, coupled to Roger Tsien et al.'s "base-by-base" sequencing method, is now implemented in Illumina (company), Illumina's Hi-Seq genome sequencers. In 1998, Phil Green and Brent Ewing of the University of Washington described their phred quality score for sequencer data analysis, a landmark analysis technique that gained widespread adoption, and which is still the most common metric for assessing the accuracy of a sequencing platform. 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 laboratories.


Basic methods


Maxam-Gilbert sequencing

Allan Maxam and
Walter Gilbert Walter Gilbert (born March 21, 1932) is an American biochemist, physicist, molecular biology pioneer, and Nobel laureate. Education and early life Walter Gilbert was born in Boston, Massachusetts, on March 21, 1932, the son of Emma (Cohen), a ch ...
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning. This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made. Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.


Chain-termination methods

The Sanger sequencing, chain-termination method developed by
Frederick Sanger Frederick Sanger (; 13 August 1918 – 19 November 2013) was an English biochemist who received the Nobel Prize in Chemistry twice. He won the 1958 Chemistry Prize for determining the amino acid sequence of insulin and numerous other pr ...

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 sequencer A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then ...
s. 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 Human Genome Project, 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 in 2011.


Large-scale sequencing and ''de novo'' sequencing

Large-scale sequencing often aims at sequencing very long DNA pieces, 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 clone (genetics), cloned into a Vector DNA, DNA vector and amplified in a bacterial host such as ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative bacteria, Gram-negative, Facultative anaerobic organism, facultative anaer ...

Escherichia coli
''. Short DNA fragments purified from individual bacterial colonies are individually sequenced and sequence assembly, assembled electronically into one long, contiguous sequence. Studies have shown that adding a size selection step to collect 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 sequencing, shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with Microsatellite (genetics), 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. A polymerase chain reaction (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 developed by Marguilis et al. (commercialized by 454 Life Sciences), Shendure and Porreca et al. (also known as "Polony (biology), polony sequencing") and ABI Solid Sequencing, SOLiD sequencing, (developed by Agencourt, later Applied Biosystems, now Life Technologies (Thermo Fisher Scientific), Life Technologies). Emulsion PCR is also used in the GemCode and Chromium platforms developed by 10x Genomics.


Shotgun sequencing

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 using the Sanger sequencing#Method, chain termination method, the sequences can be reassembled on the basis of their overlapping regions.


High-throughput methods

High-throughput sequencing, which includes next-generation "short-read" and third-generation "long-read" sequencing methods,"Next-generation" remains in broad use as of 2019. For instance, applies to exome sequencing, genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChIP-sequencing), and epigenome characterization. The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that multiplex (assay), 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 parallel. Such technologies led to the ability to sequence an entire human genome in as little as one day. , corporate leaders in the development of high-throughput sequencing products included Illumina, Inc., Illumina, Qiagen and Thermo Fisher Scientific, ThermoFisher Scientific.


Long-read sequencing methods


Single molecule real time (SMRT) 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. In 2015, 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 sequencing, third-generation" or "long-read" sequencing.


Nanopore DNA sequencing

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 does not require modified nucleotides and is performed in real time. Nanopore sequencing is referred to as "Third-generation sequencing, 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 occurred as nucleotides passed by hemolysin, alpha(α)-hemolysin pores covalently bound with cyclodextrin. However the subsequent commercial method, 'strand sequencing', sequenced DNA bases in an intact strand. 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 α-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 pore's entrance.


Short-read sequencing methods


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 Applied Biosystems#History, 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 (company), 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 sequencing
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to gene expression, ...

cDNA
for measurements of gene expression levels.


Polony sequencing

The 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 sequence an ''E. coli'' genome at an accuracy of >99.9999% and a cost approximately 1/9 that of Sanger sequencing. The technology was licensed to 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 (Thermo Fisher Scientific), Life Technologies, now part of Thermo Fisher Scientific.


454 pyrosequencing

A parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read (biology), reads. 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 (company), Illumina, was founded by Shankar Balasubramanian and 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 massively 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. 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 Fluorescent labeling, 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. 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 (genetics), coverage per hour per instrument, and 1 human genome re-sequenced (at approx. 30x) per day per instrument (equipped with a single camera).


Combinatorial probe anchor synthesis (cPAS)

This method is an upgraded modification to combinatorial probe anchor ligation technology (cPAL) described by Complete Genomics which has since become part of Chinese genomics company Beijing Genomics Institute, BGI in 2013. The two companies have refined the technology to allow for longer read lengths, reaction time reductions and faster time to results. In addition, data are now generated as contiguous full-length reads in the standard FASTQ file format and can be used as-is in most short-read-based bioinformatics analysis pipelines. The two technologies that form the basis for this high-throughput sequencing technology are DNA nanoball sequencing, DNA nanoballs (DNB) and patterned arrays for nanoball attachment to a solid surface. DNA nanoballs are simply formed by denaturing double stranded, adapter ligated libraries and ligating the forward strand only to a splint oligonucleotide to form a ssDNA circle. Faithful copies of the circles containing the DNA insert are produced utilizing Rolling Circle Amplification that generates approximately 300–500 copies. The long strand of ssDNA folds upon itself to produce a three-dimensional nanoball structure that is approximately 220 nm in diameter. Making DNBs replaces the need to generate PCR copies of the library on the flow cell and as such can remove large proportions of duplicate reads, adapter-adapter ligations and PCR induced errors. The patterned array of positively charged spots is fabricated through photolithography and etching techniques followed by chemical modification to generate a sequencing flow cell. Each spot on the flow cell is approximately 250 nm in diameter, are separated by 700 nm (centre to centre) and allows easy attachment of a single negatively charged DNB to the flow cell and thus reducing under or over-clustering on the flow cell. Sequencing is then performed by addition of an oligonucleotide probe that attaches in combination to specific sites within the DNB. The probe acts as an anchor that then allows one of four single reversibly inactivated, labelled nucleotides to bind after flowing across the flow cell. Unbound nucleotides are washed away before laser excitation of the attached labels then emit fluorescence and signal is captured by cameras that is converted to a digital output for base calling. The attached base has its terminator and label chemically cleaved at completion of the cycle. The cycle is repeated with another flow of free, labelled nucleotides across the flow cell to allow the next nucleotide to bind and have its signal captured. This process is completed a number of times (usually 50 to 300 times) to determine the sequence of the inserted piece of DNA at a rate of approximately 40 million nucleotides per second as of 2018.


SOLiD sequencing

Applied Biosystems' (now a Life Technologies (Thermo Fisher Scientific), 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. Each base in the template is sequenced twice, and the resulting data are decoded according to the 2 base encoding scheme used in this method. Before sequencing, the 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 Torrent Systems Inc. (now owned by Life Technologies (Thermo Fisher Scientific), 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 DNA polymerase, polymerisation of , 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. If the introduced nucleotide is complementarity (molecular biology), 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.


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 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 costs compared to other high-throughput sequencing platforms. However, only short sequences of DNA are determined from each 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

Heliscope sequencing is a method of single-molecule sequencing developed by 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. What made this technology especially novel was that it was the first of its class to sequence non-amplified DNA, thus preventing any read errors associated with amplification steps. In 2009 a human genome was sequenced using the Heliscope, however in 2012 the company went bankrupt.


Microfluidic Systems

There are two main microfluidic systems that are used to sequence DNA; Droplet-based microfluidics, droplet based microfluidics and digital microfluidics. Microfluidic devices solve many of the current limitations of current sequencing arrays. Abate et al. studied the use of droplet-based microfluidic devices for DNA sequencing. These devices have the ability to form and process picoliter sized droplets at the rate of thousands per second. The devices were created from Polydimethylsiloxane, polydimethylsiloxane (PDMS) and used Forster resonance energy transfer, Förster resonance energy transfer, FRET assays to read the sequences of DNA encompassed in the droplets. Each position on the array tested for a specific 15 base sequence. Fair et al. used digital microfluidic devices to study DNA pyrosequencing. Significant advantages include the portability of the device, reagent volume, speed of analysis, mass manufacturing abilities, and high throughput. This study provided a proof of concept showing that digital devices can be used for pyrosequencing; the study included using synthesis, which involves the extension of the enzymes and addition of labeled nucleotides. Boles et al. also studied pyrosequencing on digital microfluidic devices. They used an electro-wetting device to create, mix, and split droplets. The sequencing uses a three-enzyme protocol and DNA templates anchored with magnetic beads. The device was tested using two protocols and resulted in 100% accuracy based on raw pyrogram levels. The advantages of these digital microfluidic devices include size, cost, and achievable levels of functional integration. DNA sequencing research, using microfluidics, also has the ability to be applied to the RNA-Seq, sequencing of RNA, using similar droplet microfluidic techniques, such as the method, inDrops. This shows that many of these DNA sequencing techniques will be able to be applied further and be used to understand more about genomes and transcriptomes.


Methods in development

DNA sequencing methods currently under development include reading the sequence as a DNA strand transits through nanopore sequencing, 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 microscope, atomic force microscopy or Transmission electron microscopy DNA sequencing, 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 sequencing, 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.


Tunnelling currents DNA sequencing

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 different bases. The use of tunnelling currents has the potential to sequence orders of magnitude faster than ionic current methods and the sequencing of several 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), also called DNA probes, to reconstruct a target DNA sequence. Non-specific hybrids are removed by washing and the target DNA is 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 DNA sequences. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, or Matrix-assisted laser desorption/ionization, 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 indirectly sequence DNA with MS-based methods by converting it to RNA first. 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 DNA from samples in a 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 pathogenic ''Streptococcus'' strains.


Microfluidic Sanger sequencing

In microfluidic
Sanger sequencing Sanger sequencing is a method of DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bas ...
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.


Microscopy-based techniques

This approach directly visualizes the sequence of DNA molecules using electron microscopy. The first identification of DNA base pairs within intact 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 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome has been demonstrated.


RNAP sequencing

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 Interactome, protein interactions using a combination of 454 pyrosequencing and an ''in vitro'' virus mRNA display method. Specifically, this method covalently links proteins of interest to the mRNAs encoding them, then detects the mRNA pieces using reverse transcription Polymerase chain reaction, PCRs. The mRNA 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.


Sample preparation

The success of any DNA sequencing protocol relies upon the DNA or RNA sample extraction and preparation from the biological material of interest. * A successful DNA extraction will yield a DNA sample with long, non-degraded strands. * A successful RNA extraction will yield a RNA sample that should be converted to complementary DNA (cDNA) using reverse transcriptase—a DNA polymerase that synthesizes a complementary DNA based on existing strands of RNA in a PCR-like manner. Complementary DNA can then be processed the same way as genomic DNA. After DNA or RNA extraction, samples may require further preparation depending on the sequencing method. 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. Assessing the quality and quantity of nucleic acids both after extraction and after library preparation identifies degraded, fragmented, and low-purity samples and yields high-quality sequencing data. The high-throughput nature of current DNA/RNA sequencing technologies has posed a challenge for sample preparation method to scale-up. Several liquid handling instruments are being used for the preparation of higher numbers of samples with a lower total hands-on time:


Development initiatives

In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the 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.


Computational challenges

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 (software), Phred and Phrap. Other challenges have to deal with Repetitive DNA, 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 Bioinformatics, bioinformatical analysis. Yet new methods for sequencing and correcting sequencing errors were developed.


Read trimming

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:


Ethical issues

Human genetics have been included within the field of bioethics since the early 1970s and the growth in the use of DNA sequencing (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. As 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.Statement of Administration policy
Executive Office of the President, Office of Management and Budget, 27 April 2007
In 2012, the US Presidential Commission for the Study of Bioethical Issues reported that existing privacy legislation for DNA sequencing data such as GINA and the 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. In most of the United States, DNA that is "abandoned", such as that found on a licked stamp or envelope, coffee cup, cigarette, chewing gum, household trash, or hair that has fallen on a public sidewalk, may legally be collected and sequenced by anyone, including the police, private investigators, political opponents, or people involved in paternity disputes. As of 2013, eleven states have laws that can be interpreted to prohibit "DNA theft".The DNA in your garbage: up for grabs
/ref> Ethical issues have also been raised by the increasing use of genetic variation screening, both in newborns, and in adults by companies such as 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 (magazine), 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'' has shown that individuals undergoing disease risk profiling did not show increased levels of anxiety.


See also

* * * * * * * * * * * * * * * * * * * * * *


Notes


References


External links

* A Wikibooks:Next Generation Sequencing (NGS), wikibook on next generation sequencing {{DEFAULTSORT:Dna Sequencing DNA sequencing, Biotechnology DNA Genetic mapping Molecular biology Molecular biology techniques 1970 introductions 1970 in biology 1970 in biotechnology 1970 in science 1998 in technology