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{{Short description|Process of determining the nucleic acid sequence}}
{{Use dmy dates|date=June 2023}}
{{Genetics sidebar}}

'''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 bases: [[adenine]], [[thymine]], [[cytosine]], and [[guanine]]. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.<ref>{{cite web|url=https://theconversation.com/introducing-dark-dna-the-phenomenon-that-could-change-how-we-think-about-evolution-82867|title=Introducing 'dark DNA' – the phenomenon that could change how we think about evolution|date=24 August 2017 }}</ref><ref>{{cite journal | vauthors = Behjati S, Tarpey PS | title = What is next generation sequencing? | journal = Archives of Disease in Childhood: Education and Practice Edition | issue = 6 | pages = 236–8 | date = December 2013 | volume = 98 | pmid = 23986538 | pmc = 3841808 | doi = 10.1136/archdischild-2013-304340 }}</ref>

Knowledge of '''DNA sequences''' has become indispensable for basic biological research, [[Genographic Project|DNA Genographic Projects]] and in numerous applied fields such as [[medical diagnosis]], [[biotechnology]], [[forensic biology]], [[virology]] and biological [[systematics]]. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,<ref>{{cite journal | vauthors = Chmielecki J, Meyerson M | title = DNA sequencing of cancer: what have we learned? | journal = Annual Review of Medicine | volume = 65 | issue = 1 | pages = 63–79 | date = 2014-01-14 | pmid = 24274178 | doi = 10.1146/annurev-med-060712-200152 | doi-access = free }}</ref> characterize antibody repertoire,<ref name=":3" /> and can be used to guide patient treatment.<ref>{{cite journal | vauthors = Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB, Millot F, El Harrak A, Hutchison JB, Larson JW, Link DR, Laurent-Puig P, Griffiths AD, Taly V | display-authors = 6 | title = Quantitative and sensitive detection of rare mutations using droplet-based microfluidics | journal = Lab on a Chip | volume = 11 | issue = 13 | pages = 2156–66 | date = July 2011 | pmid = 21594292 | doi = 10.1039/c1lc20128j }}</ref> 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.<ref name=":3" />

The rapid advancements in DNA sequencing technology have played a crucial role in sequencing complete genomes of various life forms, including humans, as well as numerous animal, plant, and microbial species.[[File:Radioactive Fluorescent Seq.jpg|thumbnail|An example of the results of automated chain-termination DNA sequencing]]

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]]-based sequencing methods with a [[DNA sequencer]],<ref name=olsvik1993>{{cite journal | vauthors = Olsvik O, Wahlberg J, Petterson B, Uhlén M, Popovic T, Wachsmuth IK, Fields PI | title = Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae O1 strains | journal = [[J. Clin. Microbiol.]] | volume = 31 | issue = 1 | pages = 22–25 | date = January 1993 | pmid = 7678018 | pmc = 262614 | doi = 10.1128/JCM.31.1.22-25.1993 }}{{open access}}</ref> DNA sequencing has become easier and orders of magnitude faster.<ref name="pmid18992322">{{cite journal | vauthors = Pettersson E, Lundeberg J, Ahmadian A | title = Generations of sequencing technologies | journal = Genomics | volume = 93 | issue = 2 | pages = 105–11 | date = February 2009 | pmid = 18992322 | doi = 10.1016/j.ygeno.2008.10.003 | doi-access = free }}</ref><ref name="Bambara Padmanabhan Wu 1974"/>

== Applications ==
DNA sequencing may be used to determine the sequence of individual [[gene]]s, larger genetic regions (i.e. clusters of genes or [[operons]]), full chromosomes, or [[Whole genome sequencing|entire genomes]] of any organism.  DNA sequencing is also the most efficient way to indirectly sequence [[RNA]] or [[protein]]s (via their [[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]], and [[anthropology]].

=== Molecular biology ===
Sequencing is used in [[molecular biology]] to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes 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]] to study how different organisms are related and how they evolved. In February 2021, scientists reported, for the first time, the sequencing of [[DNA]] from [[Carrion|animal remains]], a [[mammoth]] in this instance, over a million years old, the oldest DNA sequenced to date.<ref name="CNN-20210217">{{cite news |last=Hunt |first=Katie |title=World's oldest DNA sequenced from a mammoth that lived more than a million years ago |url=https://www.cnn.com/2021/02/17/world/mammoth-oldest-dna-million-years-ago-scn/index.html |date=17 February 2021 |publisher=[[CNN]] |accessdate=17 February 2021 }}</ref><ref name="NAT-20210217">{{cite journal |last=Callaway |first=Ewen |title=Million-year-old mammoth genomes shatter record for oldest ancient DNA – Permafrost-preserved teeth, up to 1.6 million years old, identify a new kind of mammoth in Siberia. |date=17 February 2021 |journal=[[Nature (journal)|Nature]] |volume=590 |issue=7847 |pages=537–538 |doi=10.1038/d41586-021-00436-x |pmid=33597786 |bibcode=2021Natur.590..537C |doi-access=free }}</ref>

=== Metagenomics ===
{{Main|Metagenomics}}
The field of [[metagenomics]] involves identification of organisms present in a body of water, [[sewage]], dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in [[ecology]], [[epidemiology]], [[microbiology]], and other fields. Sequencing enables researchers to determine which types of microbes may be present in a [[microbiome]], for example.

=== Virology ===
{{Main|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.<ref name=":0c"/> 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.<ref name="Shirlee"/> Traditional [[Sanger sequencing]] 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]] of viral pathogens, and drug-resistance testing. There are more than 2.3 million unique viral sequences in [[GenBank]].<ref name=":0c" /> Recently, NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes.<ref name=":0c">{{cite journal |last1=Castro |first1=Christina |last2=Marine |first2=Rachel |last3=Ramos |first3=Edward |last4=Ng |first4=Terry Fei Fan |title=The effect of variant interference on de novo assembly for viral deep sequencing |journal=BMC Genomics |doi=10.1186/s12864-020-06801-w |biorxiv=10.1101/815480 |year=2019 |volume=21 |issue=1 |page=421 |pmid=32571214 |pmc=7306937 |doi-access=free }}</ref>

During the [[Influenza A virus subtype H5N1#Outbreaks|1997 avian influenza outbreak]], viral sequencing determined that the influenza sub-type originated through [[reassortment]] between [[quail]] and poultry. This led to legislation in [[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]] technique.<ref name="Shirlee">{{cite journal|doi=10.1146/annurev-virology-110615-035747|title=Genomic Analysis of Viral Outbreaks|year=2016|last1=Wohl|first1=Shirlee|last2=Schaffner|first2=Stephen F.|last3=Sabeti|first3=Pardis C.|author3-link=Pardis Sabeti|journal=Annual Review of Virology|volume=3|issue=1|pages=173–195|pmid=27501264|pmc=5210220}}</ref>

=== 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]], though some genetic tests may not involve DNA sequencing.

As of 2013 DNA sequencing was increasingly used to diagnose and treat rare diseases. As more and more genes are identified that cause rare genetic diseases, molecular diagnoses for patients become more mainstream. DNA sequencing allows clinicians to identify genetic diseases, improve disease management, provide reproductive counseling, and more effective therapies.<ref>{{cite journal |last1=Boycott |first1=Kym M. |last2=Vanstone |first2=Megan R. |last3=Bulman |first3=Dennis E. |last4=MacKenzie |first4=Alex E. |title=Rare-disease genetics in the era of next-generation sequencing: discovery to translation |journal=Nature Reviews Genetics |date=October 2013 |volume=14 |issue=10 |pages=681–691 |doi=10.1038/nrg3555 |pmid=23999272 |s2cid=8496181 }}</ref> Gene sequencing panels are used to identify multiple potential genetic causes of a suspected disorder.<ref>{{Cite journal |last1=Bean |first1=Lora |last2=Funke |first2=Birgit |last3=Carlston |first3=Colleen M. |last4=Gannon |first4=Jennifer L. |last5=Kantarci |first5=Sibel |last6=Krock |first6=Bryan L. |last7=Zhang |first7=Shulin |last8=Bayrak-Toydemir |first8=Pinar |date=March 2020 |title=Diagnostic gene sequencing panels: from design to report—a technical standard of the American College of Medical Genetics and Genomics (ACMG) |journal=Genetics in Medicine |volume=22 |issue=3 |pages=453–461 |doi=10.1038/s41436-019-0666-z |issn=1098-3600|doi-access=free |pmid=31732716 }}</ref>

Also, DNA sequencing may be useful for determining a specific bacteria, to allow for more [[Antimicrobial spectrum|precise antibiotics treatments]], hereby reducing the risk of creating [[antimicrobial resistance]] in bacteria populations.<ref>{{cite journal| pmid=28425484 | doi=10.1038/srep46327 | volume=7 | title=''Mycobacterium tuberculosis'' resistance prediction and lineage classification from genome sequencing: comparison of automated analysis tools | year=2017 | journal=Sci Rep | page=46327 | vauthors=Schleusener V, Köser CU, Beckert P, Niemann S, Feuerriegel S| pmc=7365310 | bibcode=2017NatSR...746327S | doi-access=free }}</ref><ref>{{cite journal| pmid=31106066 | doi=10.7717/peerj.6857 | volume=7 | title=A large scale evaluation of TBProfiler and Mykrobe for antibiotic resistance prediction in ''Mycobacterium tuberculosis'' | pmc=6500375 | year=2019 | journal=PeerJ | page=e6857 | vauthors=Mahé P, El Azami M, Barlas P, Tournoud M | doi-access=free }}</ref><ref>[https://innovation.ox.ac.uk/wp-content/uploads/2015/04/Mykrobe-predictor-Poster.pdf Mykrobe predictor –Antibiotic resistance prediction for S. aureus and M. tuberculosis from whole genome sequence data]</ref><ref>{{cite journal |last1=Bradley |first1=Phelim |last2=Gordon |first2=N. Claire |last3=Walker |first3=Timothy M. |last4=Dunn |first4=Laura |last5=Heys |first5=Simon |last6=Huang |first6=Bill |last7=Earle |first7=Sarah |last8=Pankhurst |first8=Louise J. |last9=Anson |first9=Luke |last10=de Cesare |first10=Mariateresa |last11=Piazza |first11=Paolo |last12=Votintseva |first12=Antonina A. |last13=Golubchik |first13=Tanya |last14=Wilson |first14=Daniel J. |last15=Wyllie |first15=David H. |last16=Diel |first16=Roland |last17=Niemann |first17=Stefan |last18=Feuerriegel |first18=Silke |last19=Kohl |first19=Thomas A. |last20=Ismail |first20=Nazir |last21=Omar |first21=Shaheed V. |last22=Smith |first22=E. Grace |last23=Buck |first23=David |last24=McVean |first24=Gil |last25=Walker |first25=A. Sarah |last26=Peto |first26=Tim E. A. |last27=Crook |first27=Derrick W. |last28=Iqbal |first28=Zamin |title=Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis |journal=Nature Communications |date=21 December 2015 |volume=6 |issue=1 |page=10063 |doi=10.1038/ncomms10063 |pmid=26686880 |pmc=4703848 |bibcode=2015NatCo...610063B }}</ref><ref>{{Cite web |url=https://www.tvo.org/transcript/115187X/michael-mosley-vs-the-superbugs |title=Michael Mosley vs the superbugs |access-date=21 October 2019 |archive-date=24 November 2020 |archive-url=https://web.archive.org/web/20201124174040/https://www.tvo.org/transcript/115187X/michael-mosley-vs-the-superbugs |url-status=dead }}</ref><ref>{{Citation|title=Mykrobe|date=2022-12-24|url=https://github.com/Mykrobe-tools/mykrobe|publisher=Mykrobe-tools|access-date=2023-01-02}}</ref>

=== Forensic investigation ===
{{Main|Forensic DNA analysis}}
DNA sequencing may be used along with [[DNA profiling]] methods for [[forensic identification]]<ref>{{Cite news|url=https://theconversation.com/from-the-crime-scene-to-the-courtroom-the-journey-of-a-dna-sample-82250|title=From the crime scene to the courtroom: the journey of a DNA sample|last1=Curtis|first1=Caitlin|last2=Hereward|first2=James | name-list-style = vanc |date=29 August 2017|work=The Conversation }}</ref> and [[DNA paternity testing|paternity testing]]. DNA testing has evolved tremendously in the last few decades to ultimately link a DNA print to what is under investigation. The DNA patterns in fingerprint, saliva, hair follicles, etc. uniquely separate each living organism from another. Testing DNA is a technique which can detect specific genomes in a DNA strand to produce a unique and individualized pattern.

DNA sequencing may be used along with [[DNA profiling]] methods for [[forensic identification]] and [[paternity testing]], as it has evolved significantly over the past few decades to ultimately link a DNA print to what is under investigation. The DNA patterns in fingerprint, saliva, hair follicles, and other bodily fluids uniquely separate each living organism from another, making it an invaluable tool in the field of [[forensic science]]. The process of DNA testing involves detecting specific [[genomes]] in a DNA strand to produce a unique and individualized pattern, which can be used to identify individuals or determine their relationships.

The advancements in DNA sequencing technology have made it possible to analyze and compare large amounts of [[genetic data]] quickly and accurately, allowing investigators to gather evidence and solve crimes more efficiently. This technology has been used in various applications, including forensic identification, paternity testing, and human identification in cases where traditional identification methods are unavailable or unreliable. The use of DNA sequencing has also led to the development of new forensic techniques, such as [[DNA phenotyping]], which allows investigators to predict an individual's physical characteristics based on their genetic data.

In addition to its applications in forensic science, DNA sequencing has also been used in medical research and diagnosis. It has enabled scientists to identify genetic mutations and variations that are associated with certain diseases and disorders, allowing for more accurate diagnoses and targeted treatments. Moreover, DNA sequencing has also been used in conservation biology to study the genetic diversity of endangered species and develop strategies for their conservation.

Furthermore, the use of DNA sequencing has also raised important ethical and legal considerations. For example, there are concerns about the privacy and security of genetic data, as well as the potential for misuse or discrimination based on genetic information. As a result, there are ongoing debates about the need for regulations and guidelines to ensure the responsible use of DNA sequencing technology.

Overall, the development of DNA sequencing technology has revolutionized the field of forensic science and has far-reaching implications for our understanding of genetics, medicine, and conservation biology.

== The four canonical bases ==
{{Main|Nucleotide}}
The canonical structure of DNA has four bases: [[thymine]] (T), [[adenine]] (A), [[cytosine]] (C), and [[guanine]] (G). DNA sequencing is the determination of the physical order of these bases in a molecule of DNA. However, there are many other bases that may be present in a molecule. In some viruses (specifically, [[bacteriophage]]), cytosine may be replaced by hydroxy methyl or hydroxy methyl glucose cytosine.<ref>{{cite journal | vauthors = Moréra S, Larivière L, Kurzeck J, Aschke-Sonnenborn U, Freemont PS, Janin J, Rüger W | title = High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding | journal = Journal of Molecular Biology | volume = 311 | issue = 3 | pages = 569–77 | date = August 2001 | pmid = 11493010 | doi = 10.1006/jmbi.2001.4905 }}</ref> In mammalian DNA, variant bases with [[methyl]] groups or phosphosulfate may be found.<ref>{{cite journal | vauthors = Ehrlich M, Gama-Sosa MA, Huang LH, Midgett RM, Kuo KC, McCune RA, Gehrke C | title = Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells | journal = Nucleic Acids Research | volume = 10 | issue = 8 | pages = 2709–21 | date = April 1982 | pmid = 7079182 | pmc = 320645 | doi = 10.1093/nar/10.8.2709 }}</ref><ref>{{cite journal | vauthors = Ehrlich M, Wang RY | title = 5-Methylcytosine in eukaryotic DNA | journal = Science | volume = 212 | issue = 4501 | pages = 1350–7 | date = June 1981 | pmid = 6262918 | doi = 10.1126/science.6262918 | bibcode = 1981Sci...212.1350E }}</ref> Depending on the sequencing technique, a particular modification, e.g., the 5mC ([[5-Methylcytosine]]) common in humans, may or may not be detected.<ref>{{cite journal | vauthors = Song CX, Clark TA, Lu XY, Kislyuk A, Dai Q, Turner SW, He C, Korlach J | display-authors = 6 | title = Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine | journal = Nature Methods | volume = 9 | issue = 1 | pages = 75–7 | date = November 2011 | pmid = 22101853 | pmc = 3646335 | doi = 10.1038/nmeth.1779 }}</ref>

In almost all organisms, DNA is synthesized in vivo using only the 4 canonical bases; modification that occurs post replication creates other bases like 5 methyl C. However, some bacteriophage can incorporate a non standard base directly.<ref>{{cite journal |last1=Czernecki |first1=Dariusz |last2=Bonhomme |first2=Frédéric |last3=Kaminski |first3=Pierre-Alexandre |last4=Delarue |first4=Marc |title=Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA |journal=Nature Communications |date=5 August 2021 |volume=12 |issue=1 |pages=4710 |doi=10.1038/s41467-021-25064-x |pmid=34354070 |bibcode=2021NatCo..12.4710C |s2cid=233745192 |doi-access=free |pmc=8342488 }}</ref>

In addition to modifications, DNA is under constant assault by environmental agents such as UV and Oxygen radicals. At the present time, the presence of such damaged bases is not detected by most DNA sequencing methods, although PacBio has published on this.<ref>{{Cite web |title=Direct detection and sequencing of damaged DNA bases. |url=https://www.pacb.com/publications/direct-detection-and-sequencing-of-damaged-dna-bases/ |access-date=2024-07-31 |website=PacBio |language=en-US}}</ref>

==History==
=== Discovery of DNA structure and function ===
Deoxyribonucleic acid ([[DNA]]) was first discovered and isolated by [[Friedrich Miescher]] in 1869, but it remained under-studied for many decades because [[protein]]s, rather than DNA, were thought to hold the genetic blueprint to life. This situation changed after 1944 as a result of some experiments by [[Oswald Avery]], [[Colin Munro MacLeod|Colin MacLeod]], and [[Maclyn McCarty]] demonstrating that purified DNA could change one strain of bacteria into another. This was the first time that DNA was shown capable of transforming the properties of cells.

In 1953, [[James Watson]] and [[Francis Crick]] put forward their [[double-helix]] model of DNA, based on [[X-ray crystallography|crystallized X-ray]] structures being studied by [[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.<ref name="pmid13168976">{{cite journal | vauthors = Watson JD, Crick FH | title = The structure of DNA | journal = Cold Spring Harb. Symp. Quant. Biol. | volume = 18 | pages = 123–31 | year = 1953 | pmid = 13168976 | doi = 10.1101/SQB.1953.018.01.020 }}</ref>
[[File:Frederick Sanger2.jpg|thumb|[[Frederick Sanger]], a pioneer of sequencing. Sanger is one of the few scientists who was awarded two Nobel prizes, one for the [[Protein sequencing|sequencing of proteins]], and the other for the sequencing of DNA.]]
The foundation for sequencing proteins was first laid by the work of [[Frederick Sanger]] who by 1955 had completed the sequence of all the amino acids in [[insulin]], a small protein secreted by the pancreas. This provided the first conclusive evidence that proteins were chemical entities with a specific molecular pattern rather than a random mixture of material suspended in fluid. Sanger's success in sequencing insulin 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.<ref name="whatisbiotechnology.org">{{Cite web|title=The path to DNA sequencing: The life and work of Frederick Sanger|url=http://www.whatisbiotechnology.org/exhibitions/sanger/path|access-date=2023-06-27|website=What is Biotechnology?|language=en|first=L. |last=Marks}}</ref>

===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]], identified and published by [[Walter Fiers]] and his coworkers at the [[University of Ghent]] ([[Ghent]], [[Belgium]]), in 1972<ref>{{cite journal | vauthors = Min Jou W, Haegeman G, Ysebaert M, Fiers W | title = Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein | journal = Nature | volume = 237 | issue = 5350 | pages = 82–8 | date = May 1972 | pmid = 4555447 | doi = 10.1038/237082a0 | bibcode = 1972Natur.237...82J | s2cid = 4153893 }}</ref> and 1976.<ref>{{cite journal | vauthors = Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers A, Van den Berghe A, Volckaert G, Ysebaert M | title = Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene | journal = Nature | volume = 260 | issue = 5551 | pages = 500–7 | date = April 1976 | pmid = 1264203 | doi = 10.1038/260500a0 | bibcode = 1976Natur.260..500F | s2cid = 4289674 }}</ref> Traditional RNA sequencing methods require the creation of a [[Complementary DNA|cDNA]] molecule which must be sequenced.<ref>{{cite journal | vauthors = Ozsolak F, Milos PM | title = RNA sequencing: advances, challenges and opportunities | journal = Nature Reviews Genetics | volume = 12 | issue = 2 | pages = 87–98 | date = February 2011 | pmid = 21191423 | pmc = 3031867 | doi = 10.1038/nrg2934 }}</ref>

==== Traditional RNA Sequencing Methods ====

Traditional RNA sequencing methods involve several steps:

1) '''''Reverse Transcription''''': The first step is to convert the RNA molecule into a complementary DNA (cDNA) molecule using an enzyme called [[reverse transcriptase]].

2) '''''cDNA Synthesis''''': The cDNA molecule is then synthesized through a process called PCR ([[Polymerase Chain Reaction]]), which amplifies the cDNA to produce multiple copies.

3)'''''Sequencing''''': The amplified cDNA is then sequenced using a technique such as [[Sanger sequencing]] or [[Maxam-Gilbert sequencing]].

==== Challenges and Limitations ====

Traditional RNA sequencing methods have several limitations. For example:

They require the creation of a cDNA molecule, which can be time-consuming and labor-intensive.
They are prone to errors and biases, which can affect the accuracy of the sequencing results.
They are limited in their ability to detect rare or low-abundance transcripts.

==== Advances in RNA Sequencing Technology ====

In recent years, advances in RNA sequencing technology have addressed some of these limitations. New methods such as [[next-generation sequencing]] (NGS) and [[single-molecule real-time]] (SMRT) sequencing have enabled faster, more accurate, and more cost-effective sequencing of RNA molecules. These advances have opened up new possibilities for studying gene expression, identifying new genes, and understanding the regulation of gene expression.

===Early DNA sequencing methods===
The first method for determining [[DNA sequences]] involved a location-specific primer extension strategy established by [[Ray Wu]], a geneticist, at [[Cornell University]] in 1970.<ref>{{cite web|url=http://www.mbg.cornell.edu/faculty-staff/faculty/wu.cfm|title=Ray Wu Faculty Profile|archive-url=https://web.archive.org/web/20090304121126/http://www.mbg.cornell.edu/faculty-staff/faculty/wu.cfm|archive-date=2009-03-04|publisher=Cornell University}}</ref> 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.<ref>{{cite journal | vauthors = Padmanabhan R, Jay E, Wu R | title = Chemical synthesis of a primer and its use in the sequence analysis of the lysozyme gene of bacteriophage T4 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 71 | issue = 6 | pages = 2510–4 | date = June 1974 | pmid = 4526223 | pmc = 388489 | doi = 10.1073/pnas.71.6.2510 | bibcode = 1974PNAS...71.2510P | doi-access = free }}</ref><ref>{{cite journal | vauthors = Onaga LA | title = Ray Wu as Fifth Business: Demonstrating Collective Memory in the History of DNA Sequencing | journal = Studies in the History and Philosophy of Science | volume = 46 | pages = 1–14 | date = June 2014 | pmid = 24565976 | doi = 10.1016/j.shpsc.2013.12.006 | series = Part C }}</ref><ref name="pmid4553110">{{cite journal | vauthors = Wu R | title = Nucleotide sequence analysis of DNA | journal = Nature New Biology | volume = 236 | issue = 68 | pages = 198–200 | year = 1972 | pmid = 4553110 | doi = 10.1038/newbio236198a0 }}</ref> Between 1970 and 1973, Wu, scientist Radha Padmanabhan and colleagues demonstrated that this method can be employed to determine any DNA sequence using synthetic location-specific primers.<ref name="pmid4560009">{{cite journal | vauthors = Padmanabhan R, Wu R | title = Nucleotide sequence analysis of DNA. IX. Use of oligonucleotides of defined sequence as primers in DNA sequence analysis | journal = Biochem. Biophys. Res. Commun. | volume = 48 | issue = 5 | pages = 1295–302 | year = 1972 | pmid = 4560009 | doi = 10.1016/0006-291X(72)90852-2}}</ref><ref name="pmid4358929">{{cite journal | vauthors = Wu R, Tu CD, Padmanabhan R | title = Nucleotide sequence analysis of DNA. XII. The chemical synthesis and sequence analysis of a dodecadeoxynucleotide which binds to the endolysin gene of bacteriophage lambda | journal = Biochem. Biophys. Res. Commun. | volume = 55 | issue = 4 | pages = 1092–99 | year = 1973 | pmid = 4358929 | doi = 10.1016/S0006-291X(73)80007-5}}</ref><ref name="Bambara Padmanabhan Wu 1974">{{cite journal | vauthors = Jay E, Bambara R, Padmanabhan R, Wu R | title = DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping | journal = Nucleic Acids Research | volume = 1 | issue = 3 | pages = 331–53 | date = March 1974 | pmid = 10793670 | pmc = 344020 | doi = 10.1093/nar/1.3.331 }}</ref> 

[[Walter Gilbert]], a biochemist, and [[Allan Maxam]], a molecular geneticist, at [[Harvard University|Harvard]] also developed sequencing methods, including one for "DNA sequencing by chemical degradation".<ref name="Maxam77" /><ref>Gilbert, W. [http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/gilbert-lecture.pdf DNA sequencing and gene structure]. Nobel lecture, 8 December 1980.</ref> In 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.<ref>{{cite journal | vauthors = Gilbert W, Maxam A | title = The Nucleotide Sequence of the lac Operator | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 70 | issue = 12 | pages = 3581–84 | date = December 1973 | pmid = 4587255 | pmc = 427284 | doi = 10.1073/pnas.70.12.3581 | bibcode = 1973PNAS...70.3581G | doi-access = free }}</ref> Advancements in sequencing were aided by the concurrent development of [[recombinant DNA]] technology, allowing DNA samples to be isolated from sources other than viruses.<ref>{{Cite web |title=Chapter 5: Investigating DNA |url=https://wou.edu:443/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451-biochemistry-defining-life-at-the-molecular-level/chapter-5-investigating-dna/ |access-date=2025-01-31 |website=Chemistry |language=en-US}}</ref>

Two years later in 1975, [[Frederick Sanger]], a biochemist, and [[Alan Coulson]], a genome scientist, developed a method to sequence DNA.<ref>{{Cite journal |last1=Sanger |first1=F. |last2=Coulson |first2=A. R. |date=1975-05-25 |title=A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase |url=https://www.sciencedirect.com/science/article/abs/pii/0022283675902132 |journal=Journal of Molecular Biology |volume=94 |issue=3 |pages=441–448 |doi=10.1016/0022-2836(75)90213-2 |pmid=1100841 |issn=0022-2836|url-access=subscription }}</ref> The [[Sanger sequencing|technique]] known as the "Plus and Minus" method, involved supplying all the components of the DNA but excluding the reaction of one of the four bases needed to complete the DNA.<ref>{{Cite book |last=Cook-Deegan |first=Robert |title=The gene wars: science, politics, and the human genome |date=1995 |publisher=Norton |isbn=978-0-393-31399-4 |edition=1. publ. as a Norton paperback |location=New York NY}}</ref>

In 1976, Gilbert and Maxam, invented a method for rapidly sequencing DNA while at Harvard, known as the Maxam–Gilbert sequencing.<ref>{{Cite web |last=Johnson |first=Carolyn Y. |title=A physicist, biologist, Nobel laureate, CEO, and now, artist |url=https://www.bostonglobe.com/metro/2015/03/12/wally-gilbert-physicist-biologist-nobel-laureate-ceo-and-now-artist/b3OsCNVvHZOYCi48Dz4z6H/story.html |date=2015-03-12 |access-date=2025-02-03 |work=[[The Boston Globe]] |language=en-US}}</ref> The technique involved treating radiolabelled DNA with a chemical and using a polyacrylamide gel to determine the sequence.<ref>Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics. 2016 Jan;107(1):1-8. doi: [https://pmc.ncbi.nlm.nih.gov/articles/PMC4727787/#:~:text=The%20plus%20and%20minus%20technique,before%20the%20next%20missing%20nucleotide 10.1016/j.ygeno.2015.11.003]. Epub 2015 Nov 10. PMID: 26554401; PMCID: PMC4727787.</ref>

In 1977, Sanger then adopted a primer-extension strategy to develop more rapid DNA sequencing methods at the [[Medical Research Council (United Kingdom)|MRC Centre]], [[Cambridge]], UK. This technique was similar to his "Plus and Minus" strategy, however, it was based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by [[DNA polymerase]] during in vitro [[DNA replication]].<ref>{{cite book |doi=10.1016/B978-0-12-815499-1.00013-2 |chapter=Nucleic acid analysis in the clinical laboratory |title=Contemporary Practice in Clinical Chemistry |date=2020 |last1=Deharvengt |first1=Sophie J. |last2=Petersen |first2=Lauren M. |last3=Jung |first3=Hou-Sung |last4=Tsongalis |first4=Gregory J. |pages=215–234 |isbn=978-0-12-815499-1 }}</ref><ref>{{cite journal |last1=Heather |first1=James M. |last2=Chain |first2=Benjamin |title=The sequence of sequencers: The history of sequencing DNA |journal=Genomics |date=January 2016 |volume=107 |issue=1 |pages=1–8 |doi=10.1016/j.ygeno.2015.11.003 |pmid=26554401 |pmc=4727787 }}</ref><ref>{{cite journal |last1=Elsayed |first1=Fadwa A. |last2=Grolleman |first2=Judith E. |last3=Ragunathan |first3=Abiramy |last4=Buchanan |first4=Daniel D. |last5=van Wezel |first5=Tom |last6=de Voer |first6=Richarda M. |last7=Boot |first7=Arnoud |last8=Stojovska |first8=Marija Staninova |last9=Mahmood |first9=Khalid |last10=Clendenning |first10=Mark |last11=de Miranda |first11=Noel |last12=Dymerska |first12=Dagmara |last13=Egmond |first13=Demi van |last14=Gallinger |first14=Steven |last15=Georgeson |first15=Peter |last16=Hoogerbrugge |first16=Nicoline |last17=Hopper |first17=John L. |last18=Jansen |first18=Erik A.M. |last19=Jenkins |first19=Mark A. |last20=Joo |first20=Jihoon E. |last21=Kuiper |first21=Roland P. |last22=Ligtenberg |first22=Marjolijn J.L. |last23=Lubinski |first23=Jan |last24=Macrae |first24=Finlay A. |last25=Morreau |first25=Hans |last26=Newcomb |first26=Polly |last27=Nielsen |first27=Maartje |last28=Palles |first28=Claire |last29=Park |first29=Daniel J. |last30=Pope |first30=Bernard J. |last31=Rosty |first31=Christophe |last32=Ruiz Ponte |first32=Clara |last33=Schackert |first33=Hans K. |last34=Sijmons |first34=Rolf H. |last35=Tomlinson |first35=Ian P. |last36=Tops |first36=Carli M.J. |last37=Vreede |first37=Lilian |last38=Walker |first38=Romy |last39=Win |first39=Aung K. |title=Monoallelic NTHL1 Loss-of-Function Variants and Risk of Polyposis and Colorectal Cancer |journal=Gastroenterology |date=December 2020 |volume=159 |issue=6 |pages=2241–2243.e6 |doi=10.1053/j.gastro.2020.08.042 |pmid=32860789 |pmc=7899696 |hdl=2066/228713 |hdl-access=free }}</ref> Sanger published this method in the same year. <ref name="Sanger1977" />

=== Sequencing of full genomes ===
[[File:Genome map of the bacteriophage ΦX174 showing overlapping genes.svg|thumb|300px|right|The 5,386 bp genome of [[bacteriophage φX174]]. Each coloured block represents a gene.]]{{Main|Whole genome sequencing}}
The first full DNA genome to be sequenced was that of [[bacteriophage φX174]] in 1977.<ref>{{cite journal | vauthors = Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M | title = Nucleotide sequence of bacteriophage phi X174 DNA | journal = Nature | volume = 265 | issue = 5596 | pages = 687–95 | date = February 1977 | pmid = 870828 | doi = 10.1038/265687a0 | bibcode = 1977Natur.265..687S | s2cid = 4206886 }}</ref> [[Medical Research Council (UK)|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.<ref>{{Cite web|title=The next frontier: Human viruses |url=https://www.whatisbiotechnology.org/index.php/exhibitions/sanger/sequencing|access-date=2023-06-27|website=What is Biotechnology?|language=en|first=L. |last=Marks}}</ref><ref name="Bambara Padmanabhan Wu 1974"/>

A non-radioactive method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during [[electrophoresis]] was developed by Herbert Pohl and co-workers in the early 1980s.<ref>{{cite journal | vauthors = Beck S, Pohl FM | title = DNA sequencing with direct blotting electrophoresis | journal = EMBO J | volume = 3 | issue = 12 | pages = 2905–09 | year = 1984 | pmid = 6396083 | pmc = 557787 | doi = 10.1002/j.1460-2075.1984.tb02230.x }}</ref><ref>United States Patent 4,631,122 (1986)</ref> 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]]'' chromosome II.<ref name = "Feldmann_1994"/> [[Leroy E. Hood]]'s laboratory at the [[California Institute of Technology]] announced the first semi-automated DNA sequencing machine in 1986.<ref>{{cite journal | vauthors = Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE | title = Fluorescence Detection in Automated DNA Sequence Analysis | journal = Nature | volume = 321 | issue = 6071 | pages = 674–79 | date = 12 June 1986 | pmid = 3713851 | doi = 10.1038/321674a0 | bibcode = 1986Natur.321..674S | s2cid = 27800972 }}</ref> 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<ref>{{cite journal | vauthors = Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MA, Baumeister K | title = A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides | journal = Science | volume = 238 | issue = 4825 | pages = 336–41 | date = 16 October 1987 | pmid = 2443975 | doi = 10.1126/science.2443975 | bibcode = 1987Sci...238..336P }}</ref> which used a novel fluorescent labeling technique enabling all four [[dideoxynucleotide]]s to be identified in a single lane. By 1990, the U.S. [[National Institutes of Health]] (NIH) had begun large-scale sequencing trials on ''[[Mycoplasma capricolum]]'', ''[[Escherichia coli]]'', ''[[Caenorhabditis elegans]]'', and ''[[Saccharomyces cerevisiae]]'' at a cost of US$0.75 per base. Meanwhile, sequencing of human [[cDNA]] sequences called [[expressed sequence tag]]s began in [[Craig Venter]]'s lab, an attempt to capture the coding fraction of the [[human genome]].<ref name="pmid2047873">{{cite journal | vauthors = Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF | title = Complementary DNA sequencing: expressed sequence tags and human genome project | journal = Science | volume = 252 | issue = 5013 | pages = 1651–56 | date = June 1991 | pmid = 2047873 | doi = 10.1126/science.2047873 | bibcode = 1991Sci...252.1651A | s2cid = 13436211 }}</ref> In 1995, Venter, [[Hamilton O. Smith|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]]''.  The circular chromosome contains 1,830,137 bases and its publication in the journal Science<ref>{{cite journal | vauthors = Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM | title = Whole-genome random sequencing and assembly of ''Haemophilus influenzae Rd'' | journal = Science | volume = 269 | issue = 5223 | pages = 496–512 | date = July 1995 | pmid = 7542800 | doi = 10.1126/science.7542800 | bibcode = 1995Sci...269..496F }}</ref> marked the first published use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts.

By 2003, the Human Genome Project's shotgun sequencing methods had been used to produce a draft sequence of the human genome; it had a 92% accuracy.<ref name="Lander_2001"/><ref name="Venter_2001"/><ref>{{Cite web |date=2022-04-11 |title=First complete sequence of a human genome |url=https://www.nih.gov/news-events/nih-research-matters/first-complete-sequence-human-genome |access-date=2025-02-06 |website=National Institutes of Health (NIH) |language=EN}}</ref> In 2022, scientists successfully sequenced the last 8% of the human genome. The fully sequenced standard reference gene is called GRCh38.p14, and it contains 3.1 billion base pairs.<ref>{{Cite web |date=2022-04-11 |title=First complete sequence of a human genome |url=https://www.nih.gov/news-events/nih-research-matters/first-complete-sequence-human-genome |access-date=2025-02-06 |website=National Institutes of Health (NIH) |language=EN}}</ref><ref>{{Cite web |last=Hartley |first=Gabrielle |date=2022-03-31 |title=The Human Genome Project pieced together only 92% of the DNA – now scientists have finally filled in the remaining 8% |url=https://theconversation.com/the-human-genome-project-pieced-together-only-92-of-the-dna-now-scientists-have-finally-filled-in-the-remaining-8-176138 |access-date=2025-02-06 |website=The Conversation |language=en-US}}</ref>

=== High-throughput sequencing (HTS) methods ===
[[File:History of sequencing technology.jpg|thumb|upright=2| History of sequencing technology{{hsp}}<ref>{{cite journal |doi = 10.3389/fbioe.2020.01032|title = Review on the Application of Machine Learning Algorithms in the Sequence Data Mining of DNA|year = 2020|last1 = Yang|first1 = Aimin|last2 = Zhang|first2 = Wei|last3 = Wang|first3 = Jiahao|last4 = Yang|first4 = Ke|last5 = Han|first5 = Yang|last6 = Zhang|first6 = Limin|journal = Frontiers in Bioengineering and Biotechnology|volume = 8|page = 1032|pmid = 33015010|pmc = 7498545|doi-access = free}}</ref>]]

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]]. 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]]" 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 Object|BioCompute]] standard.

On 26 October 1990, [[Roger Tsien]], Pepi Ross, Margaret Fahnestock and Allan J Johnston filed a patent describing stepwise ("base-by-base") sequencing with removable 3' blockers on DNA arrays (blots and single DNA molecules).<ref name=TsienPatent>{{cite web|url=http://worldwide.espacenet.com/publicationDetails/biblio?FT=D&date=19910516&DB=EPODOC&locale=en_EP&CC=WO&NR=9106678A1&KC=A1&ND=4|title=Espacenet – Bibliographic data|website=worldwide.espacenet.com}}</ref>
In 1996, [[Pål Nyrén]] and his student [[Mostafa Ronaghi]] at the Royal Institute of Technology in [[Stockholm]] published their method of [[pyrosequencing]].<ref name=Ronaghi>{{cite journal | vauthors = Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P | title = Real-time DNA sequencing using detection of pyrophosphate release | journal = Analytical Biochemistry | volume = 242 | issue = 1 | pages = 84–89 | year = 1996 | pmid = 8923969 | doi = 10.1006/abio.1996.0432 }}</ref>

On 1 April 1997, [[Pascal Mayer]] and Laurent Farinelli submitted patents to the World Intellectual Property Organization describing DNA colony sequencing.<ref name=DNA_colony_patents>{{cite web | last = Kawashima | first = Eric H. | author2 = Laurent Farinelli | author3 = [[Pascal Mayer]] | title = Patent: Method of nucleic acid amplification | access-date = 2012-12-22 | date = 2005-05-12 | url = http://www.patentlens.net/patentlens/patent/WO_1998_044151_A1/en/ | archive-url = https://archive.today/20130222020134/http://www.patentlens.net/patentlens/patent/WO_1998_044151_A1/en/ | archive-date = 22 February 2013 | url-status = dead }}</ref> 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,<ref>{{cite journal|vauthors=Ewing B, Green P|date=March 1998|title=Base-calling of automated sequencer traces using phred. II. Error probabilities|journal=Genome Res.|volume=8|issue=3|pages=186–94|doi=10.1101/gr.8.3.186|pmid=9521922|doi-access=free}}</ref> a landmark analysis technique that gained widespread adoption, and which is still the most common metric for assessing the accuracy of a sequencing platform.<ref>{{cite web|url=https://www.illumina.com/documents/products/technotes/technote_Q-Scores.pdf|title=Quality Scores for Next-Generation Sequencing|date=31 October 2011|website=Illumina|access-date=8 May 2018}}</ref>

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.<ref name="Brenner_2000">{{cite journal | vauthors = Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J, Corcoran K | title = Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays | journal = Nature Biotechnology | volume = 18 | issue = 6 | pages = 630–34 | year = 2000 | pmid = 10835600 | doi = 10.1038/76469 | s2cid = 13884154 }}</ref>

== Basic methods ==

=== Maxam-Gilbert sequencing ===
{{Main|Maxam-Gilbert sequencing}}
[[Allan Maxam]] and [[Walter Gilbert]] published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases.<ref name=Maxam77>{{cite journal | vauthors = Maxam AM, Gilbert W | title = A new method for sequencing DNA | journal = Proc. Natl. Acad. Sci. USA | volume = 74 | issue = 2 | pages = 560–64 | date = February 1977 | pmid = 265521 | pmc = 392330 | doi = 10.1073/pnas.74.2.560 | bibcode = 1977PNAS...74..560M | doi-access = free }}</ref> 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.<ref name=Maxam77 />

This method is mostly obsolete as of 2023.<ref name="PubMed m584">{{cite web | title=maxam gilbert sequencing | website=PubMed | url=https://pubmed.ncbi.nlm.nih.gov/?term=maxam+gilbert+sequencing&filter=years.2013-2023&sort=pubdate}}</ref>

=== Chain-termination methods ===
{{Main|Sanger sequencing}}
The [[Sanger sequencing|chain-termination method]] developed by [[Frederick Sanger]] and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability.<ref name="Sanger1977">{{cite journal | vauthors = Sanger F, Nicklen S, Coulson AR | title = DNA sequencing with chain-terminating inhibitors | journal = Proc. Natl. Acad. Sci. USA | volume = 74 | issue = 12 | pages = 5463–77 | date = December 1977 | pmid = 271968 | pmc = 431765 | doi = 10.1073/pnas.74.12.5463 | bibcode = 1977PNAS...74.5463S | doi-access = free }}</ref><ref name=Sanger75>{{cite journal | vauthors = Sanger F, Coulson AR | title = A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase | journal = J. Mol. Biol. | volume = 94 | issue = 3 | pages = 441–48 | date = May 1975 | pmid = 1100841 | doi = 10.1016/0022-2836(75)90213-2 }}</ref> 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]]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.<ref>{{cite web |last=Wetterstrand |first=Kris |title=DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) |publisher=[[National Human Genome Research Institute]] |access-date=30 May 2013 |url=https://www.genome.gov/sequencingcosts }}</ref>

=== Sequencing by synthesis ===
The objective for sequential sequencing by synthesis (SBS) is to determine the sequencing of a [[DNA]] sample by detecting the incorporation of a [[nucleotide]] by a [[DNA polymerase]]. An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The principle of real-time sequencing by synthesis was first described in 1993<ref>{{cite journal |last1=Nyren |first1=P. |last2=Pettersson |first2=B. |last3=Uhlen |first3=M. |title=Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay |journal=Analytical Biochemistry |date=January 1993 |volume=208 |issue=1 |pages=171–175 |doi=10.1006/abio.1993.1024 |pmid=8382019 }}</ref> with improvements published some years later.<ref>{{cite journal |last1=Ronaghi |first1=Mostafa |last2=Uhlén |first2=Mathias |last3=Nyrén |first3=Pål |title=A Sequencing Method Based on Real-Time Pyrophosphate |journal=Science |date=17 July 1998 |volume=281 |issue=5375 |pages=363–365 |doi=10.1126/science.281.5375.363 |pmid=9705713 |s2cid=26331871 }}</ref> The key parts are highly similar for all embodiments of SBS and includes (1) [[Solid phase sequencing|amplification of DNA]] (to enhance the subsequent signal) and attach the DNA to be sequenced to a solid support, (2) generation of single stranded DNA on the solid support, (3) incorporation of nucleotides using an engineered polymerase and (4) real-time detection of the incorporation of nucleotide The steps 3-4 are repeated and the sequence is assembled from the signals obtained in step 4. This principle of real-time sequencing-by-synthesis has been used for almost all [[massive parallel sequencing]] instruments, including [[454 Life Sciences|454]], [[Pacific Biosciences|PacBio]], [[Ion Torrent|IonTorrent]], [[Illumina, Inc.|Illumina]] and [[MGI (company)|MGI]].

== Large-scale sequencing and ''de novo'' sequencing ==
[[File:DNA Sequencing gDNA libraries.jpg|thumb|right|Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.]]
Large-scale sequencing often aims at sequencing very long DNA pieces, such as whole [[chromosome]]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]]s) 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]]''. 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.<ref name="pmid23147856">{{cite journal | vauthors = Quail MA, Gu Y, Swerdlow H, Mayho M | title = Evaluation and optimisation of preparative semi-automated electrophoresis systems for Illumina library preparation | journal = Electrophoresis | volume = 33 | issue = 23 | pages = 3521–28 | year = 2012 | pmid = 23147856 | doi = 10.1002/elps.201200128 | s2cid = 39818212 }}</ref><ref name="pmid22713159">{{cite journal | vauthors = Duhaime MB, Deng L, Poulos BT, Sullivan MB | title = Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method | journal = Environ. Microbiol. | volume = 14 | issue = 9 | pages = 2526–37 | year = 2012 | pmid = 22713159 | pmc = 3466414 | doi = 10.1111/j.1462-2920.2012.02791.x | bibcode = 2012EnvMi..14.2526D }}</ref><ref name="pmid22675423">{{cite journal | vauthors = Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE | title = Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species | journal = PLOS ONE | volume = 7 | issue = 5 | pages = e37135 | year = 2012 | pmid = 22675423 | pmc = 3365034 | doi = 10.1371/journal.pone.0037135 |bibcode = 2012PLoSO...737135P | doi-access = free }}</ref>

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 repeat]]s 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<ref name=Williams2006ePCR>{{cite journal | vauthors = Williams R, Peisajovich SG, Miller OJ, Magdassi S, Tawfik DS, Griffiths AD | title = Amplification of complex gene libraries by emulsion PCR | journal = Nature Methods | volume = 3 | issue = 7 | pages = 545–50 | year = 2006 | pmid = 16791213 | doi = 10.1038/nmeth896 | s2cid = 27459628 }}</ref> 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]]).<ref name="Margulies_2005"/><ref name=polony_sequencing>{{cite journal | vauthors = Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM | title = Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome | journal = Science | volume = 309 | issue = 5741 | pages = 1728–32 | year = 2005 | pmid = 16081699 | doi = 10.1126/science.1117389 | bibcode = 2005Sci...309.1728S | s2cid = 11405973 | doi-access = free }}</ref><ref name=solid_sequencing>{{cite web|url=http://solid.appliedbiosystems.com/|archive-url=https://web.archive.org/web/20080516181322/http://solid.appliedbiosystems.com/|archive-date=2008-05-16|title=Applied Biosystems – File Not Found (404 Error)|date=16 May 2008}}</ref> Emulsion PCR is also used in the GemCode and Chromium platforms developed by [[10x Genomics]].<ref name="10x-epcr">{{cite journal | vauthors = Goodwin S, McPherson JD, McCombie WR | title = Coming of age: ten years of next-generation sequencing technologies | journal = Nature Reviews Genetics | volume = 17 | issue = 6 | pages = 333–51 | date = May 2016 | pmid = 27184599 | doi = 10.1038/nrg.2016.49 | pmc = 10373632 | s2cid = 8295541 }}</ref>

=== Shotgun sequencing ===
{{Main|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.<ref>{{cite journal | vauthors = Staden R | title = A strategy of DNA sequencing employing computer programs. | journal = Nucleic Acids Research | volume = 6 | issue = 7 | pages = 2601–10 | date = 11 June 1979 | pmid = 461197 | pmc = 327874 | doi = 10.1093/nar/6.7.2601 }}</ref>

== High-throughput methods ==
[[File:Mapping Reads.png|thumb|right|Multiple, fragmented sequence reads must be assembled together on the basis of their overlapping areas.]]
High-throughput sequencing, which includes next-generation "short-read" and third-generation "long-read" sequencing methods,<ref group="nt">"Next-generation" remains in broad use as of 2019.  For instance, {{cite journal|vauthors=Straiton J, Free T, Sawyer A, Martin J|date=February 2019|title=From Sanger Sequencing to Genome Databases and Beyond|journal=BioTechniques|volume=66|issue=2|pages=60–63|doi=10.2144/btn-2019-0011|pmid=30744413|quote=Next-generation sequencing (NGS) technologies have revolutionized genomic research. (opening sentence of the article)|doi-access=free}}</ref> applies to [[exome sequencing]], genome sequencing, genome resequencing, [[transcriptome]] profiling ([[RNA-Seq]]), DNA-protein interactions ([[ChIP-sequencing]]), and [[epigenome]] characterization.<ref name="pmid19900591">{{cite journal | vauthors = de Magalhães JP, Finch CE, Janssens G | title = Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions | journal = [[Ageing Research Reviews]] | volume = 9 | issue = 3 | pages = 315–23 | year = 2010 | pmid = 19900591 | pmc = 2878865 | doi = 10.1016/j.arr.2009.10.006 }}</ref>

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.<ref name="pmid23856935">{{cite journal | vauthors = Grada A | title = Next-generation sequencing: methodology and application | journal = J Invest Dermatol | volume = 133 | issue = 8 | pages = e11 | date = August 2013 | pmid = 23856935 | doi = 10.1038/jid.2013.248 | doi-access = free }}</ref><ref name=hall2007>{{cite journal | vauthors = Hall N | title = Advanced sequencing technologies and their wider impact in microbiology | journal = [[J. Exp. Biol.]] | volume =  210| issue = Pt 9 | pages = 1518–25 | date = May 2007 | pmid = 17449817 | doi = 10.1242/jeb.001370 | doi-access = free | bibcode = 2007JExpB.210.1518H }}{{open access}}</ref><ref name=church2006>{{cite journal | vauthors = Church GM | title = Genomes for all | journal = [[Sci. Am.]] | volume = 294 | issue = 1 | pages = 46–54 | date = January 2006 | pmid = 16468433 | doi = 10.1038/scientificamerican0106-46 | author-link1 = George M. Church | bibcode = 2006SciAm.294a..46C | s2cid = 28769137 }}{{subscription required}}</ref> High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.<ref name="pmid18165802">{{cite journal | vauthors = Schuster SC | title = Next-generation sequencing transforms today's biology | journal = Nat. Methods | volume = 5 | issue = 1 | pages = 16–18 | date = January 2008 | pmid = 18165802 | doi = 10.1038/nmeth1156 | s2cid = 1465786 }}</ref> In ultra-high-throughput sequencing as many as 500,000 sequencing-by-synthesis operations may be run in parallel.<ref name=kalb1992>{{cite book | title = Massively Parallel, Optical, and Neural Computing in the United States | first1 = Gilbert | last1 = Kalb | first2 = Robert | last2 = Moxley | publisher = [[IOS Press]] | year = 1992 | isbn = 978-90-5199-097-3 }}{{Page needed|date=June 2013}}</ref><ref name=tenBosch2008>{{cite journal | vauthors = ten Bosch JR, Grody WW | title = Keeping Up with the Next Generation | journal = The Journal of Molecular Diagnostics | volume = 10 | issue = 6 | pages = 484–92 | year = 2008 | pmid = 18832462 | pmc = 2570630 | doi = 10.2353/jmoldx.2008.080027 }}{{open access}}</ref><ref name=Tucker2009>{{cite journal | vauthors = Tucker T, Marra M, Friedman JM | title = Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine | journal = The American Journal of Human Genetics | volume = 85 | issue = 2 | pages = 142–54 | year = 2009 | pmid = 19679224 | pmc = 2725244 | doi = 10.1016/j.ajhg.2009.06.022 }}{{open access}}</ref> Such technologies led to the ability to sequence an entire human genome in as little as one day.<ref name=":2">{{cite journal | vauthors = Straiton J, Free T, Sawyer A, Martin J | title = From Sanger sequencing to genome databases and beyond | journal = BioTechniques | volume = 66 | issue = 2 | pages = 60–63 | date = February 2019 | pmid = 30744413 | doi = 10.2144/btn-2019-0011 | publisher = Future Science | doi-access = free }}</ref>  {{As of|2019||alt=As of 2019|lc=|since=}}, corporate leaders in the development of high-throughput sequencing products included [[Illumina, Inc.|Illumina]], [[Qiagen]] and [[Thermo Fisher Scientific|ThermoFisher Scientific]].<ref name=":2" />

{| class="wikitable" style="font-size:0.9em;"
|+ Comparison of high-throughput sequencing methods<ref name=quail2012>{{cite journal | vauthors = Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP, Gu Y | title = A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and illumina MiSeq sequencers | journal = [[BMC Genomics]] | volume = 13 | issue = 1 | page = 341 | date = 1 January 2012 | pmid = 22827831 | pmc = 3431227 | doi = 10.1186/1471-2164-13-341 | doi-access = free }}{{open access}}</ref><ref name=lin2012>{{cite journal | vauthors = Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M | title = Comparison of Next-Generation Sequencing Systems | journal = Journal of Biomedicine and Biotechnology | volume = 2012 | pages = 251364 | date = 1 January 2012 | pmid = 22829749 | doi = 10.1155/2012/251364 | pmc=3398667| doi-access = free }}{{open access}}</ref>
! Method !! '''Read length''' !! '''Accuracy (single read not consensus)''' !! '''Reads per run''' !! '''Time per run''' !! '''Cost per 1 billion bases (in US$)''' !! '''Advantages''' !! '''Disadvantages''' 
|-
| '''Single-molecule real-time sequencing (Pacific Biosciences)''' ||30,000 bp ([[N50 statistic|N50]]);
maximum read length >100,000 bases<ref name="sequel21">{{cite web|url=https://www.pacb.com/blog/new-software-polymerase-sequel-system-boost-throughput-affordability/|title=New Software, Polymerase for Sequel System Boost Throughput and Affordability – PacBio|date=7 March 2018}}</ref><ref name="autogenerated1">{{cite web |url=http://www.genomeweb.com/sequencing/after-year-testing-two-early-pacbio-customers-expect-more-routine-use-rs-sequenc |title=After a Year of Testing, Two Early PacBio Customers Expect More Routine Use of RS Sequencer in 2012 |author=<!--Staff writer(s); no by-line.--> |date=10 January 2012 |publisher=GenomeWeb }}{{registration required}}</ref><ref>{{cite press release|url=http://globenewswire.com/news-release/2013/10/03/577891/10051072/en/Pacific-Biosciences-Introduces-New-Chemistry-With-Longer-Read-Lengths-to-Detect-Novel-Features-in-DNA-Sequence-and-Advance-Genome-Studies-of-Large-Organisms.html|title=Pacific Biosciences Introduces New Chemistry With Longer Read Lengths to Detect Novel Features in DNA Sequence and Advance Genome Studies of Large Organisms|year=2013}}</ref>
| 87% raw-read accuracy<ref name="pmid23644548">{{cite journal | vauthors = Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J | title = Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data | journal = Nat. Methods | volume = 10 | issue = 6 | pages = 563–69 | year = 2013 | pmid = 23644548 | doi = 10.1038/nmeth.2474 | s2cid = 205421576 }}</ref>|| 4,000,000 per Sequel 2 SMRT cell, 100–200 gigabases<ref name="sequel21" /><ref name="flxlexblog.wordpress.com">{{cite web|url=http://flxlexblog.wordpress.com/2013/07/05/de-novo-bacterial-genome-assembly-a-solved-problem/|title=De novo bacterial genome assembly: a solved problem?|date=5 July 2013}}</ref><ref name=rasko2011>{{cite journal | vauthors = Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS, Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank D, Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP, Schadt EE, Waldor MK | title = Origins of the Strain Causing an Outbreak of Hemolytic–Uremic Syndrome in Germany | journal = [[N Engl J Med]] | volume = 365 | issue = 8 | pages = 709–17 | date = 25 August 2011 | pmid = 21793740 | doi = 10.1056/NEJMoa1106920 | pmc=3168948}}{{open access}}</ref>|| 30 minutes to 20 hours<ref name="sequel21"/><ref name=tran2012>{{cite journal | vauthors = Tran B, Brown AM, Bedard PL, Winquist E, Goss GD, Hotte SJ, Welch SA, Hirte HW, Zhang T, Stein LD, Ferretti V, Watt S, Jiao W, Ng K, Ghai S, Shaw P, Petrocelli T, Hudson TJ, Neel BG, Onetto N, Siu LL, McPherson JD, Kamel-Reid S, Dancey JE | title = Feasibility of real time next generation sequencing of cancer genes linked to drug response: Results from a clinical trial | journal = [[Int. J. Cancer]] | volume = 132 | issue = 7 | pages = 1547–55 | date = 1 January 2012 | pmid = 22948899 | doi = 10.1002/ijc.27817 | s2cid = 72705 | author-link18 = Thomas J. Hudson | author-link10 = Lincoln Stein | doi-access = free }}{{subscription required}}</ref> ||$7.2-$43.3
| Fast. Detects 4mC, 5mC, 6mA.<ref>{{cite journal | vauthors = Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, Roberts RJ | title = The methylomes of six bacteria | journal = Nucleic Acids Research | volume = 40 | issue = 22 | pages = 11450–62 | date = 2 October 2012 | pmid = 23034806 | pmc = 3526280 | doi = 10.1093/nar/gks891 }}</ref> || Moderate throughput. Equipment can be very expensive.
|-
| '''Ion semiconductor (Ion Torrent sequencing)''' || up to 600 bp<ref>{{cite web|url=https://www.thermofisher.com/order/catalog/product/A30670|title=Ion 520 & Ion 530 ExT Kit-Chef – Thermo Fisher Scientific|website=thermofisher.com}}</ref> || 99.6%<ref>{{Cite web |url=http://129.130.90.13/ion-docs/GUID-C6419130-57D8-4DE2-BCF8-47157CB3C9A2.html |title=Raw accuracy |access-date=29 March 2018 |archive-url=https://web.archive.org/web/20180330075720/http://129.130.90.13/ion-docs/GUID-C6419130-57D8-4DE2-BCF8-47157CB3C9A2.html |archive-date=30 March 2018 |url-status=dead}}</ref> || up to 80 million || 2 hours || $66.8-$950 || Less expensive equipment. Fast. || Homopolymer errors.

|-
| '''Pyrosequencing (454)''' || 700 bp || 99.9% || 1 million || 24 hours || $10,000 || Long read size. Fast. || Runs are expensive. Homopolymer errors.

|-
| '''Sequencing by synthesis (Illumina)''' ||MiniSeq, NextSeq: 75–300 bp;
MiSeq: 50–600 bp;

HiSeq 2500: 50–500 bp;

HiSeq 3/4000: 50–300 bp;

HiSeq X: 300 bp
| 99.9% (Phred30) || MiniSeq/MiSeq: 1–25 Million;
NextSeq: 130-00 Million;

HiSeq 2500: 300 million – 2 billion;

HiSeq 3/4000 2.5 billion;

HiSeq X: 3 billion
| 1 to 11 days, depending upon sequencer and specified read length<ref name=vliet2010>{{cite journal | vauthors = van Vliet AH | title = Next generation sequencing of microbial transcriptomes: challenges and opportunities | journal = [[FEMS Microbiology Letters]] | volume = 302 | issue = 1 | pages = 1–7 | date = 1 January 2010 | pmid = 19735299 | doi = 10.1111/j.1574-6968.2009.01767.x | doi-access = free }}{{open access}}</ref> || $5 to $150 || Potential for high sequence yield, depending upon sequencer model and desired application. || Equipment can be very expensive. Requires high concentrations of DNA.
|-
|'''Combinatorial probe anchor synthesis (cPAS-  BGI/MGI)'''
|BGISEQ-50: 35-50bp;
MGISEQ 200: 50-200bp;

BGISEQ-500, MGISEQ-2000: 50-300bp<ref>{{cite web|url=http://en.mgitech.cn/product/30.html|title=BGI and MGISEQ|website=en.mgitech.cn|access-date=2018-07-05}}</ref>
|99.9% (Phred30)
|BGISEQ-50: 160M;
MGISEQ 200: 300M;

BGISEQ-500: 1300M per flow cell;

MGISEQ-2000: 375M FCS flow cell, 1500M FCL flow cell per flow cell.
|1 to 9 days depending on instrument, read length and number of flow cells run at a time.
|$5– $120
|
|
|-
| '''Sequencing by ligation (SOLiD sequencing)''' || 50+35 or 50+50 bp || 99.9% || 1.2 to 1.4 billion || 1 to 2 weeks || $60–130 || Low cost per base. || Slower than other methods. Has issues sequencing palindromic sequences.<ref name="Yu-Feng Huang, Sheng-Chung Chen, Yih-Shien Chiang, Tzu-Han Chen & Kuo-Ping Chiu 2012 S10">{{cite journal | vauthors = Huang YF, Chen SC, Chiang YS, Chen TH, Chiu KP | title = Palindromic sequence impedes sequencing-by-ligation mechanism | journal = [[BMC Systems Biology]] | volume = 6 | pages = S10 | year = 2012 | issue = Suppl 2 | pmid = 23281822 | doi = 10.1186/1752-0509-6-S2-S10 | pmc=3521181 | doi-access = free }}</ref>
|-
| '''Nanopore Sequencing''' || Dependent on library preparation, not the device, so user chooses read length (up to 2,272,580 bp reported<ref>{{Cite bioRxiv |biorxiv=10.1101/312256| title=Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files | date=3 May 2018| last1=Loose| first1=Matthew| last2=Rakyan| first2=Vardhman| last3=Holmes| first3=Nadine| last4=Payne| first4=Alexander}}</ref>). || ~92–97% single read || dependent on read length selected by user || data streamed in real time. Choose 1 min to 48 hrs || $7–100 || Longest individual reads. Accessible user community. Portable (Palm sized). || Lower throughput than other machines, Single read accuracy in 90s. 
|-
|'''GenapSys Sequencing'''
|Around 150 bp single-end
|99.9% (Phred30)
|1 to 16 million
|Around 24 hours
|$667
|Low-cost of instrument ($10,000)
|
|-
|'''Chain termination (Sanger sequencing)'''|| 400 to 900 bp || 99.9% || N/A || 20 minutes to 3 hours || $2,400,000 || Useful for many applications. || More expensive and impractical for larger sequencing projects. This method also requires the time-consuming step of plasmid cloning or PCR.

|}

=== Long-read sequencing methods ===
{{Further|Long-read sequencing}}

==== Single molecule real time (SMRT) sequencing ====
{{Main|Single-molecule real-time sequencing}}
SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs)&nbsp;– 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.<ref name="flxlexblog.wordpress.com"/><ref>{{cite web|url=http://www.genomeweb.com/sequencing/pacbio-sales-start-pick-company-delivers-product-enhancements|title=PacBio Sales Start to Pick Up as Company Delivers on Product Enhancements|date=12 February 2013}}</ref> 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.<ref>{{cite web|url=http://www.bio-itworld.com/2015/9/30/pacbio-announces-sequel-sequencing-system.aspx|title=Bio-IT World|website=bio-itworld.com|access-date=16 November 2015|archive-date=29 July 2020|archive-url=https://web.archive.org/web/20200729220749/http://www.bio-itworld.com/2015/9/30/pacbio-announces-sequel-sequencing-system.aspx|url-status=dead}}</ref><ref>{{cite web|url=https://www.genomeweb.com/business-news/pacbio-launches-higher-throughput-lower-cost-single-molecule-sequencing-system|title=PacBio Launches Higher-Throughput, Lower-Cost Single-Molecule Sequencing System|date=October 2015}}</ref> SMRT sequencing is referred to as "[[Third-generation sequencing|third-generation]]" or "long-read" sequencing.

==== Nanopore DNA sequencing ====
{{Main|Nanopore 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]].<ref>{{cite journal | vauthors = Clarke J, Wu HC, Jayasinghe L, Patel A, Reid S, Bayley H | title = Continuous base identification for single-molecule nanopore DNA sequencing | journal = Nature Nanotechnology | volume = 4 | issue = 4 | pages = 265–70 | date = April 2009 | pmid = 19350039 | doi = 10.1038/nnano.2009.12 | bibcode = 2009NatNa...4..265C }}</ref> 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.<ref name="Torre 2012">{{cite journal|year=2012|title=Fabrication and characterization of solid-state nanopore arrays for high-throughput DNA sequencing|journal=Nanotechnology|volume=23|issue=38|page=385308|bibcode=2012Nanot..23L5308D|doi=10.1088/0957-4484/23/38/385308|pmc=3557807|pmid=22948520|vauthors=dela Torre R, Larkin J, Singer A, Meller A}}</ref> 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.<ref name="Pathak 2012">{{cite journal|year=2012|title=Double-functionalized nanopore-embedded gold electrodes for rapid DNA sequencing|url=https://zenodo.org/record/890231|journal=Applied Physics Letters|volume=100|issue=2|page=023701|doi=10.1063/1.3673335|vauthors=Pathak B, Lofas H, Prasongkit J, Grigoriev A, Ahuja R, Scheicher RH|bibcode=2012ApPhL.100b3701P}}</ref> 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.<ref name="Torre 2012" />

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.<ref name="Pathak 2012" /> 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.<ref name="Korlach 2008">{{cite journal|year=2008|title=Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures|journal=Proceedings of the National Academy of Sciences|volume=105|issue=4|pages=1176–81|bibcode=2008PNAS..105.1176K|doi=10.1073/pnas.0710982105|pmc=2234111|pmid=18216253|vauthors=Korlach J, Marks PJ, Cicero RL, Gray JJ, Murphy DL, Roitman DB, Pham TT, Otto GA, Foquet M, Turner SW|doi-access=free}}</ref>

=== Short-read sequencing methods {{Anchor|Next-generation methods}} ===
{{Further|Short-read sequencing}} <!-- NB. Next-generation sequencing redirects to this section -->

==== Massively parallel signature sequencing (MPSS) ====
The first of the high-throughput sequencing technologies, [[massively parallel signature sequencing]] (or MPSS, also called next generation sequencing), 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]] for measurements of [[gene expression]] levels.<ref name="Brenner_2000"/>

==== Polony sequencing ====
{{Main|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.<ref name=Shendure2005>{{cite journal | vauthors = Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM | title = Accurate multiplex polony sequencing of an evolved bacterial genome. | journal = Science | volume = 309 | issue = 5741 | pages = 1728–32 | date = 9 September 2005 | pmid = 16081699 | doi = 10.1126/science.1117389 | bibcode = 2005Sci...309.1728S | s2cid = 11405973 | doi-access = free }}</ref> 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.<ref name=Shendure2005 /> 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 ====
{{Main|454 Life Sciences#Technology}}
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]].<ref name="Margulies_2005"/> This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.<ref name="pmid18165802"/>

==== Illumina (Solexa) sequencing ====
{{Main|Illumina dye 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.<ref name = "Bentley_2008"/> The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris.<ref>{{Citation|last1=Canard|first1=Bruno|last2=Sarfati|first2=Simon | name-list-style = vanc |title=Novel derivatives usable for the sequencing of nucleic acids|date=13 October 1994|url=http://www.google.ge/patents/CA2158975A1|access-date=2016-03-09}}</ref><ref>{{cite journal | vauthors = Canard B, Sarfati RS | title = DNA polymerase fluorescent substrates with reversible 3'-tags | journal = Gene | volume = 148 | issue = 1 | pages = 1–6 | date = October 1994 | pmid = 7523248 | doi = 10.1016/0378-1119(94)90226-7 }}</ref> 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.<ref name=DNA_colony_patents /> 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.

[[File:Illumina HiSeq 2500.jpg|thumb|An Illumina HiSeq 2500 sequencer]]
[[File:Illumina NovaSeq 6000 flow cell.jpg|thumb|Illumina NovaSeq 6000 flow cell]]

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.

[[File:Illumina MiSeq sequencer.jpg|thumb|An Illumina MiSeq sequencer]]

Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity.  With an optimal configuration, the ultimately reachable instrument throughput is thus dictated solely by the analog-to-digital conversion rate of the camera, multiplied by the number of cameras and divided by the number of pixels per DNA colony required for visualizing them optimally (approximately 10 pixels/colony). In 2012, with cameras operating at more than 10&nbsp;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).<ref name="pmid18576944">{{cite journal | vauthors = Mardis ER | title = Next-generation DNA sequencing methods | journal = Annu Rev Genom Hum Genet | volume = 9 | pages = 387–402 | year = 2008 | pmid = 18576944 | doi = 10.1146/annurev.genom.9.081307.164359 }}</ref>

====Combinatorial probe anchor synthesis (cPAS)====

This method is an upgraded modification to combinatorial probe anchor ligation technology (cPAL) described by [[Complete Genomics]]<ref name=":0">{{cite journal | vauthors = Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, Carnevali P, Nazarenko I, Nilsen GB, Yeung G, Dahl F, Fernandez A, Staker B, Pant KP, Baccash J, Borcherding AP, Brownley A, Cedeno R, Chen L, Chernikoff D, Cheung A, Chirita R, Curson B, Ebert JC, Hacker CR, Hartlage R, Hauser B, Huang S, Jiang Y, Karpinchyk V, Koenig M, Kong C, Landers T, Le C, Liu J, McBride CE, Morenzoni M, Morey RE, Mutch K, Perazich H, Perry K, Peters BA, Peterson J, Pethiyagoda CL, Pothuraju K, Richter C, Rosenbaum AM, Roy S, Shafto J, Sharanhovich U, Shannon KW, Sheppy CG, Sun M, Thakuria JV, Tran A, Vu D, Zaranek AW, Wu X, Drmanac S, Oliphant AR, Banyai WC, Martin B, Ballinger DG, Church GM, Reid CA | display-authors = 6 | title = Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays | journal = Science | volume = 327 | issue = 5961 | pages = 78–81 | date = January 2010 | pmid = 19892942 | doi = 10.1126/science.1181498 | bibcode = 2010Sci...327...78D | s2cid = 17309571 | doi-access = free }}</ref> which has since become part of Chinese genomics company [[Beijing Genomics Institute|BGI]] in 2013.<ref>{{cite web|url=http://www.completegenomics.com/|title=About Us – Complete Genomics|last=brandonvd|website=Complete Genomics|access-date=2018-07-02}}</ref> 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.<ref name=":1">{{cite journal | vauthors = Huang J, Liang X, Xuan Y, Geng C, Li Y, Lu H, Qu S, Mei X, Chen H, Yu T, Sun N, Rao J, Wang J, Zhang W, Chen Y, Liao S, Jiang H, Liu X, Yang Z, Mu F, Gao S | display-authors = 6 | title = A reference human genome dataset of the BGISEQ-500 sequencer | journal = GigaScience | volume = 6 | issue = 5 | pages = 1–9 | date = May 2017 | pmid = 28379488 | pmc = 5467036 | doi = 10.1093/gigascience/gix024 }}</ref>{{citation needed|date=July 2018}}

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.<ref name=":0" /> 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&nbsp;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.<ref name=":1" />{{citation needed|date=July 2018}}
[[File:MGISEQ-2000RS.jpg|thumb|A BGI MGISEQ-2000RS sequencer]]
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&nbsp;nm in diameter, are separated by 700&nbsp;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.<ref name=":0" />{{citation needed|date=July 2018}}

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.{{citation needed|date=July 2018}}

==== SOLiD sequencing ====
[[File:Library preparation for the SOLiD platform.svg|right|thumb|Library preparation for the SOLiD platform]]
{{Main|ABI Solid Sequencing}}
[[File:Two-base encoding scheme.pdf|thumb|Two-base encoding scheme. In two-base encoding, each unique pair of bases on the 3' end of the probe is assigned one out of four possible colors. For example, "AA" is assigned to blue, "AC" is assigned to green, and so on for all 16 unique pairs. During sequencing, each base in the template is sequenced twice, and the resulting data are decoded according to this scheme.]]

[[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.<ref name="pmid18477713">{{cite journal | vauthors = Valouev A, Ichikawa J, Tonthat T, Stuart J, Ranade S, Peckham H, Zeng K, Malek JA, Costa G, McKernan K, Sidow A, Fire A, Johnson SM | title = A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning | journal = Genome Res. | volume = 18 | issue = 7 | pages = 1051–63 | date = July 2008 | pmid = 18477713 | pmc = 2493394 | doi = 10.1101/gr.076463.108 }}</ref> The result is sequences of quantities and lengths comparable to Illumina sequencing.<ref name="pmid18165802"/> This [[sequencing by ligation]] method has been reported to have some issue sequencing palindromic sequences.<ref name="Yu-Feng Huang, Sheng-Chung Chen, Yih-Shien Chiang, Tzu-Han Chen & Kuo-Ping Chiu 2012 S10"/>

==== Ion Torrent semiconductor sequencing ====
{{Main|Ion 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 ion]]s that are released during the [[DNA polymerase|polymerisation]] of [[DNA]], 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.<ref name="rusk">{{cite journal | vauthors = Rusk N | year = 2011 | title = Torrents of sequence | journal = Nat Methods | volume = 8 | issue = 1| page = 44 | doi=10.1038/nmeth.f.330| s2cid = 41040192 | doi-access = free }}</ref>

[[File:From second to fourth-generation sequencing, illustration on TAGGCT template.svg|thumb|right| Sequencing of the TAGGCT template with IonTorrent, PacBioRS and GridION]]

==== DNA nanoball sequencing ====
{{Main|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.<ref name = "Drmanac_2010" /> 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.<ref>{{cite journal | vauthors = Porreca GJ | title = Genome Sequencing on Nanoballs | journal = Nature Biotechnology | volume = 28 | issue = 1 | pages = 43–44 | year = 2010 | pmid = 20062041 | doi = 10.1038/nbt0110-43 | s2cid = 54557996 }}</ref> However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a [[reference genome]] difficult.<ref name = "Drmanac_2010"/>

==== Heliscope single molecule sequencing ====
Heliscope sequencing is a method of [[Single-molecule magnetic sequencing|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.<ref>{{cite web|url=http://www.helicosbio.com/Products/HelicosregGeneticAnalysisSystem/HeliScopetradeSequencer/tabid/87/Default.aspx|archive-url=https://web.archive.org/web/20091102041828/http://www.helicosbio.com/Products/HelicosregGeneticAnalysisSystem/HeliScopetradeSequencer/tabid/87/Default.aspx|archive-date=2009-11-02|title=HeliScope Gene Sequencing / Genetic Analyzer System : Helicos BioSciences|date=2 November 2009}}</ref><ref>{{cite journal | vauthors = Thompson JF, Steinmann KE | title = Single molecule sequencing with a HeliScope genetic analysis system | journal = Current Protocols in Molecular Biology | volume = Chapter 7 | pages = Unit7.10 | date = October 2010 | pmid = 20890904 | pmc = 2954431 | doi = 10.1002/0471142727.mb0710s92 }}</ref> The reads are short, averaging 35 bp.<ref>{{cite web |url=http://seqll.com/technical-description/ |archive-url=https://web.archive.org/web/20140808055229/http://seqll.com/technical-description/ |url-status=dead |archive-date=8 August 2014 |publisher=SeqLL |access-date=9 August 2015 |title=tSMS SeqLL Technical Explanation}}</ref> 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.<ref>{{cite journal |last1=Heather |first1=James M. |last2=Chain |first2=Benjamin |title=The sequence of sequencers: The history of sequencing DNA |journal=Genomics |date=January 2016 |volume=107 |issue=1 |pages=1–8 |doi=10.1016/j.ygeno.2015.11.003 |pmc=4727787 |pmid=26554401 }}</ref> In 2009 a human genome was sequenced using the Heliscope, however in 2012 the company went bankrupt.<ref>{{cite book|author1=Sara El-Metwally |title=Next Generation Sequencing Technologies and Challenges in Sequence Assembly |volume=7 |author2=Osama M. Ouda |author3=Mohamed Helmy |publisher=Next Generation Sequencing Technologies and Challenges in Sequence Assembly, Springer Briefs in Systems Biology Volume 7 |year=2014 |pages=51–59|doi=10.1007/978-1-4939-0715-1_6 |chapter=New Horizons in Next-Generation Sequencing |series=SpringerBriefs in Systems Biology |isbn=978-1-4939-0714-4 }}</ref>

==== 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.<ref name=":3">{{cite journal | vauthors = Abate AR, Hung T, Sperling RA, Mary P, Rotem A, Agresti JJ, Weiner MA, Weitz DA | display-authors = 6 | title = DNA sequence analysis with droplet-based microfluidics | journal = Lab on a Chip | volume = 13 | issue = 24 | pages = 4864–9 | date = December 2013 | pmid = 24185402 | pmc = 4090915 | doi = 10.1039/c3lc50905b }}</ref> 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.<ref name=":3" />

Fair et al. used digital microfluidic devices to study DNA [[pyrosequencing]].<ref name=":4">{{Cite journal| vauthors = Fair RB, Khlystov A, Tailor TD, Ivanov V, Evans RD, Srinivasan V, Pamula VK, Pollack MG, Griffin PB, Zhou J |date= January 2007 |title=Chemical and Biological Applications of Digital-Microfluidic Devices |journal=IEEE Design & Test of Computers|volume=24|issue=1|pages=10–24|doi=10.1109/MDT.2007.8 |hdl= 10161/6987 |citeseerx=10.1.1.559.1440 |s2cid= 10122940 }}</ref> 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.<ref name=":4" />

Boles et al. also studied pyrosequencing on digital microfluidic devices.<ref name=":5">{{cite journal | vauthors = Boles DJ, Benton JL, Siew GJ, Levy MH, Thwar PK, Sandahl MA, Rouse JL, Perkins LC, Sudarsan AP, Jalili R, Pamula VK, Srinivasan V, Fair RB, Griffin PB, Eckhardt AE, Pollack MG | display-authors = 6 | title = Droplet-based pyrosequencing using digital microfluidics | journal = Analytical Chemistry | volume = 83 | issue = 22 | pages = 8439–47 | date = November 2011 | pmid = 21932784 | pmc = 3690483 | doi = 10.1021/ac201416j }}</ref> 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.<ref name=":5" />

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.<ref>{{cite journal | vauthors = Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, Mazutis L | title = Single-cell barcoding and sequencing using droplet microfluidics | journal = Nature Protocols | volume = 12 | issue = 1 | pages = 44–73 | date = January 2017 | pmid = 27929523 | doi = 10.1038/nprot.2016.154 | s2cid = 767782 }}</ref> 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),<ref>{{cite web |url=http://mcb.harvard.edu/branton/index.htm |archive-url=https://web.archive.org/web/20020221002907/http://mcb.harvard.edu/branton/index.htm |url-status=dead |archive-date=21 February 2002 |title=The Harvard Nanopore Group |publisher=Mcb.harvard.edu |access-date=2009-11-15}}</ref><ref name="Physorg">{{cite web |url=http://www.physorg.com/news157378086.html |title=Nanopore Sequencing Could Slash DNA Analysis Costs }}</ref> 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 (&gt;5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.<ref>{{US patent reference
|number=20060029957
|y=2005
|m=07
|d=14 |inventor=ZS Genetics
|title=Systems and methods of analyzing nucleic acid polymers and related components
}}</ref><ref>{{cite journal | vauthors = Xu M, Fujita D, Hanagata N | title = Perspectives and challenges of emerging single-molecule DNA sequencing technologies | journal = Small | volume = 5 | issue = 23 | pages = 2638–49 | date = December 2009 | pmid = 19904762 | doi = 10.1002/smll.200900976 }}</ref>
[[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.<ref>{{cite journal | vauthors = Schadt EE, Turner S, Kasarskis A | title = A window into third-generation sequencing | journal = Human Molecular Genetics | volume = 19 | issue = R2 | pages = R227–40 | year = 2010 | pmid = 20858600 | doi = 10.1093/hmg/ddq416 | doi-access = free }}</ref>

=== 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,<ref>{{cite journal | vauthors = Xu M, Endres RG, Arakawa Y  | title = The electronic properties of DNA bases | journal = Small | volume = 3 | issue = 9 | pages = 1539–43 | year = 2007 | pmid = 17786897 | doi = 10.1002/smll.200600732| doi-access = free }}</ref> allowing differentiation between different bases.<ref>{{cite journal | vauthors = Di Ventra M | title = Fast DNA sequencing by electrical means inches closer | journal = Nanotechnology | volume = 24 | issue = 34 | page = 342501 | year = 2013 | pmid = 23899780 | doi = 10.1088/0957-4484/24/34/342501 | bibcode = 2013Nanot..24H2501D | s2cid = 140101884 | url = https://zenodo.org/record/896777 }}</ref>

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.<ref name="pmid22787559">{{cite journal | vauthors = Ohshiro T, Matsubara K, Tsutsui M, Furuhashi M, Taniguchi M, Kawai T | title = Single-molecule electrical random resequencing of DNA and RNA | journal = Sci Rep | volume = 2 | page = 501 | year = 2012 | pmid = 22787559 | pmc = 3392642 | doi = 10.1038/srep00501 |bibcode = 2012NatSR...2..501O}}</ref>

=== 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.<ref>{{cite journal |author3-link=Daniel Kuritzkes | vauthors = Hanna GJ, Johnson VA, Kuritzkes DR, Richman DD, Martinez-Picado J, Sutton L, Hazelwood JD, D'Aquila RT | title = Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase | journal = J. Clin. Microbiol. | volume = 38 | issue = 7 | pages = 2715–21 | date = 1 July 2000 | pmid = 10878069 | pmc = 87006 | doi = 10.1128/JCM.38.7.2715-2721.2000 }}</ref>

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.<ref name="Morey">{{cite journal | vauthors = Morey M, Fernández-Marmiesse A, Castiñeiras D, Fraga JM, Couce ML, Cocho JA | title = A glimpse into past, present, and future DNA sequencing | journal = Molecular Genetics and Metabolism | volume = 110 | issue = 1–2 | pages = 3–24 | year = 2013 | pmid = 23742747 | doi = 10.1016/j.ymgme.2013.04.024 | hdl = 20.500.11940/2036 | hdl-access = free }}</ref> 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.<ref name="Qin">{{cite journal | vauthors = Qin Y, Schneider TM, Brenner MP | title = Sequencing by Hybridization of Long Targets | journal = PLOS ONE | volume = 7 | issue = 5 | pages = e35819 | year = 2012 | pmid = 22574124 | pmc = 3344849 | doi = 10.1371/journal.pone.0035819 | editor1-last = Gibas | bibcode = 2012PLoSO...735819Q | editor1-first = Cynthia | doi-access = free }}</ref> 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.<ref name="Morey"/>

=== 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.<ref>{{cite journal | vauthors = Edwards JR, Ruparel H, Ju J | title = Mass-spectrometry DNA sequencing | journal = Mutation Research | volume = 573 | issue = 1–2 | pages = 3–12 | year = 2005 | pmid = 15829234 | doi = 10.1016/j.mrfmmm.2004.07.021 | bibcode = 2005MRFMM.573....3E }}</ref>

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<ref>{{cite journal | vauthors = Hall TA, Budowle B, Jiang Y, Blyn L, Eshoo M, Sannes-Lowery KA, Sampath R, Drader JJ, Hannis JC, Harrell P, Samant V, White N, Ecker DJ, Hofstadler SA | title = Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: A novel tool for the identification and differentiation of humans | journal = Analytical Biochemistry | volume = 344 | issue = 1 | pages = 53–69 | year = 2005 | pmid = 16054106 | doi = 10.1016/j.ab.2005.05.028 }}</ref> and from bones found in mass graves of World War I soldiers.<ref>{{cite journal | vauthors = Howard R, Encheva V, Thomson J, Bache K, Chan YT, Cowen S, Debenham P, Dixon A, Krause JU, Krishan E, Moore D, Moore V, Ojo M, Rodrigues S, Stokes P, Walker J, Zimmermann W, Barallon R | title = Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry | journal = Forensic Science International: Genetics | volume = 7 | issue = 1 | pages = 1–9 | date = 15 June 2011 | pmid = 21683667 | doi = 10.1016/j.fsigen.2011.05.009 | doi-access = free }}</ref>

Early chain-termination and TOF MS methods demonstrated read lengths of up to 100 base pairs.<ref>{{cite journal | vauthors = Monforte JA, Becker CH | title = High-throughput DNA analysis by time-of-flight mass spectrometry | journal = Nature Medicine | volume = 3 | issue = 3 | pages = 360–62 | date = 1 March 1997 | pmid = 9055869 | doi = 10.1038/nm0397-360 | s2cid = 28386145 }}</ref> 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.<ref>{{cite journal | vauthors = Beres SB, Carroll RK, Shea PR, Sitkiewicz I, Martinez-Gutierrez JC, Low DE, McGeer A, Willey BM, Green K, Tyrrell GJ, Goldman TD, Feldgarden M, Birren BW, Fofanov Y, Boos J, Wheaton WD, Honisch C, Musser JM | title = Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics | journal = Proceedings of the National Academy of Sciences | volume = 107 | issue = 9 | pages = 4371–76 | date = 8 February 2010 | pmid = 20142485 | pmc = 2840111 | doi = 10.1073/pnas.0911295107 | bibcode = 2010PNAS..107.4371B | doi-access = free }}</ref>

=== Microfluidic Sanger sequencing ===
{{Main|Sanger sequencing}}
In microfluidic [[Sanger sequencing]] the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10&nbsp;cm in diameter) thus reducing the reagent usage as well as cost.<ref>{{cite journal | vauthors = Kan CW, Fredlake CP, Doherty EA, Barron AE | title = DNA sequencing and genotyping in miniaturized electrophoresis systems | journal = Electrophoresis | volume = 25 | issue = 21–22 | pages = 3564–88 | date = 1 November 2004 | pmid = 15565709 | doi = 10.1002/elps.200406161 | s2cid = 4851728 }}</ref> In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips.<ref>{{cite journal | vauthors = Chen YJ, Roller EE, Huang X | title = DNA sequencing by denaturation: experimental proof of concept with an integrated fluidic device | journal = Lab on a Chip | volume = 10 | issue = 9 | pages = 1153–59 | year = 2010 | pmid = 20390134 | pmc = 2881221 | doi = 10.1039/b921417h }}</ref> Research will still need to be done in order to make this use of technology effective.

=== Microscopy-based techniques ===
{{Main|Transmission electron microscopy DNA sequencing}}
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.<ref>{{cite journal | vauthors = Bell DC, Thomas WK, Murtagh KM, Dionne CA, Graham AC, Anderson JE, Glover WR | title = DNA Base Identification by Electron Microscopy | journal = Microscopy and Microanalysis | volume = 18 | issue = 5 | pages = 1049–53 | date = 9 October 2012 | pmid = 23046798 | doi = 10.1017/S1431927612012615 | bibcode = 2012MiMic..18.1049B | s2cid = 25713635 }}</ref>

=== 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.<ref>{{cite journal | vauthors = Pareek CS, Smoczynski R, Tretyn A | title = Sequencing technologies and genome sequencing | journal = Journal of Applied Genetics | volume = 52 | issue = 4 | pages = 413–35 | date = November 2011 | pmid = 21698376 | pmc = 3189340 | doi = 10.1007/s13353-011-0057-x }}</ref> A comparison is made between regions and sequence information is deduced by comparing the known sequence regions to the unknown sequence regions.<ref name="Pareek CS">{{cite journal | vauthors = Pareek CS, Smoczynski R, Tretyn A | title = Sequencing technologies and genome sequencing | journal = Journal of Applied Genetics | volume = 52 | issue = 4 | pages = 413–35 | year = 2011 | pmid = 21698376 | pmc = 3189340 | doi = 10.1007/s13353-011-0057-x }}</ref>

=== ''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|PCR]]s. 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.<ref>{{cite journal | vauthors = Fujimori S, Hirai N, Ohashi H, Masuoka K, Nishikimi A, Fukui Y, Washio T, Oshikubo T, Yamashita T, Miyamoto-Sato E | title = Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data | journal = Scientific Reports | volume = 2 | page = 691 | year = 2012 | pmid = 23056904 | pmc = 3466446 | doi = 10.1038/srep00691 | bibcode = 2012NatSR...2..691F }}</ref>

== Market share ==
While there are many different ways to sequence DNA, only a few dominate the market. In 2022, Illumina had about 80% of the market; the rest of the market is taken by only a few players (PacBio, Oxford, 454, MGI)<ref>{{cite web | url=https://sandiegomics.com/2022-sequencing-market-share-same-as-it-ever-was-for-now/ | title=2022 Sequencing Market Share – Same as It Ever Was (For Now) | date=25 June 2023 }}</ref>

== 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.<ref>{{cite journal | vauthors = Harbers M | year = 2008 | title = The Current Status of cDNA Cloning | journal = Genomics | volume = 91 | issue = 3| pages = 232–42 | doi = 10.1016/j.ygeno.2007.11.004 | pmid = 18222633 | doi-access = free }}</ref> 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.<ref>{{cite journal |vauthors=Alberti A, Belser C, Engelen S, Bertrand L, Orvain C, Brinas L, Cruaud C, etal | year = 2014 | title = Comparison of Library Preparation Methods Reveals Their Impact on Interpretation of Metatranscriptomic Data | journal = BMC Genomics | volume = 15 | issue = 1 | pages = 912–12 | doi = 10.1186/1471-2164-15-912 | pmid=25331572 | pmc=4213505 | doi-access = free }}</ref> 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.<ref>{{cite web| url= https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/library-qc-fragment-analyzer-application-note-770-2017-002.pdf|title=Scalable Nucleic Acid Quality Assessments for Illumina Next-Generation Sequencing Library Prep|access-date=2017-12-27}}</ref>

== Development initiatives ==
[[File:Historic cost of sequencing a human genome.svg|thumb|280px|Total cost of sequencing a human genome over time as calculated by the [[National Human Genome Research Institute|NHGRI]]]]
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."<ref>{{cite web|url=http://genomics.xprize.org/|title=Archon Genomics XPRIZE|website=Archon Genomics XPRIZE|access-date=9 August 2007|archive-date=17 June 2013|archive-url=https://web.archive.org/web/20130617175241/http://genomics.xprize.org/|url-status=dead}}</ref>

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.<ref>{{cite web|url=http://www.genome.gov/10000004|title=Grant Information|website=National Human Genome Research Institute (NHGRI)}}</ref>

== 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 [[chromosome]]s. The production of raw sequence data is only the beginning of its detailed [[Bioinformatics|bioinformatical]] analysis.<ref name="pmid24727769">{{cite journal | vauthors = Severin J, Lizio M, Harshbarger J, Kawaji H, Daub CO, Hayashizaki Y, Bertin N, Forrest AR | title = Interactive visualization and analysis of large-scale sequencing datasets using ZENBU | journal = Nat. Biotechnol. | volume = 32 | issue = 3 | pages = 217–19 | year = 2014 | pmid = 24727769 | doi = 10.1038/nbt.2840 | s2cid = 26575621 }}</ref> Yet new methods for sequencing and correcting sequencing errors were developed.<ref>{{cite journal | vauthors = Shmilovici A, Ben-Gal I | title = Using a VOM model for reconstructing potential coding regions in EST sequences | journal = Computational Statistics | year = 2007 | volume = 22 | issue = 1 | pages = 49–69 | doi = 10.1007/s00180-007-0021-8 | s2cid = 2737235 | url = http://www.eng.tau.ac.il/~bengal/VOM_EST.pdf | access-date = 10 January 2014 | archive-date = 31 May 2020 | archive-url = https://web.archive.org/web/20200531014400/http://www.eng.tau.ac.il/~bengal/VOM_EST.pdf | url-status = dead }}</ref>

=== 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.<ref>{{cite journal | vauthors = Del Fabbro C, Scalabrin S, Morgante M, Giorgi FM | title = An Extensive Evaluation of Read Trimming Effects on Illumina NGS Data Analysis | journal = PLOS ONE | volume = 8 | issue = 12 | pages = e85024 | year = 2013 | pmid = 24376861 | pmc = 3871669 | doi = 10.1371/journal.pone.0085024 | bibcode = 2013PLoSO...885024D | doi-access = free }}</ref> This is a partial list of the trimming algorithms currently available, specifying the algorithm class they belong to:

{| class="wikitable"
|+ Read Trimming Algorithms
! Name of algorithm !! Type of algorithm
|-
| Cutadapt<ref name=cutadapt>{{cite journal|last1=Martin|first1=Marcel|title=Cutadapt removes adapter sequences from high-throughput sequencing reads|journal=EMBnet.journal|date=2 May 2011|volume=17|issue=1|page=10|doi=10.14806/ej.17.1.200|doi-access=free}}</ref> || Running sum
|-
| ConDeTri<ref name=condetri>{{cite journal | vauthors = Smeds L, Künstner A | title = ConDeTri--a content dependent read trimmer for Illumina data | journal = PLOS ONE | volume = 6 | issue = 10 | pages = e26314 | date = 19 October 2011 | pmid = 22039460 | pmc = 3198461 | doi = 10.1371/journal.pone.0026314 | bibcode = 2011PLoSO...626314S | doi-access = free }}</ref> || Window based
|-
| ERNE-FILTER<ref name="erne-bs5">{{cite book| vauthors = Prezza N, Del Fabbro C, Vezzi F, De Paoli E, Policriti A |title=Proceedings of the ACM Conference on Bioinformatics, Computational Biology and Biomedicine |chapter=Erne-Bs5 |date=2012|volume=12|pages=12–19|doi=10.1145/2382936.2382938|isbn=9781450316705|s2cid=5673753}}</ref> || Running sum
|-
| FASTX quality trimmer || Window based
|-
| PRINSEQ<ref name=prinseq>{{cite journal | vauthors = Schmieder R, Edwards R | title = Quality control and preprocessing of metagenomic datasets | journal = Bioinformatics | volume = 27 | issue = 6 | pages = 863–4 | date = March 2011 | pmid = 21278185 | pmc = 3051327 | doi = 10.1093/bioinformatics/btr026 }}</ref> || Window based
|-
| Trimmomatic<ref name=trimmomatic>{{cite journal | vauthors = Bolger AM, Lohse M, Usadel B | title = Trimmomatic: a flexible trimmer for Illumina sequence data | journal = Bioinformatics | volume = 30 | issue = 15 | pages = 2114–20 | date = August 2014 | pmid = 24695404 | pmc = 4103590 | doi = 10.1093/bioinformatics/btu170 }}</ref> || Window based
|-
| SolexaQA<ref name=solexaqa>{{cite journal | vauthors = Cox MP, Peterson DA, Biggs PJ | title = SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data | journal = BMC Bioinformatics | volume = 11 | issue = 1 | pages = 485 | date = September 2010 | pmid = 20875133 | pmc = 2956736 | doi = 10.1186/1471-2105-11-485 | doi-access = free }}</ref> || Window based
|-
| SolexaQA-BWA || Running sum
|-
| Sickle || Window based
|}

== Ethical issues ==
{{Expand section|date=May 2015}}
{{Further|Bioethics}}
Human genetics have been included within the field of [[bioethics]] since the early 1970s<ref name="ethics-murray">{{cite journal | vauthors = Murray TH | title = Ethical issues in human genome research | journal = FASEB Journal | volume = 5 | issue = 1 | pages = 55–60 | date = January 1991 | pmid = 1825074 | doi = 10.1096/fasebj.5.1.1825074 | doi-access = free | s2cid = 20009748 }}</ref> 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.<ref name="usd1000-genome-ethics">{{cite journal|vauthors=Robertson JA|date=August 2003|title=The $1000 genome: ethical and legal issues in whole genome sequencing of individuals|journal=The American Journal of Bioethics|volume=3|issue=3|pages=W–IF1|doi=10.1162/152651603322874762|pmid=14735880|s2cid=15357657}}<!--|access-date=20 May 2015--></ref> 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.<ref name="usd1000-genome-ethics" />

As DNA sequencing becomes more widespread, the storage, security and sharing of genomic data has also become more important.<ref name="usd1000-genome-ethics" /><ref name="guardian-ethics">{{cite news|last1=Henderson|first1=Mark|title=Human genome sequencing: the real ethical dilemmas|url=https://www.theguardian.com/science/2013/sep/09/genetics-ethics-human-gene-sequencing|newspaper=The Guardian|access-date=20 May 2015|date=2013-09-09}}</ref> 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.<ref name="guardian-ethics" /><ref name="ny-times-insurance-ethics">{{cite news|last1=Harmon|first1=Amy|title=Insurance Fears Lead Many to Shun DNA Tests|url=https://www.nytimes.com/2008/02/24/health/24dna.html?pagewanted=all&_r=0|website=The New York Times|access-date=20 May 2015|date=24 February 2008}}</ref> 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.<ref name="OMB support">[http://www.genome.gov/Pages/PolicyEthics/GeneticDiscrimination/SAPonHR493.pdf Statement of Administration policy], Executive Office of the President, Office of Management and Budget, 27 April 2007</ref><ref name="Signing">{{cite news |url=http://www.genome.gov/27026050|title=President Bush Signs the Genetic Information Nondiscrimination Act of 2008|author=National Human Genome Research Institute|date=21 May 2008| access-date=17 February 2014}}</ref> 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.<ref name="nature-ethics">{{cite news |last1=Baker |first1=Monya |title=US ethics panel reports on DNA sequencing and privacy |url=https://blogs.nature.com/news/2012/10/us-ethics-panel-reports-on-dna-sequencing-and-privacy.html |work=Nature News Blog |date=11 October 2012 }}</ref><ref name="privacy-progress-report">{{cite web|title=Privacy and Progress in Whole Genome Sequencing|url=http://bioethics.gov/sites/default/files/PrivacyProgress508_1.pdf|publisher=Presidential Commission for the Study of Bioethical Issues|access-date=20 May 2015|archive-url=https://web.archive.org/web/20150612001900/http://bioethics.gov/sites/default/files/PrivacyProgress508_1.pdf|archive-date=12 June 2015|url-status=dead}}</ref>

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".<ref>{{Cite web |last1=Hartnett |first1=Kevin |date=2013-05-12 |title=The DNA in your garbage: up for grabs |url=https://www.bostonglobe.com/ideas/2013/05/11/the-dna-your-garbage-for-grabs/sU12MtVLkoypL1qu2iF6IL/story.html |url-access=subscription |access-date=2023-01-02 |website=The Boston Globe |language=en-US}}</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]].<ref name="ethics-newborns">{{cite journal | vauthors = Goldenberg AJ, Sharp RR | title = The ethical hazards and programmatic challenges of genomic newborn screening | journal = JAMA | volume = 307 | issue = 5 | pages = 461–2 | date = February 2012 | pmid = 22298675 | pmc = 3868436 | doi = 10.1001/jama.2012.68 }}</ref><ref name="ethics-hughes">{{cite web|last1=Hughes|first1=Virginia|title=It's Time To Stop Obsessing About the Dangers of Genetic Information|url=http://www.slate.com/articles/health_and_science/medical_examiner/2013/01/ethics_of_genetic_information_whole_genome_sequencing_is_here_and_we_need.html|website=Slate Magazine|access-date=22 May 2015|date=2013-01-07}}</ref> 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.<ref name="ethics-bloss">{{cite journal | vauthors = Bloss CS, Schork NJ, Topol EJ | title = Effect of direct-to-consumer genomewide profiling to assess disease risk | journal = The New England Journal of Medicine | volume = 364 | issue = 6 | pages = 524–34 | date = February 2011 | pmid = 21226570 | pmc = 3786730 | doi = 10.1056/NEJMoa1011893 }}</ref> 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.<ref name="ethics-time">{{cite magazine|last1=Rochman|first1=Bonnie|title=What Your Doctor Isn't Telling You About Your DNA|url=https://healthland.time.com/2012/10/25/what-your-doctor-isnt-telling-you-about-your-dna/|magazine=[[Time (magazine)|Time]]|access-date=22 May 2015|date=25 October 2012}}</ref> 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.<ref name="ethics-bloss" /> Also, the development of Next Generation sequencing technologies such as Nanopore based sequencing has also raised further ethical concerns.<ref>{{cite journal |last1=Sajeer P |first1=Muhammad |title=Disruptive technology: Exploring the ethical, legal, political, and societal implications of nanopore sequencing technology |journal=EMBO Reports |date=4 May 2023 |volume=24 |issue=5 |pages=e56619 |doi=10.15252/embr.202256619 |pmid=36988424 |pmc=10157308 |s2cid=257803254 }}</ref>

== See also ==
{{div col|colwidth=35em}}
* {{annotated link|Bioinformatics}}
* {{annotated link|Cancer genome sequencing}}
*[[Circular consensus sequencing]]
* {{annotated link|DNA computing}}
* {{annotated link|DNA field-effect transistor}}
* {{annotated link|DNA sequencing theory}}
* {{annotated link|DNA sequencer}}
* {{annotated link|Genographic Project}}
* {{annotated link|Genome project}}
* {{annotated link|Genome sequencing of endangered species}}
* {{annotated link|Genome skimming}}
* {{annotated link|IsoBase}}
*[[Linked-read sequencing]]
* {{annotated link|Jumping library}}
* {{annotated link|Nucleic acid sequence}}
* {{annotated link|Multiplex ligation-dependent probe amplification}}
* {{annotated link|Personalized medicine}}
* {{annotated link|Protein sequencing}}
* {{annotated link|Sequence mining}}
* {{annotated link|Sequence profiling tool}}
* {{annotated link|Sequencing by hybridization}}
* {{annotated link|Sequencing by ligation}}
* {{annotated link|TIARA (database)}}
* {{annotated link|Transmission electron microscopy DNA sequencing}}
{{div col end}}

== Notes ==
{{Reflist|group=nt}}

== References ==
{{Reflist|refs=

<ref name = "Bentley_2008">{{cite journal | vauthors = Bentley DR, Balasubramanian S, ''et al''. | title = Accurate whole human genome sequencing using reversible terminator chemistry | journal = Nature | volume = 456 | issue = 7218 | pages = 53–59 | year = 2008 | pmid = 18987734 | pmc = 2581791 | doi = 10.1038/nature07517 |bibcode = 2008Natur.456...53B }}</ref>

<ref name = "Drmanac_2010" >{{cite journal | vauthors = Drmanac R, Sparks AB, ''et al''. | title = Human Genome Sequencing Using Unchained Base Reads in Self-Assembling DNA Nanoarrays | journal = Science | volume = 327 | issue = 5961 | pages = 78–81 | year = 2010 | pmid = 19892942 | doi = 10.1126/science.1181498 | bibcode = 2010Sci...327...78D | s2cid = 17309571 | doi-access = free }}</ref>

<ref name="Feldmann_1994">{{cite journal | vauthors = Feldmann H, ''et al''. | title = Complete DNA sequence of yeast chromosome II | journal = EMBO J. | volume = 13 | issue = 24 | pages = 5795–809 | year = 1994 | pmid = 7813418 | pmc = 395553 | doi = 10.1002/j.1460-2075.1994.tb06923.x }}</ref>

<ref name="Lander_2001">{{cite journal | vauthors = Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, ''et al''. | title = Initial sequencing and analysis of the human genome | journal = Nature | volume = 409 | issue = 6822 | pages = 860–921 | date = February 2001 | pmid = 11237011 | doi = 10.1038/35057062 | bibcode = 2001Natur.409..860L | url = https://deepblue.lib.umich.edu/bitstream/2027.42/62798/1/409860a0.pdf | doi-access = free }}</ref>

<ref name="Margulies_2005">{{cite journal | vauthors = Margulies M, Egholm M, ''et al''. | title = Genome Sequencing in Open Microfabricated High Density Picoliter Reactors | journal = Nature | volume = 437 | issue = 7057 | pages = 376–80 | date = September 2005 | pmid = 16056220 | pmc = 1464427 | doi = 10.1038/nature03959 | bibcode = 2005Natur.437..376M }}</ref>

<ref name="Venter_2001">{{cite journal | vauthors = Venter JC, Adams MD, ''et al''. | title = The sequence of the human genome | journal = Science | volume = 291 | issue = 5507 | pages = 1304–51 | date = February 2001 | pmid = 11181995 | doi = 10.1126/science.1058040 | bibcode = 2001Sci...291.1304V | doi-access = free }}</ref>

}}

== External links ==
{{Library resources box
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 |1= Next Generation Sequencing (NGS)
}}
* A [[Wikibooks:Next Generation Sequencing (NGS)|wikibook on next generation sequencing]]
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