Editing DNA sequencing (section)

==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>