Editing DNA sequencing (section)

== 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 }}</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>