Editing DNA sequencing (section)

=== 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>