Editing DNA sequencing (section)

====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}}