Editing DNA sequencing (section)

==== 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>