Instead of bead-based emulsion PCR, Illumina uses bridge PCR, which we just saw in the previous page. The sequencing is conducted on a flow cell using sequencing-by-synthesis methods with fluorescent lights. This requires the user of high-resolution optical devices.
The technology was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. The two founded Solexa in 1998, commercializing their sequencing method. Illumina merged with Solexa in 2007 for $600m, together hoping to "reach and exceed the $100,000 genome." And reach and exceed they did.
Whole genomes are fragmented by nebulization or sonication. The randomly fragmented genomic DNA are then end-repaired by polymerase and exonuclease activity. The 3' ends are phosphorylated, while 5' ends are adenylated. Size selection occurs through gel electrophoresis and PCR selection.
The DNA is then placed on a flow cell, which are silica slides of eight lengthwise lengths. These are about the size of a microscope slide, and are sealed to minimize contamination and handling errors.
On the slides, the flow cells are subjected to isothermal bridge amplification, created clusters densities of up to 2000 molecules. The duplication of each genomic strand aids in amplifying the generated signals upon sequencing.
Illumina sequencing devices incorporate fluorescent reversible terminators. Each dNTP has a corresponding fluorophore attached to it.
When polymerase elongates the strand with a fluorescently-labeled dNTP, the clusters are then excited by a light source and the color recorded by an optical detector. After incorporation occurs, the fluorophore is cleaved, unblocking for the next nucleotide to be incorporated in the next cycle. Since each cycle one permits the elongation of a single dNTP at a time, homopolymers are determined precisely.
In order to elongate our reads, we may sequence starting from the other end. This would be helpful for de novo assemblies, detection of insertions/deletions and other genomic mutations.
Illumina machines generate output in FASTQ format, which gives the probability of a base call being incorrect.
Two videos outlining the overview of Illumina's Sequencing technology.
Illumina acquires Solexa
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