Séquençage à très haut débit - GAII (Illumina/Solexa)

Shendure J. & Hanlee J. (2008) Nature Biotechnology vol26 p1135-1145
" Illumina Genome Analyzer.
Commonly referred to as ’the Solexa’, this platform has its origins in work by Turcatti and colleages and the merger of four companies-Solexa (Essex, UK), Lynx Therapeutics (Hayward, CA, USA), Manteia Predictive Medicine (Coinsins, Switzerland) and Illumina. Libraries can be constructed by any method that gives rise to a mixture of adaptor-flanked fragments up to several hundred base-pairs (bp) in length. Amplified sequencing features are generated by bridge PCR. In this approach, both forward and reverse PCR primers are tethered to a solid substrate by a flexible linker, such that all amplicons arising from any single template molecule during the amplification remain immobilized and clustered to a single physical location on an array. On the Illumina platform, the bridge PCR is somewhat unconventional in relying on alternating cycles of extension with Bst polymerase and denaturation with formamide. The resulting ’clusters’ each consist of ~1,000 clonal amplicons. Several million clusters can be amplified to distinguishable locations within each of eight independent are on a single flow-cell (such that eight independent libraries can be sequenced in parallel during the same instrument run). After cluster generation, the amplicons are single stranded (linearization) and a sequencing primer is hybridized to a universal sequence flanking the region of interest. Each cycle of sequence interrogation consists of single-base extension with a modified DNA polymerase and a mixture of four nucleotides. These nucleotides are modified in two ways. They are ’reversible terminators’, in that a chemically cleavable moiety at the 3’hydroxyl position allows only a single-base incorporation to occur in each cycle; and one of four fluorescent labels, also chemically cleavable, corresponds to the identity of each nucleotide. After single-base extension and acquisition of images in four channels, chemical cleavage of both groups sets up for the next cycle. Read-lengths up to 36 bp are currently routine; longer reads are possible but may incur a higher error rate."