Illumina sequencing or also known as Sequencing by Synthesis (SBS) sequencing is the most popular next-generation technology. About 90% of the sequencing is performed on illumina sequencing platforms. The speed, accuracy and cost effectiveness of illumina sequencing makes it the most popular choice for genomics community.
Illumina sequencing is based on the “sequencing-by-synthesis” (SBS) technique where a modified DNA polymerase incorporates fluorescently labeled dNTPs into a DNA template during controlled cycles of DNA synthesis. The dNTPs are specially designed bases that have a fluorescent reversible terminator that allows addition of a single base at a time. Then this terminator is cleaved off it allows for additional of another dNTP while at the same time fluorophore on the cleaved terminator is excited by a laser beam. Though Illumina’s camera are unable to detect a single fluorescence event, a bridge amplification method allows to grow a lawn of millions of DNA template at a given location of a glass flow cell amplifying the signal in each cycle of DNA synthesis. Illumina sequencing workflow has 3 key steps:
Genomics DNA is randomly fragmented into shorter fragments of ~200 bps followed by 5’ and 3’ end adapter ligations. These short fragments are then PCR amplified and purified.
Amplified library is loaded on to a flow cell where a high density lawn of immobilized oligos complementary to adapters capture short fragments of DNA. Bridge amplification process generates clonal clusters as shown in the figure below ready for sequencing.