Illumina technology is based on "base-per-base" sequencing of DNA fragments, so called Sequencing by synthesis (SBS technology) and its workflow is made up of 4 steps:

Samples preparation

All preparation methods add adapters to the end of the DNA fragments. To enrich the sequence diversity, each adapter is compost of unique DNA motif and, thanks to the complementary sequence, they anneal to the oligo attached to the surface of the FC.

Generation of clusters

Clustering is the isothermal process where each fragment is amplified. The flow cell is a glass surface composed of lanes that contain millions of clusters.

Hybridization occurs when one of the two adapters of the DNA fragment hybrids to the complementary oligo present in FC: the polymerase produces the strand complementary to the hybrid DNA fragments, the double strand gets denaturated and the original complementary fragment washed away.
Bridge amplification: The remaining strand hybridizes to the second oligo of the flow cell and it is clonally amplifying by bridge amplification. Polymerase creates the secondary strand, forming a double stranded bridge.
Clonal strand: the bridge can denaturated, resulting in two independent strand copies of the DNA fragment that can tether to the FC again. The process is repeated millions of times simultaneously, over millions of clusters.
Reverse strand cleavage: After amplification the reverse strand is washed away, leaving only the forward strand whose 3’end is blocked to prevent unwanted priming.

Sequencing

Sequencing begins from the extension of the first sequencing primer. During each cycle, fluorescent-tagged nucleotides compete for addition to the strand elongation: only the nucleotide complementary to the base of the DNA template is selectively added. After each cycle, the fluorescent tag of the nucleotide is excited and the corresponding signal is emitted.

The number of cycles determines the length of the read, the emission wavelength and the intensity of the signal determine the base call.

Data analysis

Millions of reads generated are divided and sorted by the unique sequence of the index introduced during the library preparation.

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