Overview of Illumina Sequencing by Synthesis Workflow | Standard SBS chemistry

DNA fragments are prepared through various methods that add adapters to fragment ends. Reduced cycle amplification introduces sequencing binding sites, indices, and regions complementary to flow cell oligos.

Prepared DNA fragments hybridize to oligos on the flow cell surface and undergo isothermal clonal amplification. The process involves repeated cycles of polymerase extension, strand denaturation, and bridge formation, resulting in millions of identical sequence clusters attached to the flow cell.

A sequencing primer extends along the template strand while fluorescently tagged nucleotides are incorporated one at a time based on template sequence. After each nucleotide addition, clusters are illuminated and characteristic fluorescent signals are detected to determine base identity.

After washing away the first read product, index 1 primer is introduced and hybridized for reading. Following similar wash and extension steps, the template folds to bind the second flow cell oligo, enabling index 2 sequencing.

The reverse strand is used as template after the forward strand is cleaved and washed away. Read 2 sequencing primer is introduced and extended through multiple cycles to achieve the desired read length, with fluorescent detection at each base incorporation step.