Overview of Illumina Sequencing by Synthesis Workflow | XLEAP SBS chemistry

DNA fragments are prepared through library preparation methods that add adapters to the ends of DNA. These adapters contain sequencing initiation sites, molecular barcodes (indexes), and regions that will bind to complementary sequences on the flow cell surface.

Library fragments hybridize to the flow cell surface and are clonally amplified using Exclusion Amplification (ExAmp) chemistry. This process creates identical copies of each DNA fragment tethered to the flow cell surface, resulting in millions of clusters.

A sequencing primer is extended using fluorescently tagged nucleotides in multiple cycles. After each nucleotide incorporation, clusters are excited by light and emit characteristic fluorescent signals that determine the base call, with the number of cycles determining read length.

After washing away the first read product, an index one sequencing primer is introduced and hybridized to generate the index read. The template then folds over to bind the second oligo on the flow cell, and polymerases extend to form a double-stranded bridge.

The double-stranded DNA is linearized and the original forward strand is removed, leaving only the reverse strand. Index two is read using a sequencing primer, then the reverse strand serves as template for the second read using the same sequencing by synthesis process.

Billions of reads representing all DNA fragments are generated and pooled library sequences are separated based on unique indexes. Downstream analysis processes can assemble sequences de novo, map sequences to a reference genome, or count molecules to assess gene expression.