Overview of Illumina Sequencing by Synthesis Workflow | XLEAP SBS chemistry

The Illumina sequencing workflow comprises four fundamental steps: library preparation, cluster generation, sequencing, and data analysis. These steps transform a sample into meaningful genomic data.

Library preparation involves adding adapters to DNA fragments, which contain essential regions for sequencing initiation, barcoding (indexes), and binding to the flow cell's complementary sequences. Different methods exist based on sample type and application.

Cluster generation amplifies each library fragment into a clonally identical cluster on the flow cell surface. Illumina utilizes patterned flow cells for higher density and efficiency. The process involves DNA fragments hybridizing to oligos on the flow cell and undergoing clonal amplification through Exclusion Amplification (ExAmp) chemistry, creating millions of copies.

Sequencing begins with the extension of a primer. Fluorescently tagged nucleotides are added one by one, with only a complementary base incorporated. Each addition emits a characteristic fluorescent signal, detected to determine the base. Illumina's proprietary Sequencing by Synthesis (SBS) uses two-channel detection, with the advanced XLEAP SBS chemistry offering faster and more accurate nucleotide incorporation due to an improved block, linker, and polymerase.

After the first read, the product is washed away, and index primers are introduced to read the molecular barcodes. The DNA then forms a double-stranded bridge, is linearized, and the original forward strand is removed, allowing for the second index read and subsequent second read of the DNA fragment, ensuring highly multiplexed and accurate sequencing.

Billions of reads are generated, and sequences from pooled libraries are separated using their unique indexes. Downstream analysis can involve assembling sequences, mapping them to a reference genome to identify variations, or counting molecules for abundance and gene expression studies. This omic data can be centralized within the Illumina connected software ecosystem for efficient analysis.