Illumina sequencing
Learning Objectives
- Understand patterned flow cell architecture and Exclusion Amplification (ExAmp) kinetics.
- Trace the cycle of fluorescent reversible terminators: incorporation, imaging, cleavage, and unblocking.
- Compare 4-color optical detection (HiSeq) vs 2-color optical detection (NovaSeq) vs 1-color (iSeq).
- Troubleshoot index hopping, optical duplication, and phasing/pre-phasing quality score decay.
1. The Four-Step Cyclic Reversible Terminator Chemistry
Illumina Sequencing by Synthesis (SBS) relies on proprietary modified nucleotides that possess two key biochemical features:
- A Cleavable Fluorophore: Attached to the nitrogenous base via an enzymatically or chemically labile linker.
- A 3'-O-Azidomethyl Blocking Group: Temporarily caps the 3'-OH group of the ribose sugar. Because no free 3'-OH is available, DNA polymerase can incorporate exactly one single nucleotide per cycle across all DNA strands within a cluster, completely preventing homopolymer over-runs.
Each sequencing cycle executes in four synchronized physical steps:
- Step 1 (Incorporation): DNA polymerase extends the primed strand by incorporating the complementary blocked nucleotide. Excess unbound nucleotides are washed away.
- Step 2 (Fluorescence Imaging): Lasers excite the fluorophores. High-resolution CCD/CMOS optical cameras image the flow cell tiles, recording the fluorescent wavelength and intensity at each cluster coordinate.
- Step 3 (Cleavage & Unblocking): A cleavage reagent (tris(2-carboxyethyl)phosphine, TCEP) cleaves both the fluorescent dye and the 3'-O-azidomethyl protecting group, regenerating a native 3'-OH group.
- Step 4 (Wash & Repeat): The flow cell is thoroughly washed, preparing the template for the next cycle.
2. Patterned Flow Cells & Exclusion Amplification (ExAmp)
Traditional non-patterned flow cells relied on stochastic bridge amplification, resulting in clusters of random sizes and variable distances. Modern high-output systems (HiSeq X, NovaSeq 6000, NovaSeq X) employ patterned flow cells containing billions of pre-etched nanowells (sub-micron wells spaced at uniform distances).
Using Exclusion Amplification (ExAmp) chemistry, amplification of a single library molecule occurs faster than the binding of a second molecule. Once a molecule enters a nanowell, it rapidly replicates to fill the well, chemically excluding competing molecules. This enables exceptionally high cluster density without optical overlapping.