What is Illumina sequencing?
Illumina is a widely used example of next-generation sequencing technology. Its sequencing-by-synthesis (SBS) method reads the order of DNA bases through repeated cycles of synthesis and signal detection.
Preparing the DNA library
The workflow begins with preparing DNA libraries by fragmenting DNA into smaller pieces, mechanically or using enzymes. Specific, known adapters are then attached to both ends of each fragment. These adapters connect the fragments to the sequencing workflow.
Binding to the flow cell and forming clusters
Adapters bind to complementary sequences on a flow cell. Amplification creates clusters of copies of each DNA fragment, supporting many parallel readouts. Cluster generation is a preparation step; it is distinct from reading the DNA sequence itself.
How sequencing by synthesis reads the bases
During each SBS cycle, a fluorescently labeled reversible terminator is incorporated into a growing DNA strand and imaged. The label and blocking group are removed so the next base can be added. Repeating the cycle produces a base-by-base readout. The fluorescent signal comes from nucleotide incorporation, not from labeling whole cells.
Why the sequence matters
Whole-genome sequencing can support comparisons between bacterial genomes and investigations of genetic variation in research samples, including cancer-related studies. The biological question, sample quality, sequencing strategy, and analysis determine what can be concluded from the resulting data.
References
Quainoo S, Coolen JPM, van Hijum SAFT, Huynen MA, Melchers WJG, van Schaik W, Wertheim HFL. 2017. Whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis. Clinical Microbiology Reviews 30:1015-1063. Read the paper.