Illumina-Sequencing

Illumina Sequencing (also known as Sequencing by Synthesis, SBS) is a widely used next-generation sequencing (NGS) technology that can determine DNA or RNA sequences quickly, accurately and cost-effectively. It was developed by Illumina Inc. and is now one of the standard methods in genome research, medicine and biotechnology.

1. sample preparation (library preparation). The DNA or RNA is first:

- broken down into many small fragments

- Adapter sequences are attached to the ends of these fragments (short synthetic DNA pieces that are later used for binding to the chip and sequencing).

2. attachment to the flow cell: The DNA fragments with their adapters are placed on a glass surface, the so-called flow cell. The flow cell contains complementary oligonucleotides that bind to the adapters.

3. cluster generation (bridge amplification): Each individual DNA fragment is amplified in situ (directly on the flow cell) to create thousands of identical copies in one place - a so-called cluster. This step is important to sufficiently amplify the signal during sequencing.

4. sequencing by synthesis (SBS): This is where the actual reading process takes place:

- A DNA polymerase adds complementary bases (A, T, C, G) to the growing strand.

- Each base is marked with a fluorescent dye - each base in a different color.

- After each incorporation, a photo is taken to record which base has been incorporated.

- The dye is then chemically removed and the next cycle begins. This process happens millions of times in parallel - each cluster provides a sequence.

5. data evaluation (base calling & alignment)

The many fluorescent signals are translated into digital data (A, T, C, G).

The resulting reads (typically 50-300 bases long) are then:

- quality checked,

- assembled (e.g. into a genome or transcriptome),

- and compared with reference sequences.

Advantages of Illumina Sequencing

• High accuracy (error rate < 0.1 %)
• High throughput (billions of bases per run)
• Low cost per base
• Well standardized and widely used

Disadvantages

• Relatively short reads (up to ~600 bp with paired-end sequencing)
• Complex data analysis
• Devices and reagents are expensive