09. Illumina Sequencing-By-Synthesis (SBS) Technology

Instead of bead-based emulsion PCR, Illumina uses bridge PCR, which we just saw in the previous page. The sequencing is conducted on a flow cell using sequencing-by-synthesis methods with fluorescent lights. This requires the user of high-resolution optical devices.

History

The technology was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. The two founded Solexa in 1998, commercializing their sequencing method. Illumina merged with Solexa in 2007 for $600m, together hoping to "reach and exceed the $100,000 genome." And reach and exceed they did.

Procedure

1) Library Preparation

Whole genomes are fragmented by nebulization or sonication. The randomly fragmented genomic DNA are then end-repaired by polymerase and exonuclease activity. The 3' ends are phosphorylated, while 5' ends are adenylated. Size selection occurs through gel electrophoresis and PCR selection.

2) Clonal colony cluster creation

The DNA is then placed on a flow cell, which are silica slides of eight lengthwise lengths. These are about the size of a microscope slide, and are sealed to minimize contamination and handling errors.

An Illumina flowcell
What an Illumina flowcell looks like with a US quarter for scale. Very similar to a microscope slide.

On the slides, the flow cells are subjected to isothermal bridge amplification, created clusters densities of up to 2000 molecules. The duplication of each genomic strand aids in amplifying the generated signals upon sequencing.

An Illumina flowcell with clusters.
Several clusters are formed on the Illumina flow cell like the above.

3) Sequencing

Illumina sequencing devices incorporate fluorescent reversible terminators. Each dNTP has a corresponding fluorophore attached to it.

When polymerase elongates the strand with a fluorescently-labeled dNTP, the clusters are then excited by a light source and the color recorded by an optical detector. After incorporation occurs, the fluorophore is cleaved, unblocking for the next nucleotide to be incorporated in the next cycle. Since each cycle one permits the elongation of a single dNTP at a time, homopolymers are determined precisely.

Sequencing by Synthesis. dNTP fluorescence is translated to a base call.
Sequencing by Synthesis. dNTP fluorescence is translated to a base call.

4) Paired-end reads

In order to elongate our reads, we may sequence starting from the other end. This would be helpful for de novo assemblies, detection of insertions/deletions and other genomic mutations.

  1. After the forward strand is sequenced, we remove it by denaturation.
  2. Index 1 primer attaches, and index 1 is read off the template.
  3. 3' ends are unblocked and index 2 is then read.
  4. dsDNA clusters are regenerated by bridge amplification and DNA is denatured.
  5. The forward base strand is cleaved.
  6. Use the newly synthesized strands to sequence and produce the paired end sequence data.

5) Output

Illumina machines generate output in FASTQ format, which gives the probability of a base call being incorrect.

Videos

Two videos outlining the overview of Illumina's Sequencing technology.

References

Illumina acquires Solexa

Illumina Website

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