Polymerization-based sequencing

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Illumina relies on polymerization-based sequencing.

Bridge amplification -- blocking
Single-stranded clusters with terminated 3' ends and annealed sequencing primers are prepared on Cluster station (or on Genome Analyser for second stage of paired-end sequencing).

[править] Cycle sequencing

Sequencing reaction performed by repeating of identical cycles. During one cycle one nucleotide is determined for each cluster and sequencing primer became one nucleotide longer.

Sequencing -- extension

[править] Extension mixture

DNA polymerase and all four nucleotides are added to template. Each nucleotide has
-- distinguishable fluorophore (four nucleotides ⇒ four colours);
-- reversible terminator. The terminator prevents incorporation of several nucleotides one after another. Illumina does not provide information if fluorophore works as a terminator or they are two different chemical groups.

Sequencing -- extension
Polymerase add one nucleotide to the sequencing primer. Cluster consists from identical molecules, so the same nucleotide (same colour) incorporated within the cluster. Different clusters incorporates different nucleotides.

Sequencing -- imaging

[править] Imaging

Non-incorporated fluorescent nucleotides washed out from the channel. Imaging detect incorporated nucleotides.

Sequencing -- removing of fluorophore and terminator

[править] Cleavage

Fluorescent group and terminator are removed. It is possible to make a next cycle: sequencing primer became one nucleotide longer.

[править] Dephasing

Sequencing -- dephaising
Dephaising is a main reason of a limited read length. This picture illustrates the possible reasons for dephasing:
  • 1st oligonucleotide: normal extension // no dephasing;
  • 2nd oligonucleotide: nucleotide does not incorporated // negative dephasing on next cycle;
  • 3rd oligonucleotide: incorporated nucleotide have no terminator // positive dephasing;
  • 4th oligonucleotide: incorporated nucleotide have no fluorophore and terminator // positive dephasing;
  • it is also possible, that terminator was not removed during cleavage, it will result in negative dephasing on a next cycle.

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