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Polymerization-based sequencing

Cycle sequencing Dephasing  Comments

Illumina relies on polymerization-based sequencing.


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).






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.


Extension mixture
DNA polymerase and all four nucleotides are added to template. Each nucleotide triphosphate has (i) distinguishable fluorophore (four nucleotides ⇒ four colours) and (ii) 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.


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.


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


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








Dephasing

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