Ligation-based sequencing

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[править] Structure of detector oligonucleotides

Structure of detector oligonucleotides

Detector oligonucleotides (DO) are 8-mers fluorescently labeled on 3' end. DO's can't be too short, otherwise T4 ligase would not recognize them as a substrate.

Altogether, there are 1024 different detection oligos: (dinucleotide + 3 degenerate)4 = 54 = 1024. First two nucleotides determine the colour of the fluorophore. Colour table show the relationship between dinucleotides and fluorophores. Four different dinucleotides (256 different oligonucleotides) correspond to each fluorophore. If first or second nucleotide (in dinucleotide) is known, colour is unambiguously related with the other nucleotide.

Three next positions — degenerate nucleotides: 64 different versions for each particular dinucleotide. When ligated to the sequencing primer, only one from these 64 versions would fit to the position.

Three last positions: universal bases, they are the same for all detector oligonucleotides.

Dark oligonucleotides have the same internal structure, but have no fluorophores.

[править] Sequencing: ligation step

Structure of detector oligonucleotides
Three main operations during lgation-based sequencing are:
  1. ligation of detector oligonucleotides: only one from 1024 possible types of oligonucleotides is suitable for ligation. Both "XY-dinucleotide" and degenerate part should be complementary to the template for the successful ligation;
  2. scanning: unincorporated oligonucleotides washed out, bead fluorescence registered in four spectral intervals;
  3. digestion of ligated DO
    • remover fluorophore,
    • expose phosphate on 5'-end,
    • shift sequencing primer to a new position.

[править] Ligation accuracy

Two factors provide specificity of ligation:

  • hybridization stability: 8-mer oligonucleotide should be very sensitive to any mismatches;
  • T4 ligase accuracy: enzyme is particularly sensitive to mismatches on 3'-side of the gap (sensitivity drop down fast with increasing of a distance to the gap). Presence of Sodium Acetate in the ligation buffer increases specificity and decreases activity of ligase. In the first version of the SOLiD protocol detector oligonucleotides had the following structure: nnnXYzzz*. Such structure relies practically only on hybridization-based specificity, which was probably not high enough.

[править] Sequencing: example of 35-base sequencing

Structure of detector oligonucleotides

Five primers & seven ligations for each primer: 35 reactions altogether.

Each ligation reaction provides information about colour of particular dinucleotide. According to colour code table (bottom-right) four different dinucleotides correspond to the same colour. To resolve this ambiguity one ligation reaction analyses dinucleotide with one known nucleotide in the first ligation with primer "B", dinucleotide overlaps with known adaptor sequence. Starting from the first known nucleotide it is possible to determine the whole sequence.

[править] Sequencing: anti-dephasing measures

Sequencing: anti-dephasing measures

There are two anti-dephasing measures in SOLiD protocol:

  1. phosphatase treatment: if sequencing primer does not ligate to the detector oligonucleotide it is better to remove phosphate from it. Otherwise it would have a chance to ligate DO on the next ligation step (out of phase). Dephosphorilated sequencing primer will not participate in next steps of the sequencing reaction. This results in decreasing of fluorescent signals.
  2. ligation of dark oligos: signal would drop too fast if all unligated sequencing primers would be removed from the reaction by phosphatase treatment. Dark detector oligos help to keep unligated templates for the following sequencing step. Dark oligos have practically the same structure as normal detector oligonucleotides, but they do not have fluorescent group.

  • XYnnnzzz* & ligase: some sequencing primers remain unligated;

  • XYnnnzzz & 2xligase: some of phosphorilated sequencing primers are ligated with dark detector oligos. Two times more ligase is used for this step to accelerate reaction;

  • phosphatase: remaining phosphates are removed. Dephosphorilated templates do not further participate in sequencing reaction;

  • scanning and digestion: imaging detects incorporated fluorophores/oligonucleotides. After digestion dephosphorilated sequencing primers remain dephosphorilated, detector oligonucleotides were digested and 5' phosphates were exposed.

  • XYnnnzzz* & ligase: next cycle of ligation

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