Helicos

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Helicos single-molecule sequencing utilises sequencing-by-synthesis, i.e. sequencing using DNA polymerases, of individual nucleic acid molecules that have a ligated poly-A tail. The Helicos flow cell surface is coated with oligo-dT-50 oligonucleotides that can capture the poly-A tailed nucleic acid molecules. The binding between the poly-A tail and the oligo-dT-50 oligos may leave A's unbound; for example:

TTTTTTTTTTTTTTTTTTTT

            AAAAAAAAAAAAAAAAAAAAAXXXXX

Therefore dTTP and a polymerase are added to fill the remaining nucleotides of the poly-A tail. Following the fill, the nucleic acid molecules are locked in place by the addition of fluorescently labelled dCTP, dGTP, and dATP Virual Terminator nucleotides, which are incorporated as a single nucleotide and act like the chain-terminating base of ddNTPs, i.e. further extension is prohibited. This fill and lock" step ensures that each template becomes available for the sequencing-by-synthesis reaction. One drawback of this procedure is that the first base of the template is unknown.

Sequencing then proceeds through the addition of fluorescently labelled Virtual Terminator nucleotides, laser illumination, and fluorescence capture via a CCD camera.

Known issues

~5% error rate of reads