Patent ID: 7291460

Claim:
A method for determining the sequence of a polynucleotide comprising, a) providing at least one population of polynucleotides wherein members of the population share a nucleotide sequence; b) providing 4 x sets of sequencing adaptors, wherein each set of sequencing adaptors comprises one of 4 x possible combinations of nucleotides in a single-stranded region of x nucleotides long, a nuclease recognition site for a nuclease that produces a single-stranded region of x nucleotides long, and a detectable optical marker that is unique to that set of sequencing adaptors, where x is an integer of 3 or more; c) attaching a first end of said polynucleotides to a substrate and ligating to a second end of said polynucleotides a set of initial adaptors comprising a recognition site for a nuclease; d) treating said polynucleotides with a nuclease specific for the recognition site of the initial adaptors, wherein said nuclease cleaves the polynucleotides but not the recognition site to produce digested polynucleotides comprising a single-stranded region of x nucleotides; e) simultaneously contacting the digested polynucleotides with the 4 x sets of sequencing adaptors under conditions such that the sequencing adaptors hybridize to the digested polynucleotides with minimal mismatches; f) ligating the hybridized sequencing adaptors to the polynucleotides to produce adaptor-ligated polynucleotides; g) removing unhybridized sequencing adaptors; h) determining the identity of the hybridized sequencing adaptors by their unique marker thus simultaneously determining the identity and order of x nucleotides in said polynucleotides; i) treating the adaptor-ligated polynucleotides with a nuclease specific for the recognition site of the sequencing adaptors, wherein said nuclease cleaves the polynucleotides but not the recognition site to produce digested polynucleotides comprising a single-stranded region of x nucleotides and a sequencing adaptor fragment; and j) repeating e) through i) at least once, or until the sequence of the polynucleotide has been determined wherein said optical markers are selected using the formula: min Cmap ⁢ ( ∑ correct adaptorss ⁢ ( ∑ incorrrect adaptors ⁢ Δ ⁢ ⁢ G ^ ⁡ ( adaptor , overhang , T ) ⁢ ( Color anyother adaptor · Color correct adaptor ) ) ) .