Methods for sequencing polynucleotides

A method for determining a probability of whether the sequence of a first portion of one of a plurality of double-stranded polynucleotide molecules and the sequence of a second portion of the one of the plurality of double-stranded polynucleotide molecules are located on the same target polynucleotide molecule, comprising the steps of:

• a) providing a plurality of double-stranded polynucleotide molecules having a known length;
• b) attaching the plurality of double-stranded polynucleotide molecules to a surface, wherein a first portion on a first strand of each of the plurality of double-stranded polynucleotide molecules is attached to a first location of the surface and a second portion on a second strand of each of the plurality of double-stranded polynucleotide molecules is attached to a second location of the surface, wherein the first portion and the second portion are located at the opposite ends of two complementary strands of each of the plurality of double-stranded polynucleotide molecules;
• c) separating the first portion on the first strand of each of the plurality of double-stranded polynucleotide molecules from the second portion on the second strand of each of the plurality of double-stranded polynucleotide molecules by denaturing the two complementary strands of each of the plurality of double-stranded polynucleotide molecules, thereby generating a plurality of denatured single polynucleotide strands, each of the plurality of denatured single polynucleotide strands is immobilized on the first location or the second location of the surface;
• d) after step c), amplifying the first portion of each of the plurality of denatured single polynucleotide strands immobilized on the first location and amplifying the second portion of each of the plurality of denatured single polynucleotide strands immobilized on the second location , thereby producing a plurality of nucleic acid clusters on the surface;
• e) after step d), identifying the locations of the plurality of nucleic acid clusters on the surface using an imaging device, and determining the distance from one cluster of the plurality of nucleic acid clusters to another cluster of the plurality of nucleic acid clusters on the surface;
• f) after step e), determining the sequences of the first portion and the second portion of the plurality of nucleic acid clusters;
• and
• g) comparing the location of each of the plurality of nucleic acid clusters on the surface, wherein the distance from one of the plurality of nucleic acid clusters to another of the plurality of nucleic acid clusters determines the probability of whether the sequence of the first portion of the one of the plurality of double-stranded polynucleotide molecules and the sequence of the second portion of the one of the plurality of double-stranded polynucleotide molecules are located on the same target polynucleotide molecule.