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Abstract

Methods and compositions for forming ligation product hybridized to a nucleic acid template. The ligation product is formed by contacting a nucleic acid template with a primer capable of hybridizing to the template to form a primed template. The primed template is then contacted with a pool of random extension oligonucleotides of length N and an enzyme system. The enzyme system is capable of covalently linking the primer to an extension oligonucleotide adjacently hybridized to it and one or more other extension oligonucleotides adjacently hybridized to the ligation product defined by covalently linked ligation primer and one or more extension oligonucleotides. When covalently linked, the ligation product is hybridized to the nucleic acid template. Modifications permit the determination of the nucleotide sequence of one or more members of a first set of target nucleotide residues in the nucleic acid template that are spaced at intervals of N nucleotides. In such sequencing methods, labeled ligation product is formed wherein the position and type of label incorporated into the labeled ligation product provides information concerning the nucleotide residue in the nucleic acid template with which the labeled nucleotide residue is base paired. The compositions comprise a pool of oligonucleotides of a predetermined length N containing at least one differentially labeled sequencing nucleotide residue at a predetermined position and random nucleotide residues at each of the other nucleotide positions.

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First Claim