Patent ID: 8563478

Claim:
A method of generating a library of different polynucleotide molecules, the method comprising: (a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs, wherein the extension products comprise nucleic acid sequences that differ from the nucleic acid sequences of either strand of the adapter-target constructs and the extension products collectively provide a library of different template polynucleotide molecules which have common sequences at their 5′ ends and common sequences at their 3′ ends; wherein the polynucleotide adapters are forked adapters formed by annealing of partially complementary first and second polynucleotide strands; and wherein a sequence of 5 or more consecutive nucleotides at 3′ end of the first strand of the forked adapters is complementary to a sequence of 5 or more consecutive nucleotides the 5′ end of the second strand of the forked adapters such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands.