Methods for preparing a library of polynucleotide molecules

A method for generating a library of different polynucleotide molecules, the method comprising:

• (a) providing a plurality of different target double-stranded polynucleotides;
• (b) providing polynucleotide adapters, wherein each polynucleotide adapter comprises:
• (i) a double-stranded annealed region comprising a first strand and a second strand annealed to each other at a first strand region of complementarity and a second strand region of complementarity, respectively, wherein said second strand comprises a plurality of cleavable or excisable bases in said second strand region of complementarity;
• and
• (ii) a 5′ overhang region on said first strand;
• (c) ligating said polynucleotide adapters to both ends of said different target double-stranded polynucleotides to form adapter-target constructs;
• (d) subjecting said adapter-target constructs to conditions sufficient to cleave or excise respective pluralities of cleavable or excisable bases, thereby dissociating respective second strands from respective first strands of said polynucleotide adapters;
• and
• (e) annealing an oligonucleotide to said first strand region of complementarity of said adapter-target constructs;
• thereby generating a library of different polynucleotide molecules.