Methods and Enzymatic Compositions for Forming Libraries of Adapter Ligated Nucleic Acid Molecules

A method for generating a double-stranded region in a target polynucleotide, comprising:

• (a) ligating a 3′ adaptor to a target polynucleotide in a reaction mix, wherein the 3′ adaptor is partially double-stranded, and the target polynucleotide is single-stranded,
• wherein the 3′ adaptor comprises a top strand and a bottom strand, and wherein:
• the top strand comprises a nucleic acid sequence that is complementary to a portion of the nucleic acid sequence of the bottom strand, such that the top strand and bottom strand form a double-stranded region by complementary base-pairing;
• and
• the bottom strand comprises:
• (i) a non-complementary 3′ single-stranded extension comprising at least 4 degenerate nucleotides, wherein the at least 4 degenerate nucleotide sequence is a random sequence, and (ii) a site-specific cleavable sequence or nucleotide at or near the junction between the double-stranded region and the extension;
• (b) cleaving the 3′ adaptor at the site-specific cleavable sequence or nucleotide after ligation to remove at least a portion of the extension,
• wherein a double-stranded region is formed at the 3′ end of the target polynucleotide.