High efficiency construction of DNA libraries

A method for adaptor ligation to one or more DNA fragments comprising:

• (a) removing the terminal phosphate residues of the one or more DNA fragments;
• (b) treating the dephosphorylated DNA fragments with one or more end-repair enzymes to generate end-repaired DNA fragments;
• (c) ligating one or more double-stranded DNA (dsDNA) pre-adaptors to the 3′ end of each strand of the end-repaired DNA fragments to form pre-adaptor/end-repaired DNA complexes, wherein each dsDNA pre-adaptor comprises (i) a ligation strand oligonucleotide that is ligated to the 3′ end of each strand of the end-repaired DNA fragments and comprises an anchor sequence;
• and (ii) a non-ligation partner strand oligonucleotide;
• (d) displacing the non-ligation partner strand oligonucleotide from the pre-adaptor/end-repaired DNA complexes with a repair oligonucleotide, wherein the repair oligonucleotide is longer than the ligation strand oligonucleotide, and wherein the 3′ end of the repair oligonucleotide is complementary to the 5′ end of the ligation strand oligonucleotide;
• (e) ligating the repair oligonucleotide to the 5′ end of the end-repaired DNA fragments using a kinase ligase strategy to form adaptor/end-repaired DNA complexes, wherein each adaptor comprises the ligation strand oligonucleotide and the repair oligonucleotide;
• wherein the kinase ligase strategy comprises contacting the adaptor/end repaired DNA complexes with a kinase and a ligase, wherein the kinase adds a phosphate group to the 5′ terminal nucleotide of each strand of the end repaired DNA fragments, and wherein the ligase ligates the repair oligonucleotide to the phosphorylated 5′ end of each strand of the end repaired DNA fragments;
• and
• (f) extending the ligation strand oligonucleotide at the 3′ end using the repair oligonucleotide as a template to form one or more contiguous dsDNA fragments each comprising an adaptor molecule ligated to each end of the DNA fragment.