Compositions and methods for enhanced adapter ligation

A next generation sequencing adaptor system comprising:

• a 3′ adaptor polynucleotide comprising a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises the second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5′ phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3′ end, wherein the blocking group is not competent for ligation;
• a phosphatase for removing a phosphate from a 5′ terminus of a substrate nucleic acid molecule;
• a DNA polymerase with 3′ to 5′ exonuclease activity for filling in of 3′ recessed ends and removal of 3′ overhangs of the substrate nucleic acid molecule;
• at least one 2′-deoxynucleoside 5′ triphosphate (dNTP);
• and
• a first ligase for ligating the 3′ adaptor polynucleotide to a 3′ terminus of the substrate nucleic acid molecule.

A method of producing a processed substrate nucleic acid molecule, the method comprising:

• (i) applying a phosphatase enzyme to a sample comprising a substrate nucleic acid molecule, wherein the substrate nucleic acid molecule is double stranded;
• (ii) incubating the sample under conditions sufficient to permit the phosphatase enzyme to remove a phosphate from a 5′ terminus of the substrate nucleic acid molecule;
• (iii) applying a DNA polymerase with 3′ to 5′ exonuclease activity and dNTPs to the sample;
• (iv) incubating the sample under conditions sufficient to permit the DNA polymerase with 3′ to 5′ exonuclease activity to produce a blunt end on the substrate nucleic acid molecule;
• (v) applying a 3′ adaptor polynucleotide and a first ligase to the sample, wherein the 3′ adaptor polynucleotide comprises a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises the second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5′ phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3′ end, wherein the blocking group is not competent for ligation;
• and
• (vi) incubating the sample under conditions sufficient to permit ligation of the first oligonucleotide to a 3′ terminus of the substrate nucleic acid molecule to yield a first processed substrate nucleic acid molecule.

A next generation sequencing adaptor system comprising:

• a 3′ adaptor polynucleotide comprising a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises the second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5′ phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3′ end, wherein the blocking group is not competent for ligation;
• a 5′ adaptor polynucleotide comprising a first sequence that is partially or fully complementary to a segment of the first oligonucleotide of the 3′ adaptor polynucleotide;
• a first ligase for ligating the 3′ adaptor polynucleotide to a 3′ terminus of a substrate nucleic acid molecule;
• a second ligase for ligating the 5′ adaptor polynucleotide to a 5′ terminus of the substrate nucleic acid molecule;
• and
• a DNA polymerase having nick translation activity.

A method of producing a processed substrate nucleic acid molecule, the method comprising:

• (i) applying a 3′ adaptor polynucleotide and a first ligase to a sample comprising a substrate nucleic acid molecule, wherein the substrate nucleic acid molecule is double stranded, wherein the 3′ adaptor polynucleotide comprises a first oligonucleotide, a second oligonucleotide, and a double-stranded portion, wherein the double-stranded portion comprises the second oligonucleotide hybridized to the first oligonucleotide, wherein the first oligonucleotide comprises a 5′ phosphate, and wherein the second oligonucleotide comprises a blocking group at its 3′ end, wherein the blocking group is not competent for ligation;
• (ii) incubating the sample under conditions sufficient to permit ligation of the first oligonucleotide to a 3′ terminus of the substrate nucleic acid molecule to yield a first processed substrate nucleic acid molecule
• (iii) applying a 5′ adaptor polynucleotide, a second ligase, at least one dNTP, and a DNA polymerase having nick translation activity to the sample, wherein the 5′ adapter polynucleotide comprises a first sequence that is partially or fully complementary to a segment of the first oligonucleotide of the 3′ adaptor polynucleotide;
• and
• (iv) incubating the sample under conditions sufficient to:
• (a) promote annealing of the 5′ adapter polynucleotide to the first oligonucleotide of the 3′ adaptor;
• (b) permit nick translation activity by the DNA polymerase;
• and (c) permit ligation of the 5′ adaptor polynucleotide to a 5′ terminus of the substrate molecule, wherein upon completion of step (viii), the first processed substrate nucleic acid molecule is a second processed substrate nucleic acid molecule comprising the 3′ adaptor polynucleotide and the 5′ adaptor polynucleotide ligated to the 3′ terminus and 5′ terminus, respectively, of both strands of the substrate nucleic acid molecule.