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1. (WO2018140695) CONSTRUCTION OF NEXT GENERATION SEQUENCING (NGS) LIBRARIES USING COMPETITIVE STRAND DISPLACEMENT
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WHAT IS CLAIMED IS:

A method of preparing a target nucleic acid fragment for sequencing, the method comprising:

a. ligation of a first adaptor sequence to the 3 ' end of the target nucleic acid fragment with a first ligase; and

b. ligation of a second adaptor sequence to the 5 ' end of said target nucleic acid fragment with a second ligase

c. whereby said first adaptor sequence is adenylated on its 5' end and d. whereby said first ligase is an adenylation-deficient ATP-dependent ligase.

The method of claim 1 wherein the first ligase is T4 DNA ligase with an amino acid substitution at K.159.

The method of claim 2 wherein the amino acid substitution is a K1S9S.

The method of claim 1 wherein the second ligase is an ATP-dependent ligase. The method of claim 1 wherein the second ligase is a non-ATP-dependent ligase.

The method of claim 5 wherein the second ligase is an NAD-dependent ligase.

A method of preparing a sample of target nucleic acid fragments for sequencing, the method comprising:

a. ligation of a plurality of first adaptor sequences to the 3' ends of the target nucleic acid fragments with a first ligase, whereby the first adaptor sequences comprise

i. variable tag sequences on the 5' ends which serve to

independently label the sense and antisense strands of each target fragment on the 3 ' ends and

ii. a constant sequence located 3 ' of the variable tag sequences, b. annealing of a second adaptor sequence to the constant sequence of each of the ligated first adaptor sequences, whereby the second adaptor sequence comprises a constant sequence on its 3' end that is complementary to the constant sequence of each first adaptor sequence,

c. filling in resulting gaps spanning the first variable tag sequences with a polymerase, creating a plurality of second variable tag sequences, each complementary to its corresponding first variable tag sequence, which serve to independently label the sense and antisense strands of the target fragments on their 5' ends and,

d. ligating the 3' ends of the second tag sequences to the 5' ends of the target sequence with a second ligase,

e. whereby said first adaptor sequence is adenylated on its 5' end and f. whereby said first ligase is an adenylation-deficient ATP-dependent ligase.

8. The method of claim 7 wherein the first ligase is T4 DNA ligase with an amino acid substitution at K.159.

9. The method of claim 8 wherein the amino acid substitution is a K159S.

10. The method of claim 7 wherein the second ligase is an ATP-dependent ligase.

11. The method of claim 7 wherein the second ligase is a non-ATP-dependent ligase.

12. The method of claim 11 wherein the second ligase is an NAD-dependent ligase.

13. The method of claim 7 where the bases of the tag sequences are degenerate.

14. The method of claim 7 where the bases of the tag sequences are fixed.

15. The method of claim 7 where the bases of the tag sequences are both

degenerate and fixed.

16. The method of claim 7 where the tag sequences are between 3 and 20

nucleotides in length.

17. A method of preparing a sample of target nucleic acid fragments for

sequencing, the method comprising:

a. ligation of a plurality of first adaptor sequences to the 3' ends of the target nucleic acids with a first ligase, whereby the first adaptor sequences comprise

i. variable first tag sequences on their 5 ' ends which serve to independently label the sense and antisense strands of each target fragment on their 3' ends and

ii. a constant sequence located 3' of the variable tag sequences, b. annealing of a plurality of second adaptor sequences to the constant and variable tag sequences of the ligated first adaptor sequences, whereby the second adaptor sequences comprise

i. variable second tag sequences on their 3' ends which are complementary to their corresponding first tag sequences and serve to independently label the sense and antisense strands of each target fragment on their 5' ends and

ii. a second constant sequence located 5' of the variable second tag sequences that is complementary to the first constant sequence of each first adaptor sequence,

c. ligating the 3' ends of the second tag sequences to the 5' ends of the target sequence with a second ligase,

d. whereby said first adaptor sequences are adenylated on their 5' ends and

e. whereby said first ligase is an adenylation-deficient ATP-dependent ligase.

18. The method of claim 17 wherein the first ligase is T4 DNA ligase with an amino acid substitution at Kl 59.

19. The method of claim 18 wherein the amino acid substitution is a K159S.

20. The method of claim 17 wherein the second ligase is an ATP-dependent ligase.

21. The method of claim 17 wherein the second ligase is a non- ATP-dependent ligase.

22. The method of claim 21 wherein the second ligase is an NAD-dependent ligase.

23. The method of claim 17 where the bases of the tag sequences are degenerate.

24. The method of claim 17 where the bases of the tag sequences are fixed.

25. The method of claim 17 where the bases of the tag sequences are both

degenerate and fixed.

26. The method of claim 17 where the tag sequences are between 3 and 20

nucleotides in length.