Method of making a paired tag library for nucleic acid sequencing

• US 9,657,291 B2
• Filed: 07/14/2015
• Issued: 05/23/2017
• Est. Priority Date: 01/09/2008
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Li, Bin, Xi, Lei, Ranade, Swati, Wang, Yangzhou
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/798,558
• Publication Number:
  US 20150315639A1
• Time in Patent Office: 679 Days
• Field of Search: 435 912
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The present disclosure relates to methods and compositions for making paired tags and paired tag libraries.

Claims

1. A plurality of circular nucleic acid molecules, the individual circular molecules in the plurality comprising:

(a) a first and a second adapter sequence common to the plurality of circular nucleic acid molecules, wherein each of the first and the second adapter sequences are less than 100 bases in length; and

(b) a target sequence region that differs between different molecules of the plurality of circular molecules, wherein each target sequence region comprises a fragment of genomic DNA comprising between 200 bp and 15 kb, wherein each of the circular nucleic molecules comprise a first and second nick in the target sequence, one on each strand of the circular nucleic acid molecule, wherein the first and second nick are each located at different positions within the target sequence region.

2. The molecules of claim 1, wherein the first and the second adapter sequences comprise a double-stranded nucleic acid.

3. The molecules of claim 1, wherein the first and the second adapter sequences further comprise a binding moiety.

4. The molecules of claim 3, wherein the binding moiety comprises biotin.

5. The molecules of claim 3, wherein the binding moiety comprises streptavidin.

6. The molecules of claim 1, further comprising a nick translating enzyme bound to the nucleic acid molecules.

7. The molecules of claim 6, wherein the nick translating enzyme comprises DNA polymerase I.

15. The molecules of claim 1, wherein target sequence region is derived from the human genome.

17. The molecules of claim 1, wherein the target sequence region is derived from random fragments generated from a genomic library.

19. The molecules of claim 17, wherein the random fragments are generated from an entire genome.

21. The molecules of claim 1, wherein the first nick is located between 10 and 2000 nucleotides from an end of the first adapter sequence and the second nick is located between 10 and 2000 nucleotides from an end of the second adapter sequence.

22. The molecules of claim 1, wherein the first nick is located between 10 and 500 nucleotides from an end of the first adapter sequence and the second nick is located between 10 and 500 nucleotides from an end of the second adapter sequence.

23. The molecules of claim 1, wherein the first nick is located between 10 and 200 nucleotides from an end of the first adapter sequence and the second nick is located between 10 and 200 nucleotides from an end of the second adapter sequence.

24. The molecules of claim 1, wherein the first nick is located between 10 and 100 nucleotides from an end of the first adapter sequence and the second nick is located between 10 and 100 nucleotides from an end of the second adapter sequence.

8. A plurality of circular nucleic acid molecules, the individual circular molecules in the plurality comprising:

(a) a first and a second adapter sequence common to the plurality of circular nucleic acid molecules, wherein each of the first and the second adapter sequences are less than 100 bases in length;

(b) a target sequence region that differs between different molecules of the plurality of circular molecules, wherein each target sequence region comprises a fragment of genomic DNA comprising between 200 bp and 15 kb; and

(c) a first and second nick, one on each strand of the circular nucleic acid molecule, located at different positions between an end of the first adapter sequence and an end of the second adapter sequence.

9. The molecules of claim 8, wherein the first adapter sequence comprises a double-stranded nucleic acid.

10. The molecules of claim 8, wherein the first adapter sequence further comprises a binding moiety.

11. The molecules of claim 10, wherein the binding moiety comprises biotin.

12. The molecules of claim 10 wherein the binding moiety comprises streptavidin.

13. The molecules of claim 8, further comprising a nick translating enzyme bound to the plurality of circular nucleic acid molecules.

14. The molecules of claim 13, wherein the nick translating enzyme comprises DNA polymerase I.

16. The molecules of claim 8, wherein target sequence region is derived from the human genome.

18. The molecules of claim 8, wherein the target sequence region is derived from random fragments generated from a genomic library.

20. The molecules of claim 18, wherein the random fragments are generated from an entire genome.