MATE PAIR LIBRARY CONSTRUCTION

• US 20180044668A1
• Filed: 10/13/2015
• Published: 02/15/2018
• Est. Priority Date: 10/14/2014
• Status: Abandoned Application

• Current Assignee: MGI Tech Co., Ltd (BGI Genomics Co., Ltd.)
• Original Assignee: BGI Shenzhen
• Inventors: JIANG, Yuan, DRMANAC, Radoje, HUROWITZ, Evan, ALEXEEV, Andrei, ZHAO, Xia, RUAN, Jie
• Application Number:
  US15/519,149
• Publication Number:
  US 20180044668A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• 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 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/50 Detection characterised by immobilisation to a surface
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention provides a novel method for ligating an adapter to a target polynucleotide and methods of generating a library of mate-pair polynucleotide constructs that employ such a ligation method. Libraries and arrays comprising mate-pair polynucleotide constructs, and methods of sequencing libraries and arrays comprising mate-pair polynucleotide constructs, are also provided.

Claims

1-7. -7. (canceled)

8. A method of making a mate pair polynucleotide library comprising:

providing a plurality of double-stranded target polynucleotides;

producing circular constructs, each comprising a target polynucleotide, a first adapter, and a nick or gap in the first adapter;

performing controlled nick translation to produce nick translation products, each comprising the target polynucleotide, the first adapter, and a nick or gap a first selected distance within the target polynucleotide;

performing 3′

branch ligation to ligate a 3′

branch adapter to each nick translation product at the nick or gap to produce gap ligation products;

performing controlled primer extension to produce primer extension products by hybridizing a primer to the 3′

branch adapter of the gap ligation products and extending the primer a second selected distance within the target polynucleotides; and

adding a 5′

adapter to a 5′

end of the primer extension products to produce a mate pair library, each member of the library comprising;

the 5′

adapter, a first end portion of a target polynucleotide, the first adapter, a second end portion of the target polynucleotide, and the 3′

branch adapter.

9. The method of claim 8 wherein the first adapter comprises two half adapter arms, the method comprising:

ligating to each end of the target polynucleotides a half adapter arm of the first adapter to produce a ligation product; and

ligating the half adapter arms together to produce the circular construct.

10. The method of claim 8 wherein the first adapter comprises one or more uracil residues, the method comprising excising said one or more uracil residues to produce the nick or gap in the first adapter.

11. The method of claim 8 wherein performing nick translation comprises performing controlled nick translation.

12. The method of claim 11 wherein controlled nick translation is ttCNT orntCNT.

13. The method of claim 8 comprising denaturing the gap ligation products to produce linear single strands and hybridizing the primer to the linear single strands.

14. The method of claim 8 wherein the 3′

branch adapter comprises a 5′

end comprising a top strand comprising a 5′

-phosphate that is ligatable to a 3′

-hydroxyl of the nick translation product at the nick or gap and a 3′

end that is blocked from ligation.

15. The method of claim 8 wherein the mate pair library is a double-stranded mate pair library, the method comprising producing single strands from the mate pair library and ligating ends of the single strands to produce single-stranded library circles.

16. The method of claim 15 comprising amplifying the library circles by rolling circle replication to produce DNA nanoballs.

17. The method of claim 9 comprising disposing the DNA nanoballs in an array on a solid support to produce a DNA nanoball array.

18. The method of claim 8 wherein the mate pair library is a double-stranded mate pair library, the method comprising:

producing single strands from the mate pair library;

disposing the single strands on a surface of a solid support in an array; and

amplifying the single strands on the array to produce an amplified array.

19. The method of claim 18 comprising amplifying the single strands on the array by bridge PCR.

20. A mate pair polynucleotide library made by the method of claim 8.

21. A kit for constructing a mate pair polynucleotide library for performing the method of claim 8, the kit comprising:

5′ and

3′

half adapter arms of a first adapter;

a 3′

branch adapter;

a 5′

adapter; and

instructions for use.

22. The kit of claim 21 wherein at least one of said 5′

and 3′

half adapter arms of said first adapter comprises at least one uracil residue.

23. The kit of claim 21 comprising a single stranded splint oligonucleotide.

24. The kit of claim 21 comprising one or more members of the group consisting of:

a uracil-excising enzyme;

a DNA ligase; and

a DNA polymerase.