TARGETED SEQUENCING LIBRARY PREPARATION BY GENOMIC DNA CIRCULARIZATION

• US 20120003657A1
• Filed: 06/30/2011
• Published: 01/05/2012
• Est. Priority Date: 07/02/2010
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Myllykangas, Samuel, Ji, Hanlee P.
• Application Number:
  US13/174,297
• Publication Number:
  US 20120003657A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

Certain embodiments provide a method of sequencing that comprises: a) contacting, under hybridization conditions, a target genomic fragment with: i. a vector oligonucleotide comprising a binding site for a sequencing primer; and ii. a splint oligonucleotide that hybridizes to the vector oligonucleotide and to the nucleotide sequences at the ends of a target genomic fragment, to produce a circular nucleic acid; b) contacting the circular nucleic acid with a ligase, thereby ligating the ends of the vector oligonucleotide to the ends of the target genomic fragment to produce a circular DNA molecule; c) separating the circular DNA molecule from the splint oligonucleotide; and d) sequencing the target genomic fragment of the circular DNA molecule using the first sequencing primer.

Claims

1. A method of sequencing comprising:

a) digesting a sample comprising genomic DNA using a restriction enzyme to produce a digested sample;

b) producing a circular nucleic acid comprising i. a splint oligonucleotide, ii. a vector oligonucleotide comprises a binding site for a first sequencing primer iii. a target genomic fragment, and iv. a duplex region in which the 5′

end of said vector oligonucleotide is ligatably adjacent to the 3′

end of the target genomic fragment, and the 3′

end of said vector oligonucleotide is ligatably adjacent to the 5′

end of said target genomic fragment by;

contacting, under hybridization conditions, said digested sample with;

i. said vector oligonucleotide; and

ii. said splint oligonucleotide, wherein said splint oligonucleotide comprises;

a central region that hybridizes to the entirety of said vector oligonucleotide;

a 5′

region that hybridizes to a first region in a target genomic fragment in said digested sample, and

a 3′

region that hybridizes to a second region in said target genomic fragment;

and, optionally enzymatic treatment remove any 5′

overhang from said target genomic fragment to make the 3′

end of said vector oligonucleotide ligatably adjacent to the 5′

end of said target genomic fragment;

c) contacting said circular nucleic acid with a ligase, thereby ligating the 5′

end of said vector oligonucleotide to the 3′

end of the target genomic fragment and ligating the 3′

end of said vector oligonucleotide to the 5′

end of the target genomic fragment to produce a circular DNA molecule;

d) separating said circular DNA molecule from said splint oligonucleotide; and

e) sequencing the target genomic fragment of said circular DNA molecule using said first sequencing primer.

2. The method of claim 1, wherein said vector oligonucleotide further comprises a second binding site for a second sequencing primer and said sequencing step e) comprises sequencing the target genomic fragment of said circular DNA molecule using said first and second sequencing primers.

3. The method of claim 1, further comprising, prior to said sequencing set e), amplifying the target genomic fragment of said circular DNA molecule by polymerase chain reaction (PCR) using a pair of primers that bind to primer sites that are also present in said vector oligonucleotide in addition to said sequencing primer site.

4. The method of claim 1, further comprising linearizing the circular DNA molecule prior to said sequencing step e).

5. The method of claim 1, wherein said contacting steps b) and c) are done in single vessel without the addition of further reagents.

6. The method of claim 1, wherein steps d) and e) are done in the absence of amplifying said circular DNA.

7. The method of claim 1, wherein step b) comprises enzymatic treatment to remove any 5′

overhang from said target genomic fragment to make the 3′

end of said vector oligonucleotide ligatably adjacent to the 5′

end of said target genomic fragment.

8. The method of claim 7, wherein said enzymatic treatment comprises contacting with a FLAP endonuclease.

9. The method of claim 8, wherein said FLAP endonuclease is Taq.

10. The method of claim 5, wherein said contacting steps b) and c) are done in a single vessel in which said genomic fragment, said vector oligonucleotide, said splint oligonucleotide and a thermostable ligase are thermally cycled through multiple rounds of a temperature suitable for denaturation and a temperature suitable for hybridization and ligation.

11. The method of claim 3, wherein said amplifying is clonal amplification in which said circular DNA molecules are amplified in separate reactions that are spatially distinct from one another.

12. The method of claim 11, wherein said clonal amplification is done by bridge PCR.

13. The method of claim 11, wherein said clonal amplification is done by emulsion PCR.

14. The method of claim 3, wherein said amplifying is a bulk amplification in which said circular DNA molecules are amplified in a single reaction containing a plurality of said circular DNA molecules.

15. The method of claim 1, wherein said method isolates and provides the nucleotide sequence of known loci of a genome.

16. The method of claim 1, wherein said method isolates and provides the nucleotide sequence of a partitioned genome.

17. The method of claim 1, wherein said sequencing is done by sequencing is by a next generation sequencing method.

18. A kit comprising:

i. a vector oligonucleotide comprising a first binding site for a sequencing primer and a second binding site for a second sequencing primer; and

ii. a splint oligonucleotide that hybridizes to said the vector oligonucleotide and to the nucleotide sequences at the ends of a plurality of restriction fragments in a mammalian genome,

wherein said vector and splint oligonucleotides are characterized in that, when hybridized with said restriction fragment, they produce a circular nucleic acid comprising a duplex region in which at least the 5′

end of said vector oligonucleotide is ligatably adjacent to the 3′

end of the genomic fragment.

19. The kit of claim 18, further comprising a ligase.

20. The kit of claim 18, further comprising primers that bind to sites in said vector oligonucleotide and that can amplify said genomic fragments, once ligated to said vector oligonucleotide.