METHODS OF TARGETED SEQUENCING

• US 20130045872A1
• Filed: 09/10/2010
• Published: 02/21/2013
• Est. Priority Date: 09/10/2009
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Mei, Rui, Zhou, Wei, Matsuzaki, Hajime
• Granted Patent: US 10,072,287 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

The present invention provides methods for targeted sequencing of polynucleotide. In one aspect, the present invention provides a method of sequencing a target polynucleotide with fewer probes. In another aspect, the present invention provides a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing method are also provided in the present invention. The methods of the present invention allow low-cost, high-throughput and accurate sequencing of nucleic acids.

Claims

1. A method of sequencing comprising:

(a) forming a hybridization complex comprising;

(i) a first set of capture probes;

(ii) a target polynucleotide; and

(iii) a first solution probe,

wherein at least one of the first set of capture probes binds a first region of the target polynucleotide, the first solution probe binds a second region of the target polynucleotide, the first solution probe has a base at the 3′

end complementary to a first loci site of the target polynucleotide, therefore when hybridization occurs said at least one of the first set of capture probes and the first solution probe can be ligated, wherein said first solution probe comprises a cleavable first cap;

(b) ligating said at least one of the first set of capture probes and said first solution probe;

(c) adding a second cap to capture probes of said first set of capture probes not ligated to said first solution probe;

(d) removing said first cap; and

(e) sequencing said target polynucleotide.

2. The method of claim 1, further comprising:

(f) forming a hybridization complex comprising a second solution probe that hybridizes to the target polynucleotide, wherein the second solution probe binds to a third region of the target polynucleotide, wherein the second solution probe has a base complementary to a second loci site of the target polynucleotide;

(g) ligating the second solution probe to the first solution probe;

(h) forming a hybridization complex comprising a third solution probe that hybridizes to the target polynucleotide, wherein the third solution probe binds to a fourth region of the target polynucleotide, wherein the third solution probe has a base complementary to a third loci site of the target polynucleotide, wherein the third solution probe comprises a universal primer sequence;

(i) ligating the third solution probe to the second solution probe;

(j) removing the target polynucleotide; and

(k) sequencing with a primer that hybridizes to said universal primer sequence of said third solution probe.

3. The method of any of claims 1-2, wherein sequencing comprises placing the target-probe hybridization complex under nucleic acid synthesis conditions in the presence of free nucleotides to allow extension of the probe that is complementary to the target polynucleotide.

4. The method of any of claims 1-3, further comprising removing non-specific hybridization.

5. The method of any of claims 1-4, wherein said first cap comprises a capping dye and said first solution probe comprises a cleavage site.

6. The method of any of claims 1-5, wherein said second cap comprises a protection group.

7. The method of any of claims 1-6, wherein said first set of capture probes is attached to a flat surface or a bead.

8. The method of claim 7, wherein said first set of capture probes is synthesized or spotted on said flat surface.

9. The method of claim 8, wherein said first set of capture probes is spotted randomly at a controlled density.

10. The method of claim 8, wherein said first set of capture probes is spotted at known locations on the flat surface.

11. The method of any of claims 1-10, wherein said first set of capture probes comprise polynucleotides.

12. The method of claim 11, wherein each capture probe of said first set of capture probes contains 50-150 polynucleotides.

13. The method of any of claims 1-12, wherein said first, second, or third solution probe comprises a polynucleotide.

14. The method of claim 13, wherein said first, second, or third solution probe contains 5 to 50 nucleotides.

15. The method of any of claims 1-14, wherein said target polynucleotide comprises a single nucleotide polymorphism (SNP), an insertion or deletion, or a copy number variation.

16. The method of claim 2, further comprising removal of the target polynucleotide from the hybridization duplex.

17. The method of any of claims 1-16, wherein said first set of capture probes comprises allele-specific capture probes.

18. The method of claim 17, wherein said allele-specific capture probes are known alleles of said first loci.

19. The method of any of claims 1-18, wherein said first cap comprises a nucleotide reversible terminator.

20. The method of claim 19, wherein the nucleotide reversible terminator comprises an amino-2-hydroxypropyl group.

21. The method of any of claims 1-20, wherein at least 50% of a genome is sequenced using no more than 500 million probes.

22. The method of claim 21, wherein 99.5% of the genome is sequenced.

23. The method of any of claims 1-22, wherein the nucleic acid sequence of said first, second or third solution probe is known.

24. The method of any of claims 1-23, wherein the number of total probes used is about 5 million to 500 million.

25. The method of any of claims 1-24, wherein the number of duplicate probes is less than 5.

26. The method of any of claims 1-25, wherein multiple loci in the genome are sequenced.

27. The method of any of claims 1-26, wherein the sequencing avoids SNP with minor allele frequency of greater than 0.05 at the extreme 3′

end of said first, second, third or fourth solution probe or first or second capture probe sequences.

28. The method of any of claims 1-27, wherein sequencing said target polynucleotide comprises generating reads that are greater than 5-50 bases.

29. The method of claim 7, wherein the flat surface is a flow cell.

30. The method of any of claims 1-29, wherein the first, second, third or fourth solution probe is labeled.

31. The method of any of claims 1-30, wherein the first, second, third or fourth solution probe has at least one base that is degenerate.

32. The method of any of claims 1-31, further comprising obtaining sequence information of the target polynucleotide or genome via a computer system.

33. The method of claim 32, wherein the computer system comprises an algorithm for detecting, processing, analyzing and/or assembling nucleotide sequence information.