Methods and Compositions for Large-Scale Analysis of Nucleic Acids Using DNA Deletions

• US 20080171331A1
• Filed: 11/09/2007
• Published: 07/17/2008
• Est. Priority Date: 11/09/2006
• Status: Abandoned Application

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje T.
• Application Number:
  US11/938,106
• Publication Number:
  US 20080171331A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are constructs that include pairs of target sequences which are separated by a known distance in the polynucleotide from which they are derived.

Claims

1. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first linear construct, wherein the linear construct comprises a first adaptor interposed between a first target polynucleotide fragment and a second target polynucleotide fragment, and wherein the first target polynucleotide fragment and the second target polynucleotide fragment are contiguous nucleic acids within a target polynucleotide;

(b) ligating a deletion adaptor to the first linear construct to form a second linear construct, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease, and wherein the restriction endonuclease cleaves at a known distance from said recognition site;

(c) cleaving the second linear construct with the restriction endonuclease to form a third linear construct,

thereby forming the polynucleotide comprising a deletion mate pair.

2. The method of claim 1, further comprising:

(d) providing a second adaptor to the cleaved second linear construct; and

(e) ligating the adaptor to one end of the cleaved second linear construct to form a fourth linear construct.

3. The method of claim 2, further comprising:

(f) providing a third adaptor to the end of the fourth linear construct opposite the end with the ligated second adaptor; and

(g) ligating the third adaptor to the fourth linear construct.

4. The method of claim 3, further comprising;

(h) circularizing the fourth linear construct.

5. The method of claim 1, wherein the cleaving step (c) comprises deleting a known number of bases from the second target polynucleotide fragment.

6. The method of claim 1, wherein at least one adaptor comprises two recognition sites for the restriction endonuclease on each end.

7. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first circular construct, wherein the construct comprises a first adaptor and a target polynucleotide, wherein the first adaptor comprises a recognition site for a first restriction endonuclease that cleaves at a known distance from the recognition site and a recognition site for a second restriction endonuclease that cleaves within the first adaptor;

(b) cleaving the first circular construct with the first restriction endonuclease to form a first linear construct,

(c) cleaving the first linear construct with the second restriction endonuclease to form a second linear construct; and

(d) circularizing the second linear construct to create a second circular construct;

thereby forming the polynucleotide comprising a deletion mate pair.

8. The method of claim 7, further comprising repeating steps (b) through (d) on the second circular construct, thereby forming a third circular construct comprising a deletion mate pair.

9. The method of claim 8, wherein steps (b) through (d) are repeated at least three times to create a series of constructs.

10. The method of claim 8, wherein different second restriction endonucleases are used in cleaving step (c).

11. The method of claim 8, wherein the same second restriction endonuclease is used to cleave in step (c).

12. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first linear construct comprising a target polynucleotide and an adaptor, wherein a first adaptor is attached to one end of the target polynucleotide;

(b) ligating a deletion adaptor to the end of the first linear construct opposite the first adaptor, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site; and

(c) cleaving the first linear construct with the restriction endonuclease to form a second linear construct,

thereby forming the polynucleotide comprising a deletion mate pair.

13. The method of claim 12, further comprising:

(d) providing a second adaptor to the cleaved second linear construct; and

(e) ligating the second adaptor to the end of the second linear construct that is opposite of the first adaptor.

14. The method of claim 12, further comprising;

(f) circularizing the second linear construct.

15. The method of claim 13, wherein steps (b) and (c) are repeated on the second linear construct.

16. The method of claim 15, wherein steps (b) and (c) are repeated three or more times to create a series of constructs.

17. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first linear construct comprising a target polynucleotide;

(b) ligating a deletion adaptor to one end of the linear construct, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site; and

(c) cleaving the first linear construct with the restriction endonuclease to form a second linear construct,

thereby forming the polynucleotide comprising a deletion mate pair.

18. The method of claim 17, further comprising

(d) providing a first adaptor;

and

(e) ligating the first adaptor to the second linear construct to create a third linear construct.

19. The method of claim 18, further comprising circularizing the third linear construct.

20. The method of claim 18, further comprising:

(d) providing a second adaptor; and

(e) ligating the second adaptor to the second linear construct on the end opposite of the first adaptor.

21. The method of claim 17, further comprising repeating steps (b) and (c) on the second linear construct, thereby forming a third linear construct with a deletion mate pair.

22. The method of claim 21, wherein steps (b) and (c) are repeated at least three times to create a series of constructs.

23. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first circular construct, wherein the construct comprises a first adaptor and a target polynucleotide, wherein the first adaptor comprises a recognition site for a first restriction endonuclease that cleaves at a known distance from the recognition site;

(b) cleaving the first circular construct with the first restriction endonuclease to form a first linear construct,

(c) providing a second adaptor, wherein the second adaptor comprises a recognition site for a second restriction endonuclease that cleaves at a known distance from the recognition site;

(d) ligating the second adaptor to one end of the first linear construct to create a second linear construct; and

(e) circularizing the second linear construct to form a second circular construct;

thereby forming the polynucleotide comprising a deletion mate pair.

24. The method of claim 23, further comprising repeating steps (b) through (e) on the second circular construct, thereby forming a third circular construct with a deletion mate pair.

25. The method of claim 24, wherein steps (b) through (e) are repeated at least three times to create a series of constructs.

26. A method for analyzing a polynucleotide sequence, the method comprising:

(a) providing a deletion mate pair construct, wherein the deletion mate pair construct comprises;

i) a first adaptor,

ii) a second adaptor,

iii) a first target sequence, and

iv) a second target sequence,

wherein the first target sequence and the second target sequence span a portion of the polynucleotide sequence;

(b) identifying at least one nucleotide of the first target sequence and at least one nucleotide of the second target sequence,

thereby analyzing the polynucleotide sequence.

27. The method of claim 26, wherein the at least one nucleotide of the first target sequence and the at least one nucleotide of the second target sequence are separated by a known distance in the polynucleotide sequence.

28. The method of claim 26, wherein one or more of the first adaptor and the second adaptor comprises an anchor probe hybridization site, and the identifying step (b) comprises:

i) hybridizing an anchor probe to the anchor probe hybridization site;

ii) hybridizing sequencing probes to the first target sequence and the second target sequence;

iii) ligating adjacent hybridized sequencing and anchor probes to form ligated probes; and

iv) detecting the ligated probes to identify nucleotides of one or more of the first target sequence and the second target sequence.

29. The method of claim 26, wherein one or more of the first adaptor and the second adaptor comprises an anchor probe hybridization site, and the identifying step (b) comprises:

i) hybridizing an anchor probe to the anchor probe hybridization site;

ii) adding a polymerase and at least one dNTP comprising a label under conditions wherein if the dNTP is perfectly complementary to a detection position, the dNTP is added to the anchor probe to form an extended probe, thereby creating an interrogation position of the extended probe; and

iii) determining the nucleotide at the interrogation position of the extended probe.

30. The method of claim 26, wherein the identifying step (b) comprises:

(a) contacting the deletion mate pair construct with a set of sequencing probes, wherein each of the sequencing probes comprises;

i) a first domain complementary to the first adaptor or to the second adaptor;

ii) a unique nucleotide at a first interrogation position; and

iii) a label;

under conditions wherein if the unique nucleotide is complementary to a first nucleotide in the first target sequence or the second target sequence, a sequencing probe hybridizes to the deletion mate pair construct; and

(b) detecting the label of the hybridized sequencing probe, thereby identifying a nucleotide in the first target sequence or the second target sequence.

31. A method for forming a library of a plurality of circularized deletion mate pair constructs, the method comprising:

(a) ligating a deletion adaptor to each of a plurality of first linear constructs, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease, and wherein the restriction endonuclease cleaves at a known distance from the recognition site;

(b) cleaving at least a portion of the plurality of first linear constructs with the restriction endonuclease to provide a plurality of second linear constructs; and

(c) circularizing a plurality of the second linear constructs, thereby forming the library of circularized deletion mate pair constructs.

32. The method of claim 31, wherein each of the plurality of first linear constructs comprises a circularization adaptor, wherein the circularization adaptor comprises a recognition site for a restriction endonuclease.

33. The method of claim 32, wherein the plurality of first linear constructs is formed by a method comprising:

(a) providing a plurality of circularized polynucleotides, wherein each of the circularized polynucleotides comprises a circularization adaptor, and wherein the circularization adaptor comprises a recognition site for a restriction endonuclease;

(b) cleaving the plurality of circularized polynucleotides with the restriction endonuclease,

thereby forming the plurality of first linear constructs.

34. The method of claim 32 or 33, wherein the recognition site in the circularization adaptor is identical to the recognition site in the deletion adaptor.

35. The method of claim 32 or 33, wherein the recognition site in the circularization adaptor is different from the recognition site in the deletion adaptor.

36. The method of claim 31, wherein the known distance in step (a) is at least 20 bases.

37. The method of claim 31, wherein step (b) is performed without purifying the product of step (a), and step (c) is performed without purifying the second linear constructs formed in step (b).

54. The substrate of claim 37, wherein the plurality of immobilized concatemers represents at least about 80% of a genome.

38. A method for forming a random array, the method comprising:

(a) providing a support comprising a surface;

(b) providing a plurality of deletion mate pair constructs;

(c) immobilizing the plurality of deletion mate pair constructs, thereby forming the random array.

39. The method of claim 38, wherein:

(i) the surface comprises capture probes;

(ii) each of the plurality of deletion mate pair constructs comprises at least one adaptor, wherein the at least one adaptor comprises a sequence complementary to at least one capture probe;

(iii) the immobilizing step comprises forming a duplex between the adaptor and the complementary capture probe,

40. The method of claim 38, wherein:

(i) the surface comprises reactive functionalities;

(ii) each of the plurality of deletion mate pair constructs comprises at least one functionality complementary to at least one reactive functionality; and

(iii) the immobilizing step comprises forming one or more linkages between at least one reactive functionality and a functionality complementary to that reactive functionality.

41. The method of claim 38, wherein the immobilizing step occurs through non-covalent interactions between the plurality of deletion mate pair constructs and the surface.

42. The method of claim 38, wherein the plurality of deletion mate pair constructs is formed by:

(a) providing a plurality of circularized polynucleotides, wherein each of the circularized polynucleotides comprises a first adaptor, and wherein the first adaptor comprises a first recognition site for a first restriction endonuclease,

(b) cleaving the plurality of circularized polynucleotides with the first restriction endonuclease to provide a plurality of first linear constructs;

(c) ligating a second adaptor to the first linear constructs, wherein the second adaptor comprises a recognition site for a second restriction endonuclease, and wherein the second restriction endonuclease cleaves at a known distance from the recognition site;

(d) cleaving the plurality of first linear constructs with the second restriction endonuclease to provide a plurality of second linear constructs,

thereby forming the plurality of deletion mate pair constructs.

43. An array made according to the method of claim 38.

44. A library comprising a plurality of deletion mate pair constructs, wherein the plurality of deletion mate pair constructs comprises target sequences, and wherein the target sequences together represent at least about 80% of a genome.

45. The library of claim 44, wherein each of the deletion mate pair constructs further comprises an adaptor, and the adaptor comprises a recognition site for a restriction endonuclease.

46. The library of claim 45, wherein the adaptor further comprises a recognition site for a sequencing primer.

47. The library of claim 45, wherein the adaptor is interposed between a first target sequence and a second target sequence.

48. The library of claim 47, wherein the first target sequence and the second target sequence are of the same length.

49. The library of claim 47, wherein the first target sequence and the second target sequence are of different lengths.

50. The library of claim 47, wherein the first target sequence and the second target sequence each have a length of about 3 nucleotides to about 100 nucleotides.

51. The library of claim 47, wherein the first target sequence and the second target sequence each have a length of about 8 nucleotides to about 24 nucleotides.

52. A library comprising a plurality of circularized deletion mate pair constructs, wherein:

(a) the plurality of deletion mate pair constructs comprises target sequences, and wherein the target sequences represent at least about 80% of a genome;

(b) each of the plurality of circularized deletion mate pair constructs comprises a first adaptor, a first target sequence, and a second target sequence; and

(c) the first target sequence and the second target sequence are separated by a known number of bases within the genome.

53. A substrate comprising a plurality of immobilized concatemers, wherein:

(a) each unit of the concatemer comprises a deletion mate pair construct, wherein the deletion mate pair construct comprises a first target sequence and a second target sequence;

(b) the first target sequence and the second target sequence are derived from a target polynucleotide; and

(c) the first target sequence and the second target sequence are separated by a known distance within the target polynucleotide.

55. An amplicon made by amplification of a circular library construct comprising target nucleic acid interspersed with a plurality of adaptors, wherein at least two sets of the adaptors are positioned on either side of a target polynucleotide of known length.

56. The amplicon of claim 55, wherein the target polynucleotide between the two sets of adaptors are of the same known length.

57. The amplicon of claim 55, wherein one or more of the adaptors comprises a restriction endonuclease recognition site.

58. The amplicon of claim 57, wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site.

59. The amplicon of claim 55, wherein each adaptor of the plurality of adaptors further comprises a different anchor primer binding site at a 5′

and 3′

end of each of the plurality of adaptors.

60. A plurality of amplicons of circular library constructs, wherein each amplicon comprises target nucleic acid interspersed with a plurality of adaptors, wherein at least two sets of the adaptors are positioned on either side of a target polynucleotide of known length.

61. The plurality of amplicons of claim 60, wherein each of the plurality of adaptors in each amplicon has been selected to have a desired orientation with respect to at least one other of the plurality of adaptors.

62. The plurality of amplicons of claim 60, wherein the target nucleic acid is genomic DNA, cDNA or RNA, and wherein the plurality of amplicons comprises substantially all of genomic DNA, cDNA or RNA of interest.

63. The plurality of amplicons of claim 60, wherein one or more of the adaptors comprises a restriction endonuclease recognition site.

64. The plurality of amplicons of claim 63, wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site.

65. The plurality of amplicons of claim 63, wherein each adaptor of the plurality of adaptors further comprise a different anchor primer binding site at a 5′

and 3′

end of each of the plurality of adaptors.

66. A kit for selecting for desired orientations of multiple adaptors in library constructs, wherein said kit comprises:

(a) a first double-stranded adaptor, wherein the first double-stranded adaptor comprises a recognition site for a first Type IIs restriction endonuclease;

(b) a second double-stranded adaptor, wherein the second double-stranded adaptor comprises a restriction site for a second Type IIs restriction endonuclease; and

(c) primers complementary to both ends of each of said first and second adaptors.