Methods for detecting genome-wide sequence variations associated with a phenotype

• US 20040002090A1
• Filed: 03/04/2003
• Published: 01/01/2004
• Est. Priority Date: 03/05/2002
• Status: Abandoned Application

• Current Assignee: Solexa Incorporated (Illumina Incorporated)
• Original Assignee: Solexa Incorporated (Illumina Incorporated)
• Inventors: Farinelli, Laurent, Mayer, Pascal, Osteras, Magne, Leviev, Ilia
• Application Number:
  US10/378,688
• Publication Number:
  US 20040002090A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The invention provides methods for determining genome-wide sequence variations associated with a phenotype of a species in a hypothesis-free manner. In the methods of the invention, a set of restriction fragments for each of a sub-population of individuals having the phenotype are generated by digesting nucleic acids from the individual using one or more different restriction enzymes. A set of restriction sequence tags for the individual is then determined from the set of restriction fragments. The restriction sequence tags for the sub-population of organisms are compared and grouped into one or more groups, each of which comprising restriction sequence tags that comprise homologous sequences. The obtained one or more groups of restriction sequence tags identify the sequence variations associated with the phenotype. The methods of the invention can be used for, e.g., analysis of large numbers of sequence variants in many patient samples to identify subtle genetic risk factors.

Claims

1. A method for determining genome-wide sequence variations associated with a phenotype of one or more individual organisms, comprising

I) generating a set of restriction sequence tags for each individual organism of said one or more individual organisms by a method comprising

A) digesting nucleic acids from each said individual organism using one or more first restriction enzymes to generate a set of restriction fragments;

and

B) determining a set of restriction sequence tags for each said individual organism, wherein said set of restriction sequence tags comprises one or more restriction sequence tags for each of said restriction fragments, each said one or more restriction sequence tags comprising a sequence in the corresponding restriction fragment; and

II) grouping restriction sequence tags for said one or more individual organisms into one or more groups of restriction sequence tags, each said group comprising restriction sequence tags that are homologous;

wherein said one or more groups of restriction sequence tags identify sequence variations associated with said phenotype.

2. The method of claim 1, wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first circular nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence comprising one or more recognition sites of a second restriction enzyme, said recognition sites being located and oriented such that said second restriction enzyme cut in said restriction fragments;

B2) digesting said first circular nucleic acid fragments with said second restriction enzyme;

B3) modifying the ends generated by said second restriction enzyme to permit ligation;

B4) linking said ends generated by said second restriction enzyme to produce a set of second circular nucleic acid fragments; and

B5) sequencing at least a portion of each of said restriction fragments in said second circular nucleic acids to determine said set of restriction sequence tags.

3. The method of any one of claims 2, wherein each said one or more recognition sites are located close to an end of said first engineered nucleic acid.

4. The method of claim 3, wherein each of said one of more recognition sites is located less than 25 nucleotides apart from an end of said first engineered nucleic acid.

5. The method of claim 4, wherein each of said one or more recognition sites is located zero to 5 nucleotides apart from an end of said first engineered nucleic acid.

6. The method of claim 2, wherein said second restriction enzyme is a type IIS endonuclease.

7. The method of claim 6, further comprising before said step B5) a step of fixing and amplifying nucleic acid fragments comprised in said second circular nucleic acid fragments on a solid surface.

8. The method of claim 7, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said second circular nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules comprising one of said nucleic acid fragments in said second circular nucleic acid fragments.

9. The method of claim 8, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) providing a solid surface comprising a plurality of colony primers immobilized on said solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linearized fragments;

iii) denaturing said linearized fragments to generate single stranded fragments;

iv) annealing said single stranded fragments to said immobilized colony primers;

v) carrying out a primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers;

viii) repeating said steps v) through vii) such that said colonies are generated, each at a particular location on said solid surface.

10. The method of claim 8, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) denaturing said immobilized linearized fragments to generate immobilized single-stranded fragments;

v) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

vi) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vii) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

viii) annealing said immobilized single stranded fragments to immobilized colony primers; and

ix) repeating said steps v) through viii) such that said colonies are generated, each at a particular location on said solid surface.

11. The method of claim 8, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted linearized fragments can occur;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

12. The method of any one of claims 9-11, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of the nucleotide sequence of each of said colonies.

13. The method of claim 12, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

14. The method of claim 1, wherein said first restriction enzyme cuts at both sides of its recognition site in such a manner that the cutting sites enclose a part of sequence that is not part of the recognition site, and wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) modifying the ends generated by said first restriction enzyme to permit ligation;

B2) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first circular nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence; and

B3) sequencing at least a portion of each of said restriction fragments in said first circular nucleic acids to determine said set of restriction sequence tags.

15. The method of claim 14, further comprising before said step B3) a step of fixing and amplifying nucleic acid fragments comprised in said second circular nucleic acid fragments on a solid surface.

16. The method of claim 15, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said first circular nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said first circular nucleic acid fragments.

17. The method of claim 16, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) providing a solid surface comprising a plurality of colony primers immobilized at the 5′

end on said solid surface, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linearized fragments;

iii) denaturing said linearized fragments to generate single stranded fragments;

iv) annealing said single stranded fragments to said immobilized colony primers;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers;

viii) repeating said steps v) through vii) such that said colonies are generated, each at a particular location on said solid surface.

18. The method of claim 16, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) denaturing said immobilized linearized fragments to generate immobilized single-stranded fragments;

v) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

vi) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vii) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

viii) annealing said immobilized single stranded fragments to immobilized colony primers; and

ix) repeating said steps v) through viii) such that said colonies are generated, each at a particular location on said solid surface.

19. The method of claim 16, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted linearized fragments can occur;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

20. The method of any one of claims 17-19, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

21. The method of claim 20, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

22. The method of claim 1, wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme, said recognition site being located and oriented such that said second restriction enzyme cut in said restriction fragments;

B2) digesting said first nucleic acid fragments with said second restriction enzyme;

B3) modifying the ends generated by said second restriction enzyme to permit ligation;

B4) linking said ends generated by said second restriction enzyme with a second engineered nucleic acid to produce second nucleic acid fragments, said second engineered nucleic acid comprising a predetermined nucleotide sequence; and

B5) sequencing at least a portion of each of said restriction fragments in said second nucleic acid fragments to determine said set of restriction sequence tags.

23. The method of claim 22, wherein said recognition site of said second restriction enzyme is located close to an end of said first engineered nucleic acid.

24. The method of claim 23, wherein said recognition site is located less than 25 nucleotides apart from said end of said first engineered nucleic acid.

25. The method of claim 24, wherein said recognition site is located zero to 5 nucleotides apart from said end of said first engineered nucleic acid.

26. The method of claim 22, wherein said second restriction enzyme is a type IIs endonuclease.

27. The method of claim 26, further comprising before said step B5) a step of fixing and amplifying nucleic acid fragments in said second nucleic acid fragments on a solid surface.

28. The method of claim 27, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said second nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said second nucleic acid fragments.

29. The method of claim 28, wherein said colonies are generated by a method comprising

i) providing a solid surface comprising a plurality of colony primers immobilized on said solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) denaturing said second nucleic acid fragments to generate single stranded fragments;

iii) annealing said single stranded fragments to said immobilized colony primers;

iv) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

v) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vi) annealing said immobilized single stranded fragments to immobilized colony primers;

vii) repeating said steps iv) through vi) such that said colonies are generated, each at a particular location on said solid surface.

30. The method of claim 28, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized nucleic acid fragments and immobilized colony primers;

iii) denaturing said immobilized nucleic acid fragments to generate immobilized single-stranded fragments;

iv) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers; and

viii) repeating said steps iv) through vii) such that said colonies are generated, each at a particular location on said solid surface.

31. The method of claim 28, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted second nucleic acid fragments can occur;

iii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized second nucleic acid fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

32. The method of any one of claims 29-31, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

33. The method of claim 32, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

34. The method of claim 1, wherein said first restriction enzyme is a rare cutter and wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence;

B2) digesting said first nucleic acid fragments with one or more second restriction enzymes to obtain second restriction fragments, wherein said second restriction enzymes are different from said first restriction enzyme and does not cut in said first engineered nucleic acid;

B3) linking the ends of said second restriction fragments with a second engineered nucleic acid to produce a set of second nucleic acid fragments, said second engineered nucleic acid comprising a predetermined nucleotide sequence; and

B4) sequencing at least a portion of each of said restriction fragments in said second nucleic acid fragments to determine said set of restriction sequence tags.

35. The method of claim 34, wherein said rare cutter recognizes a 6-base recognition sequence.

36. The method of claim 34, wherein said rare cutter recognizes an 8-base or a more than 8-base recognition sequence.

37. The method of claim 34, further comprising before said step B4) a step of fixing and amplifying nucleic acid fragments in said second nucleic acid fragments on a solid surface.

38. The method of claim 37, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said second nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said second nucleic acid fragments.

39. The method of claim 38, wherein said colonies are generated by a method comprising

i) providing a solid surface comprising a plurality of colony primers immobilized on said solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) denaturing said second nucleic acid fragments to generate single stranded fragments;

iii) annealing said single stranded fragments to said immobilized colony primers;

iv) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

v) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vi) annealing said immobilized single stranded fragments to immobilized colony primers;

vii) repeating said steps iv) through vi) such that said colonies are generated, each at a particular location on said solid surface.

40. The method of claim 38, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized second nucleic acid fragments and immobilized colony primers;

iii) denaturing said immobilized second nucleic acid fragments to generate immobilized single-stranded fragments;

iv) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers; and

iii) repeating said steps iv) through vii) such that said colonies are generated, each at a particular location on said solid surface.

41. The method of claim 38, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted second nucleic acid fragments can occur;

iii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized second nucleic acid fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

42. The method of any one of claims 39-41, wherein said sequencing is carried out by method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

43. The method of claim 42, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

44. The method of claim 1, wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence;

B2) digesting said first nucleic acid fragments with a second restriction enzyme to obtain second restriction fragments, wherein said second restriction enzyme is different from said first restriction enzyme and does not cut in said first engineered nucleic acid;

B3) linking the ends of said second restriction fragments with a second engineered nucleic acid to produce a set of second nucleic acid fragments, said second engineered nucleic acid comprising a predetermined nucleotide sequence; and

B4) sequencing at least a portion of each of said restriction fragments in said second nucleic acid fragments to determine said set of restriction sequence tags.

45. The method of claim 44, further comprising repeating said steps B2) through B4) for each of a plurality of different second restriction enzymes.

46. The method of claim 45, further comprising before said step B5) a step of fixing and amplifying nucleic acid fragments in said second nucleic acid fragments on a solid surface.

47. The method of claim 46, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said second nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said second nucleic acid fragments.

48. The method of claim 47, wherein said colonies are generated by a method comprising

i) providing a solid surface comprising a plurality of colony primers immobilized on aid solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) denaturing said second nucleic acid fragments to generate single stranded fragments;

iii) annealing said single stranded fragments to said immobilized colony primers;

iv) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

v) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vi) annealing said immobilized single stranded fragments to immobilized colony primers;

vii) repeating said steps iv) through vi) such that said colonies are generated, each at a particular location on said solid surface.

49. The method of claim 47, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments;

ii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized second nucleic acid fragments and immobilized colony primers;

iii) denaturing said immobilized second nucleic acid fragments to generate immobilized single-stranded fragments;

iv) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers; and

viii) repeating said steps iv) through vii) such that said colonies are generated, each at a particular location on said solid surface.

50. The method of claim 47, wherein said colonies are generated by a method comprising

i) mixing said second nucleic acid fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said second nucleic acid fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted second nucleic acid fragments can occur;

iii) grafting said second nucleic acid fragments and colony primers on a solid surface at the 5′

end to generate immobilized second nucleic acid fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

51. The method of any one of claims 48-50, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

52. The method of claim 51, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

53. The method of claim 1, wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first circular nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme and two recognition sites of a third restriction enzyme, said recognition site of said second restriction enzyme being located between said two recognition sites of said third restriction enzyme, said recognition sites of said third restriction enzyme being located and oriented such that said third restriction enzyme cut in said restriction fragments, wherein said second restriction enzyme and said third restriction enzyme are different from each other;

B2) digesting said first nucleic acid fragments with said second restriction enzyme to obtain a set of second nucleic acid fragments;

B3) linking the ends of said second restriction fragments to produce a set of second circular nucleic acid fragments; and

B4) sequencing at least a portion of each of said restriction fragments in said third circular nucleic acid fragments to determine said set of restriction sequence tags.

54. The method of claim 53, further comprising after said step 3) the steps of

B5) digesting said second circular nucleic acid fragments with said third restriction enzyme to produce a set of third nucleic acid fragments;

B6) modifying the ends generated by said third restriction enzyme to permit ligation; and

B7) linking the ends of said third nucleic acid fragments to produce a set of third circular nucleic acid fragments.

55. The method of claim 53, further comprising repeating said steps B1) through B4) for each of a plurality of different second restriction enzymes.

56. The method of claim 55, wherein each of said recognition site is located close to an end of said first engineered nucleic acid.

57. The method of claim 56, wherein each of said recognition site is located less than 25 nucleotides apart from an end of said first engineered nucleic acid.

58. The method of claim 57, wherein each of said recognition site is located zero to 5 nucleotides apart from an end of said first engineered nucleic acid.

59. The method of claim 53, wherein said second restriction enzyme is a type IIs endonuclease.

60. The method of claim 59, further comprising before said step B4) a step of fixing and amplifying nucleic acid fragments in said second circular nucleic acid fragments on a solid surface.

61. The method of claim 60, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said second circular nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said second circular nucleic acid fragments.

62. The method of claim 61, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) providing a solid surface comprising a plurality of colony primers immobilized on said solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linearized fragments;

iii) denaturing said linearized fragments to generate single stranded fragments;

iv) annealing said single stranded fragments to said immobilized colony primers;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers;

viii) repeating said steps v) through vii) such that said colonies are generated, each at a particular location on said solid surface.

63. The method of claim 61, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) denaturing said immobilized linearized fragments to generate immobilized single-stranded fragments;

v) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

vi) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vii) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

viii) annealing said immobilized single stranded fragments to immobilized colony primers; and

ix) repeating said steps v) through viii) such that said colonies are generated, each at a particular location on said solid surface.

64. The method of claim 61, wherein said colonies are generated by a method comprising

i) linearizing said second circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted linearized fragments can occur;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

65. The method of any one of claims 62-64, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

66. The method of claim 65, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

67. The method of claim 1, wherein said step of determining said set of restriction sequence tags is carried out by a method comprising

B1) linking said restriction fragments in said set of restriction fragments with a first engineered nucleic acid to obtain a set of first nucleic acid fragments, said first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme different from said first restriction enzyme;

B2) digesting said first nucleic acid fragments with said second restriction enzyme to obtain a set of second nucleic acid fragments;

B3) linking the ends of said second restriction fragments to produce a set of first circular nucleic acid fragments;

B4) sequencing at least a portion of each of said fourth nucleic acid fragments, thereby determining said set of restriction sequence tags.

68. The method of claim 67, further comprising after said step 3) the steps of

B5) digesting said first circular nucleic acid fragments with a third restriction enzyme to produce a set of third nucleic acid fragments, wherein said third restriction enzyme is different from said first and second restriction enzyms;

B6) modifying the ends generated by said third restriction enzyme to permit ligation; and

B7) linking the ends of said third nucleic acid fragments to produce a set of second circular nucleic acid fragments.

69. The method of claim 67, further comprising repeating said steps B1) through B4) for each of a plurality of different second restriction enzymes.

70. The method of claim 69, further comprising before said step B4) a step of fixing and amplifying nucleic acid fragments in said first circular nucleic acid fragments on a solid surface.

71. The method of claim 70, wherein said step of fixing and amplifying is carried out by generating colonies of said nucleic acid fragments in said first circular nucleic acid fragments on said solid surface, wherein each of said colonies comprises a plurality of immobilized single stranded DNA molecules of one of said nucleic acid fragments in said first circular nucleic acid fragments.

72. The method of claim 71, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) providing a solid surface comprising a plurality of colony primers immobilized on said solid surface at 5′

end, wherein each said colony primer comprises a sequence that is hybridizable to a sequence at the 3′

end of said linearized fragments;

iii) denaturing said linearized fragments to generate single stranded fragments;

iv) annealing said single stranded fragments to said immobilized colony primers;

v) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vi) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

vii) annealing said immobilized single stranded fragments to immobilized colony primers;

viii) repeating said steps v) through vii) such that said colonies are generated, each at a particular location on said solid surface.

73. The method of claim 71, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) denaturing said immobilized linearized fragments to generate immobilized single-stranded fragments;

v) annealing said immobilized single stranded fragments to immobilized colony primers to obtain annealed single-stranded fragments;

vi) carrying out primer extension reaction using said annealed single stranded fragments as templates to generate immobilized double stranded nucleic acid fragments;

vii) denaturing said immobilized double stranded nucleic acid fragments to generate immobilized single stranded fragments;

viii) annealing said immobilized single stranded fragments to immobilized colony primers; and

ix) repeating said steps v) through viii) such that said colonies are generated, each at a particular location on said solid surface.

74. The method of claim 71, wherein said colonies are generated by a method comprising

i) linearizing said first circular nucleic acid fragments to generate linearized fragments;

ii) mixing said linearized fragments with colony primers, wherein each said colony primers comprises a sequence that is hybridizable to a sequence at the 3′

end of said linerarized fragments, and wherein the concentration of said colony primers is adjusted such that amplification of grafted linearized fragments can occur;

iii) grafting said linearized fragments and colony primers on a solid surface at the 5′

end to generate immobilized linearized fragments and immobilized colony primers;

iv) applying an amplification solution containing a polymerase and nucleotides to said solid surface such that said colonies are generated isothermally, each at a particularly location on said solid surface.

75. The method of any one of claims 72-74, wherein said sequencing is carried out by a method comprising

i) hybridizing sequencing primers to said colonies;

ii) carrying out primer extension with one labeled nucleotide;

iii) detecting the amount of the labeled nucleotide which is incorporated into extended primers for each said location; and

iv) repeating steps ii) and iii) to determine a portion of sequence of each of said colony.

76. The method of claim 75, wherein said labeled nucleotide is a fluorecently-labeled nucleotide, and wherein said detecting involves detecting the fluorescence intensity of said labeled nucleotide.

77. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, further comprising in said step A) digesting said set of restriction fragments with a plurality of different first restriction enzymes.

78. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said group consists of restriction sequence tags that are at least 60% homologous.

79. The method of claim 78, wherein each said group consists of restriction sequence tags that are at least 70% homologous.

80. The method of claim 79, wherein each said group consists of restriction sequence tags that are at least 80% homologous.

81. The method of claim 80, wherein each said group consists of restriction sequence tags that are at least 90% homologous.

82. The method of claim 81, wherein each said group consists of restriction sequence tags that are at least 99% homologous.

83. A method for determining genome-wide sequence variations among a plurality of different phenotypes, comprising,

A) determining for each of a population of organisms a set of restriction sequence tags by the method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, said population of organisms comprising for each of said plurality of different phenotypes one or more organisms;

B) comparing said sets of restriction sequence tags among organisms of different phenotypes so as to determine one or more sequence variations that associate with different phenotypes.

84. The method of claim 83, further comprising after said step B) a step of mapping said one or more restriction sequence tags to the genomic sequence of said organism so as to identify genomic locations of said one or more restriction sequence tags.

85. The method of any one of claims 45, 55, and 69, wherein said plurality of different second restriction enzymes comprises at least 3 different restriction enzymes.

86. A method for determining genome-wide sequence variations among a plurality of different phenotypes, comprising

A) determining for each of a population of organisms a set of restriction sequence tags by the method of claim 85, said population of organisms comprising for each of said plurality of different phenotypes one or more organisms;

B) comparing said sets of restriction sequence tags among organisms of different phenotypes so as to determine one or more sequence variations that associate with different phenotypes.

87. The method of claim 86, further comprising after said step B) a step of mapping said one or more restriction sequence tags to the genomic sequence of said organism so as to identify genomic locations of said one or more restriction sequence tags.

88. The method of any one of claims 45, 55, and 69, wherein said plurality of different second restriction enzymes comprises at least 10 different restriction enzymes.

89. A method for determining genome-wide sequence variations among a plurality of different phenotypes, comprising

A) determining for each of a population of organisms a set of restriction sequence tags by the method of claim 88, said population of organisms comprising for each of said plurality of different phenotypes one or more organisms;

B) comparing said sets of restriction sequence tags among organisms of different phenotypes so as to determine one or more sequence variations that associate with different phenotypes.

90. The method of claim 89, further comprising after said step B) a step of mapping said one or more restriction sequence tags to the genomic sequence of said organism so as to identify genomic locations of said one or more restriction sequence tags.

91. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein said one or more individual organisms are humans.

92. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 10 different restriction fragments.

93. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 100 different restriction fragments.

94. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 1000 different restriction fragments.

95. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 10,000 different restriction fragments.

96. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 100,000 different restriction fragments.

97. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 10⁶ different restriction fragments.

98. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 10⁷ different restriction fragments.

99. The method of any one of claims 1, 2, 14, 22, 34, 44, 53, and 67, wherein each said set of restriction fragments comprises at least 10⁸ different restriction fragments.

100. The method of claim 1, wherein said step I) is carried out for one individual.

101. The method of claim 1, wherein said step II) of grouping restriction sequence tags further comprises comparing said restriction sequence tags to reference sequences.

102. The method of claim 101, wherein said reference sequences comprise the genomic sequence of the organism.