Fixed address analysis of sequence tags

• US 6,261,782 B1
• Filed: 04/06/2000
• Issued: 07/17/2001
• Est. Priority Date: 04/06/1999
• Status: Expired due to Fees

• Current Assignee: Apothecary Products Incorporated, Agilix Corp. (Revvity, Inc.), Yale University
• Original Assignee: Yale University
• Inventors: Kaufman, Joseph C., Lizardi, Paul M., Feng, Li, Latimer, Darin R., Roth, Matthew E., Guerra, Cesar E., Weber, Shane C.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/544,713
• Time in Patent Office: 467 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.2, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Fixed Address Analysis of Sequence Tags (FAAST), involves generation of a set of nucleic acid fragments having a variety of sticky end sequences; indexing of the fragments into sets based on the sequence of sticky ends; associating a detector sequence with the fragments; sequence-based capture of the indexed fragments on a detector array; and detection of the fragment labels. Generation of the multiple sticky end sequences is accomplished by incubating the nucleic acid sample with one or more nucleic acid cleaving reagents. The indexed fragments are captured by hybridization and coupling, preferably by ligation, to a probe. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner. One form of the method allows determination of associations, in a nucleic acid molecule, of different combinations of known or potential sequences. Another form of the method assesses modification of sequences in nucleic acid molecules by basing cleavage of the molecules on the presence or absence of modification.

Claims

1. A method of identifying nucleic acid fragments in a nucleic acid sample, the method comprising

(a) incubating a nucleic acid sample with one or more nucleic acid cleaving reagents that collectively generate sticky ends having a plurality of different sequences to produce nucleic acid fragments with sticky ends,

(b) mixing a plurality of adaptor-indexers with the nucleic acid sample and covalently coupling the adaptor-indexers to the nucleic acid fragments, wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexers is compatible with a sticky end generated by the nucleic acid cleaving reagents,

(c) hybridizing a plurality of ligator-detectors with the nucleic acid sample, wherein each ligator-detector comprises sequence matching or complementary to all or part of sequence including, and adjacent to, the sticky end of at least one of the adaptor-indexers,

(d) hybridizing a plurality of detector probes with the nucleic acid sample and covalently coupling the ligator-detectors to the detector probes, wherein each detector probe has a different sequence, and

(e) detecting, directly or indirectly, coupling of ligator-detectors to the detector probes.

2. The method of claim 1 wherein each adaptor-indexer comprises a sticky end portion and a detection portion, wherein the detector portion of each adaptor-indexer is separately detectable,

wherein the method further comprises detecting, directly or indirectly, detector portions of the adaptor-indexers.

3. The method of claim 2 wherein each ligator-detector comprises a detector portion, wherein the detector portion of each ligator-detector corresponds to the detector portion of one of the adaptor-indexers, wherein detection of the detection portion of a ligator-detector is an indirect detection of the detector portion of the corresponding adaptor-indexer.

4. The method of claim 2 wherein detection of the detection portion of an adaptor-indexer is an indirect detection of the detector portion of the corresponding ligator-detector.

5. The method of claim 2 wherein the detection portion of at least one adaptor-indexer is a label.

6. The method of claim 1 wherein the adaptor-indexers are covalently coupled to the nucleic acid fragments by ligation, and wherein the ligator-detectors are covalently coupled to the detector probes by ligation.

7. The method of claim 1 further comprising, prior to step (b),

dividing the sample into a plurality of index samples,

wherein a different adaptor-indexer is mixed with each index sample, wherein a different ligator-detector is hybridized with each index sample, wherein the ligator-detector in each index sample comprises sequence matching or complementary to all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer in that index sample.

8. The method of claim 6 wherein the ligator-detector comprises sequence matching or complementary to all or part of the sticky end of the adaptor-indexer and to all or part of the adjacent sequence of the adaptor-indexer used in the index sample.

9. The method of claim 8 further comprising, following step (b),

separating the strands of the nucleic acid fragments and proceeding with step (c) using only one of the strands.

10. The method of claim 9 wherein the strands are separated using a capture tag associated with one of the strands.

11. The method of claim 10 wherein the capture tag is associated with the adaptor-indexers.

12. The method of claim 9 wherein the concentration of the various nucleic acid fragments in the index samples are normalized.

13. The method of claim 12 wherein the strands of the nucleic acid fragments are separated and the concentration of the nucleic acid fragments is normalized by immobilizing one strand of the nucleic acid fragments, denaturing the nucleic acid fragments, renaturing the nucleic acid fragments for a time greater than the c₀t_½

for abundant nucleic acid fragments and less than the c₀t_½

for rare nucleic acid fragments, and collecting the un-renatured nucleic acid fragments.

14. The method of claim 8 wherein ligation of the ligator-detector is detected by rolling circle replication of an amplification target circle wherein replication is primed by the ligator-detector.

15. The method of claim 8 wherein the nucleic acid cleaving reagents generate sticky ends having N different sequences, and wherein the sample is divided into N index samples.

16. The method of claim 8 wherein the detector probes are all of the same length.

17. The method of claim 16 wherein the detector probes are six, seven, or eight nucleotides long.

18. The method of claim 8 wherein the detector probes all have similar hybrid stability.

19. The method of claim 8 wherein the nucleic acid cleaving reagents are restriction enzymes.

20. The method of claim 19 wherein the nucleic acid sample is digested with one restriction enzyme, wherein the restriction enzyme generates sticky ends having a plurality of different sequences.

21. The method of claim 20 wherein the restriction enzyme is a type II-S restriction enzyme that cleaves at a site different from its recognition sequence.

22. The method of claim 6 wherein each ligator-detector comprises sequence matching all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer.

23. The method of claim 6 wherein each ligator-detector comprises sequence complementary to all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer.

24. The method of claim 6 wherein at least one ligator-detector comprises sequence matching all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer and at least one ligator-detector comprises sequence complementary to all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer.

25. The method of claim 6 wherein the concentration of the various nucleic acid fragments in the nucleic acid sample are normalized.

26. The method of claim 25 wherein the concentration of the nucleic acid fragments is normalized by immobilizing one strand of the nucleic acid fragments, denaturing the nucleic acid fragments, renaturing the nucleic acid fragments for a time greater than the c₀t_½

for abundant nucleic acid fragments and less than the c₀t_½

for rare nucleic acid fragments, and collecting the un-renatured nucleic acid fragments.

27. The method of claim 8 wherein each adaptor-indexer, ligator-detector, or detector probe is immobilized on a substrate.

28. The method of claim 27 wherein the detector probes are immobilized on a substrate.

29. The method of claim 27 wherein all of the adaptor-indexers, ligator-detectors, or detector probes are immobilized on the same substrate.

30. The method of claim 27 wherein all of the adaptor-indexers, ligator-detectors, or detector probes are immobilized on a different substrate.

31. The method of claim 30 wherein the substrates are beads.

32. The method of claim 27 wherein the adaptor-indexers, ligator-detectors, or detector probes are immobilized on a plurality of different substrates such that at least one adaptor-indexer, ligator-detector, or detector probe is immobilized on one substrate and at least one other adaptor-indexer, ligator-detector, or detector probe, respectively, is immobilized on a different substrate.

33. The method of claim 27 wherein the adaptor-indexers, ligator-detectors, or detector probes are in an array.

34. The method of claim 33 wherein the detector probes are in an array.

35. The method of claim 8 wherein each adaptor-indexer, ligator-detector, or detector probe is associated with a capture tag, sorting tag, or both.

36. The method of claim 35 wherein the ligator-detectors are associated with capture tags or sorting tags.

37. The method of claim 35 wherein the adaptor-indexers, ligator-detectors, or detector probes are captured via the capture tags.

38. The method of claim 35 wherein the adaptor-indexers, ligator-detectors, or detector probes are sorted via the sorting tags.

39. The method of claim 35 wherein the ligator-detectors are associated with sorting tags, wherein the adaptor-indexers are associated with capture tags.

40. The method of claim 35 wherein the ligator-detectors are associated with capture tags, wherein the adaptor-indexers are associated with sorting tags.

41. The method of claim 35 wherein the detector probes are associated with sorting tags, wherein the adaptor-indexers are associated with capture tags.

42. The method of claim 35 wherein the adaptor-indexers, ligator-detectors, or detector probes are associated with a plurality of different capture tags or a plurality of different sorting tags.

43. The method of claim 8 further comprising determining the sequence of a portion of at least one of the nucleic acid fragments in the nucleic acid sample.

44. The method of claim 43 wherein the nucleic acid cleaving reagents are restriction enzymes,

wherein the nucleic acid sample is digested with one restriction enzyme, wherein the restriction enzyme generates sticky ends having a plurality of different sequences,

wherein the restriction enzyme is a type II-S restriction enzyme that cleaves at a site different from its recognition sequence, and

wherein the portion of the nucleic acid fragments corresponds to the sticky end sequence, the sequence adjacent to the sticky end sequence to which the detector probe hybridized, and the recognition sequence of the restriction enzyme.

45. The method of claim 44 wherein the portion includes a gap of known length but unknown sequence between the sequence adjacent to the sticky end and the recognition sequence of the restriction enzyme.

46. The method of claim 45 wherein the portion has the structure

A-B-C-D

wherein A is the recognition sequence of the restriction enzyme, B is the gap of unknown sequence, C is the sequence to which the detector probe hybridized, and D is the sticky end sequence.

47. The method of claim 44 further comprising detecting or amplifying a nucleic acid corresponding to a nucleic acid fragment in the nucleic acid sample using a probe or primer based on the determined sequence of the portion of the nucleic acid fragment.

48. The method of claim 8 wherein the detector probes are in an array, wherein each detector probe is immobilized at a different location in the array, and wherein detecting ligation of ligator-detectors to detector probes is accomplished by detecting the presence of ligator-detector at different locations in the arrays.

49. The method of claim 48 wherein the location, amount, or location and amount of ligator-detectors in the arrays constitutes a pattern of ligator-detectors in the arrays,

the method further comprising comparing the pattern of ligator-detectors in the arrays with the pattern of ligator-detectors in arrays determined in a separate procedure using a second nucleic acid sample.

50. The method of claim 49 further comprising

comparing the pattern of ligator-detectors in the arrays with the pattern of ligator-detectors in arrays determined in a plurality of separate procedures using a plurality of different nucleic acid samples.

51. The method of claim 8 further comprising, following step (b),

incubating the index samples with one or more second nucleic acid cleaving reagents, and

mixing a second adaptor with each index sample and ligating the second adaptors to the nucleic acid fragments, wherein each second adaptor has an end compatible with the end generated by one of the second nucleic acid cleaving reagents.

52. The method of claim 51 further comprising, prior to step (c),

dividing each index sample into one or more restricted index samples and a non-restricted index sample,

incubating each restricted index sample with a different third nucleic acid cleaving reagent,

wherein steps (c), (d), and (e) are performed with both the restricted and non-restricted index samples.

53. The method of claim 51 further comprising, prior to incubation with the second nucleic acid cleaving reagents,

dividing each index sample into a set of two or more of secondary index samples,

wherein each secondary index sample in each set of secondary index samples is incubated with a different set of one or more second nucleic acid cleaving reagents.

54. The method of claim 53 wherein steps (c), (d), and (e) are performed with each secondary index sample.

55. The method of claim 53 further comprising, prior to step (c),

dividing each secondary index sample into one or more restricted index samples and a non-restricted index sample,

incubating each restricted index sample with a different third nucleic acid cleaving reagent,

wherein steps (c), (d), and (e) are performed with both the restricted and non-restricted index samples.

56. The method of claim 55 wherein the first, second, and third nucleic acid cleaving reagents are restriction enzymes.

57. The method of claim 51 further comprising, following incubation with the second nucleic acid cleaving reagents,

dividing each index sample into a set of two or more of secondary index samples.

58. The method of claim 57 wherein steps (c), (d), and (e) are performed with each secondary index sample.

59. The method of claim 51 wherein the first and second nucleic acid cleaving reagents are restriction enzymes.

60. The method of claim 51 further comprising, following ligation of second adaptors to the nucleic acid fragments,

amplifying the nucleic acid fragments in the index samples to which adaptor-indexers and second adaptors have been ligated.

61. The method of claim 60 wherein the nucleic acid fragments are amplified by PCR.

62. The method of claim 61 wherein the primers used for PCR are complementary to sequence in the adaptor-indexers and second adaptors.

63. The method of claim 8 further comprising, prior to step (a),

dividing the nucleic acid sample into a plurality of index samples,

wherein more than one nucleic acid cleaving reagent is used in step (a), wherein each index sample is incubated with a different one of the nucleic acid cleaving reagents,

wherein a different adaptor-indexer is mixed with each index sample, wherein a different ligator-detector is hybridized with each index sample, wherein the ligator-detector in each index sample comprises sequence matching or complementary to all or part of sequence including, and adjacent to, the sticky end of the adaptor-indexer in that index sample.

64. The method of claim 8 further comprising, following step (b),

amplifying the nucleic acid fragments in the index samples to which adaptor-indexers have been ligated.

65. The method of claim 64 further comprising, following amplification,

separating the strands of the amplified nucleic acid fragments and proceeding with step (c) using only one of the strands.

66. The method of claim 65 wherein the strands are separated using a capture tag incorporated into one of the strands.

67. The method of claim 66 wherein the capture tag is associated with a primer used for amplification of the nucleic acid fragments.

68. The method of claim 64 wherein the nucleic acid fragments are amplified by PCR.

69. The method of claim 8 further comprising, following ligation in steps (b) and (d),

incubating the index samples with a reagent that destroys or alters base mismatches.

70. The method of claim 69 wherein the base mismatch reagent is (1) T4 endonuclease VII, (2) MutH, MutL, and MutS together, (3) T7 endonuclease I, (4) MutY, (5) glycosylase, or (6) topoisomerase I.

71. The method of claim 8 further comprising, prior to ligation in steps (b) and (d),

mixing the index samples with a reagent that binds to base mismatches.

72. The method of claim 71 wherein the base mismatch reagent is (1) Mec1, (2) MutS, (3) MSH2-MSH6, (4) MLH1-PMS1, or (5) MSH2-MSH3.

73. The method of claim 8 wherein each adaptor-indexer, ligator-detector, or detector probe contains a label, wherein ligation of the ligator-detectors to the detector probes is detected via the label.

74. The method of claim 73 wherein each ligator-detector contains a label,

wherein detecting ligation of the ligator-detectors to the detector probes is accomplished by

separating ligated ligator-detectors from unligated ligator-detectors, and

detecting the labels of the ligated ligator-detectors.

75. The method of claim 74 wherein each different ligator-detector contains a different label, wherein each detector probe is associated with a capture tag or a sorting tag, wherein separating ligated ligator-detectors from unligated ligator-detectors is accomplished by separating the detector probes from the unligated ligator-detectors using the capture tags or sorting tags, wherein the ligated ligator-detectors separate with the detector probes.

76. The method of claim 75 wherein the sorting tag is a fluorescent label, and wherein separating the detector probes from the unligated ligator-detectors is accomplished using a fluorescent label sorter.

77. The method of claim 73 wherein the labels are fluorescent, phosphorescent, or chemiluminescent labels.

78. The method of claim 77 wherein at least two of the labels are distinguished temporally via different fluorescent, phosphorescent, or chemiluminescent emission lifetimes.

79. The method of claim 73 wherein the labels are detectable by nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination.

80. The method of claim 79 wherein the label is detected using nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination.

81. The method of claim 73 wherein the labels are beads comprising a label.

82. The method of claim 81 wherein the label is a molecular barcode.

83. The method of claim 73 wherein the labels are mass labels.

84. The method of claim 8 further comprising

performing steps (a) through (e) on a control nucleic acid sample,

identifying differences between the nucleic acid sample and the control nucleic acid sample in the pattern of ligator-detectors ligated to different detector probes.

85. The method of claim 84 wherein the ligator-detectors used with the control nucleic acid sample contain a different label from the label of the ligator-detectors used with the nucleic acid sample,

wherein the control index samples are mixed with corresponding index samples prior to step (d).

86. The method of claim 8 further comprising performing steps (a) through (e) on a plurality of nucleic acid samples.

87. The method of claim 86 further comprising

performing steps (a) through (e) on a control nucleic acid sample,

identifying differences between the nucleic acid samples and the control nucleic acid sample in the pattern of ligator-detectors ligated to different detector probes.

88. The method of claim 86 further comprising

identifying differences between the nucleic acid samples in the pattern of ligator-detectors ligated to different detector probes.

89. The method of claim 8 wherein the pattern of the presence, amount, presence and amount, or absence of ligator-detectors ligated to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample.

90. The method of claim 89 further comprising preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog.

91. The method of claim 90 further comprising identifying or preparing nucleic acid fragments corresponding the nucleic acid fragments present at a threshold amount in the first nucleic acid sample but not present at the threshold amount in the second nucleic acid sample.

92. The method of claim 90 wherein the second nucleic acid sample is a sample from the same type of organism as the first nucleic acid sample.

93. The method of claim 90 wherein the second nucleic acid sample is a sample from the same type of tissue as the first nucleic acid sample.

94. The method of claim 90 wherein the second nucleic acid sample is a sample from the same organism as the first nucleic acid sample.

95. The method of claim 94 wherein the second nucleic acid sample is obtained at a different time than the first nucleic acid sample.

96. The method of claim 90 wherein the second nucleic acid sample is a sample from a different organism than the first nucleic acid sample.

97. The method of claim 90 wherein the second nucleic acid sample is a sample from a different type of tissue than the first nucleic acid sample.

98. The method of claim 90 wherein the second nucleic acid sample is a sample from a different species of organism than the first nucleic acid sample.

99. The method of claim 90 wherein the second nucleic acid sample is a sample from a different strain of organism than the first nucleic acid sample.

100. The method of claim 90 wherein the second nucleic acid sample is a sample from a different cellular compartment than the first nucleic acid sample.

101. The method of claim 90 further comprising identifying or preparing nucleic acid fragments corresponding the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample.

102. The method of claim 101 further comprising using the nucleic acid fragments as probes.

103. The method of claim 102 wherein using the nucleic acid fragments as probes is accomplished by repeating steps (a) through (e) with a different nucleic acid sample, wherein the nucleic acid fragments are used as detector probes in steps (d) and (e).

104. The method of claim 8 wherein the pattern of the amount of ligator-detectors ligated to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample,

wherein the pattern is compared to a predicted pattern based on probabilities of base mismatches of sequences hybridized to the detector probes.

105. The method of claim 8 wherein each adaptor-indexer comprises a nucleic acid comprising a single-stranded portion and a double-stranded portion, wherein the single-stranded portion is the sticky end, and wherein the double-stranded portion has the same sequence in each adaptor-indexer.

106. The method of claim 8 wherein detecting ligation of the ligator-detectors to the detector probes is accomplished by detecting the adaptor-indexers, the ligated ligator-detectors, mass labels associated with the adaptor-indexers, mass labels associated with the ligated ligator-detectors, or a combination, by mass spectroscopy.

107. The method of claim 106 wherein the adaptor-indexers, ligated ligator-detectors, mass labels associated with the adaptor-indexers, and mass labels associated with the ligated ligator-detectors are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy.

108. The method of claim 106 wherein the composition of the adaptor-indexers, ligated ligator-detectors, mass labels associated with the adaptor-indexers, and mass labels associated with the ligated ligator-detectors are determined by analyzing the fragmentation pattern.

109. The method of claim 106 wherein unligated ligator-detectors are washed away from the detector probes prior to detection of the ligated adaptor-indexers.

110. The method of claim 106 further comprising, following step (b), incubating the index samples with one or more second nucleic acid cleaving reagents.

111. The method of claim 106 wherein the adaptor-indexers, the detector probes, or both, contain a photocleavable nucleotide, wherein the method further comprises, following ligation of the ligator-detectors to the detector probes,

photocleavage of the photocleavable nucleotides, and

detection of one or both strands of the ligated ligator-detector by mass spectroscopy.

112. The method of claim 106 further comprising, following ligation of the ligator-detectors to the detector probes,

incubation of the ligated ligator-detectors and detector probes with one or more nucleic acid cleaving reagents, and

detection of one or both strands of the ligated ligator-detector by mass spectroscopy.

113. The method of claim 8 further comprising, following step (b),

incubating the index samples with one or more second nucleic acid cleaving reagents, and

mixing a second adaptor with each index sample and ligating the second adaptors to the nucleic acid fragments, wherein each second adaptor has an end compatible with the end generated by one of the second nucleic acid cleaving reagents,

wherein at least one of the second nucleic acid cleaving reagents is sensitive to modification of its recognition site.

114. The method of claim 113 wherein the first and second nucleic acid cleaving reagents are restriction enzymes.

115. The method of claim 114 wherein the modification to the recognition site is methylation, alkylation, dimerization, derivatization, depurination, or ADP-ribosylation.

116. The method of claim 114 wherein the modification is present in the nucleic acid fragments when isolated or is introduced to the nucleic acid fragments after isolation.

117. The method of claim 114 further comprising, prior to step (c),

dividing each index sample into one or more restricted index samples and a non-restricted index sample,

digesting each restricted index sample with a different third restriction enzyme,

wherein steps (c), (d), and (e) are performed with both the restricted and non-restricted index samples.

118. The method of claim 114 further comprising, following ligation of the second adaptors to the nucleic acid fragments,

amplifying the nucleic acid fragments in the index samples to which adaptor-indexers and second adaptors have been ligated.

119. The method of claim 118 further comprising determining the sequence of a portion of at least one of the nucleic acid fragments in the nucleic acid sample.

120. The method of claim 114 further comprising, following ligation of the second adaptors to the nucleic acid fragments,

separating nucleic acid fragments ligated to second adaptors from nucleic acid fragments not ligated to second adaptors, wherein only nucleic acid fragments ligated to second adaptors are used in step (c).

121. The method of claim 114 wherein at least one of the second restriction enzymes (1) is insensitive to modification of its recognition site and (2) has the same recognition site as the second restriction enzyme that is sensitive to modification of its recognition site,

the method further comprising, prior to digestion with the second restriction enzymes,

dividing each index sample into a set of two or more of secondary index samples,

wherein each secondary index sample in each set of secondary index samples is digested with a different second restriction enzyme,

wherein steps (c) through (e) are performed with each of the secondary index samples.

122. The method of claim 121 further comprising,

comparing the pattern of the presence or absence of ligator-detectors ligated to different detector probes involving the second restriction enzyme that is sensitive to modification of its recognition site with the pattern of the presence or absence of ligator-detectors ligated to different detector probes involving the second restriction enzyme that is insensitive to modification of its recognition site and that has the same recognition site as the second restriction enzyme that is sensitive to modification of its recognition site,

wherein differences in the patterns indicate modification of nucleic acids in the nucleic acid sample.

123. The method of claim 114 wherein the pattern of the presence, amount, presence and amount, or absence of ligator-detectors ligated to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample.

124. The method of claim 123 further comprising preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog, wherein differences in the first and second catalogs indicate differences in modification of the first and second nucleic acid samples.

125. The method of claim 123 wherein the second nucleic acid sample is a sample from the same type of cells as the first nucleic acid sample except that the cells from which the first nucleic acid sample is derived are modification-deficient relative to the cells from which the second nucleic acid sample is derived.

126. The method of claim 123 wherein the second nucleic acid sample is a sample from a different type of cells than the first nucleic acid sample, and wherein the cells from which the first nucleic acid sample is derived are modification-deficient relative to the cells from which the second nucleic acid sample is derived.

127. The method of claim 6 wherein the nucleic acid cleaving reagents are restriction enzymes, wherein the restriction enzymes are type II-S restriction enzymes that cleave at sites different from their recognition sequences,

the method further comprising, prior to step (a),

introducing a recognition site for the restriction enzyme adjacent to a region of interest in one or more nucleic acid molecules in the nucleic acid sample,

wherein the ligator-detector comprises sequence matching or complementary to all or part of the sequence of, and adjacent to, the recognition site for the restriction enzyme.

128. The method of claim 127 wherein the recognition site is introduced by amplifying the nucleic acid molecules using a primer containing the recognition sequence.

129. The method of claim 128 wherein the nucleic acid molecules are amplified using PCR.

130. The method of claim 127 wherein the detector probes include sequence matching or complementary to known or potential sequence in the region of interest.

131. The method of claim 130 wherein there are a plurality of known or potential sequences in the region of interest, wherein the set of detector probes includes probes that include sequence matching or complementary to each known or potential sequence.

132. The method of claim 131 the nucleic acid molecules in the nucleic acid sample are immunoglobulin or T cell receptor nucleic acid molecules, wherein the nucleic acid molecules have a variable region, an N region, and a joining region,

wherein the recognition site for the restriction enzyme is introduced into the variable region of the nucleic acid molecules adjacent to the N region of the nucleic acid molecules,

wherein the ligator-detector comprises sequence matching or complementary to all or part of the sequence of the recognition site for the restriction enzyme and all or a part of the sequence of the variable region sequence adjacent to the recognition site for the restriction enzyme, up to, but not including, the N region,

wherein the plurality of known or potential sequences are sequences matching or complementary to all or a part of the sequence of the joining region of the nucleic acid molecules adjacent to the N region of the nucleic acid molecules.

133. A kit comprising

a set of adaptor-indexers wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexes is compatible with a sticky end generated by one or more nucleic acid cleaving reagents that collectively generate sticky ends having a plurality of different sequences,

a set of ligator-detectors wherein each ligator-detector comprises sequence matching or complementary to all or part of the sticky end of a different adaptor-indexer and to all or part of the adjacent sequence of the adaptor-indexer, and

a plurality of detector probes, wherein each detector probe has a different sequence.

134. The kit of claim 133 wherein each sticky end of the adaptor-indexes is compatible with a sticky end generated by a restriction enzyme.

135. The kit of claim 133 wherein the kit includes at least one ligator-detector corresponding to each adaptor-indexer.

136. The kit of claim 135 wherein at least one adaptor-indexer, at least one ligator-detector, at least one detector probe, or a combination, contains a label.

137. The kit of claim 136 wherein the labels are fluorescent, phosphorescent, or chemiluminescent labels.

138. The kit of claim 137 wherein at least two of the labels are distinguished temporally via different fluorescent, phosphorescent, or chemiluminescent emission lifetimes.

139. The kit of claim 136 wherein the labels are detectable by nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination.

140. The kit of claim 136 wherein the labels are beads comprising a label.

141. The method of claim 140 wherein the label is a molecular barcode.

142. The kit of claim 136 wherein the labels are mass labels.

143. The kit of claim 133 wherein the nucleic acid cleaving reagent generates sticky ends having N different sequences, and wherein the kit includes N different adaptor-indexers.

144. The kit of claim 133 wherein the detector probes are six, seven, or eight nucleotides long.

145. The kit of claim 133 wherein each ligator-detector contains a label.

146. The kit of claim 145 wherein the label is a fluorescent label.

147. The kit of claim 145 wherein the kit includes at least two ligator-detectors corresponding to each adaptor-indexer, wherein each ligator-detector corresponding to a given adaptor-indexer contains a different label.

148. The kit of claim 133 wherein the detector probes are all of the same length.

149. The kit of claim 133 wherein the detector probes all have similar hybrid stability.

150. The kit of claim 133 wherein each adaptor-indexer, ligator-detector, or detector probe is immobilized on a substrate.

151. The kit of claim 150 wherein the detector probes are immobilized on a substrate.

152. The kit of claim 133 wherein each adaptor-indexer, ligator-detector, or detector probe is associated with a capture tag, sorting tag, or both.

153. The kit of claim 152 wherein the ligator-detectors are associated with capture tags or sorting tags.

154. The kit of claim 133 wherein the detector probes are nucleic acid fragments prepared by

(a) incubating a nucleic acid sample with one or more nucleic acid cleaving reagents that collectively generate sticky ends having a plurality of different sequences to produce nucleic acid fragments with sticky ends,

(b) mixing a plurality of adaptor-indexers with the nucleic acid sample and covalently coupling the adaptor-indexers to the nucleic acid fragments, wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexers is compatible with a sticky end generated by the nucleic acid cleaving reagents,

(c) hybridizing a plurality of ligator-detectors with the nucleic acid sample, wherein each ligator-detector comprises sequence matching or complementary to all or part of sequence including, and adjacent to, the sticky end of at least one of the adaptor-indexers,

(d) hybridizing the nucleic acid sample with a plurality of detector probes and covalently coupling the ligator-detectors to the detector probes, wherein each detector probe has a different sequence, and

(e) detecting, directly or indirectly, coupling of ligator-detectors to the detector probes, wherein the pattern of ligator-detectors coupled to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample,

(f) preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog, and

(g) preparing nucleic acid fragments corresponding the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample.