High throughput genome sequencing on DNA arrays
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Abstract
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.
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Citations
80 Claims
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1-42. -42. (canceled)
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43. A method of inserting multiple adaptors in a target nucleic acid comprising:
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a) ligating a first adaptor to one terminus of said target nucleic acid to form a first linear construct, wherein said adaptor comprises a recognition site for a first restriction enzyme; b) circularizing first linear construct to create a first circular polynucleotide; c) cleaving said first circular polynucleotide with said first restriction enzyme to form a second linear construct, wherein said first restriction enzyme is able to bind to said recognition site within said first adaptor; d) ligating a second adaptor to said second linear construct to form a third linear construct, wherein said second adaptor comprises a recognition site for a second restriction enzyme; e) circularizing said third linear construct to create a second circular polynucleotide. - View Dependent Claims (44, 45, 46, 47)
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48. A method of inserting multiple adaptors in a target nucleic acid comprising:
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a) ligating a first adaptor to one terminus of said target nucleic acid to form a first linear construct, wherein said adaptor comprises; i. a recognition site for a first restriction enzyme, and ii. a first secondary structure sequence; b) circularizing first linear construct to create a first circular polynucleotide; c) cleaving said first circular polynucleotide with said first restriction enzyme to form a second linear construct, wherein said first restriction enzyme is able to bind to said recognition site within said first adaptor; d) ligating a second adaptor to said second linear construct to form a third linear construct, wherein said second adaptor comprises; iii. a recognition site for a second restriction enzyme, and iv. a second secondary structure sequence; e) circularizing said third linear construct to create a second circular polynucleotide. - View Dependent Claims (49, 50, 51, 52)
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53. A method of identifying a nucleotide at a detection position of a target nucleic acid, wherein said target nucleic acid comprises a plurality of detection positions, said method comprising:
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a) providing a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises; i) a first target domain of said target nucleic acid comprising a first set of target detection positions; ii) a first adaptor comprising a Type IIs endonuclease restriction site; iii) a second target domain of said target nucleic acid comprising a second set of target detection positions; and iv) a second interspersed adaptor comprising a Type IIs endonuclease restriction site; b) identifying said nucleotide. - View Dependent Claims (54)
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55. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:
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a) providing a plurality of immobilized concatemers, wherein each concatemer comprises; i) multiple copies of a fragment of said target nucleic acid, ii) a plurality of interspersed adaptors at predetermined sites, wherein each interspersed adaptor comprises a secondary structure sequence; b) identifying a sequence of at least a portion of each fragment adjacent to at least one interspersed adaptor in at least one concatemer; thereby identifying a nucleotide sequence of the target nucleic acid. - View Dependent Claims (56, 57, 58, 59, 60)
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61. A method for identifying a nucleotide sequence of a target nucleic acid, said method comprising:
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a) providing a target nucleic acid comprising a plurality of interspersed adaptors, wherein each interspersed adaptor has at least one boundary with said target nucleic acid; and b) identifying at least one nucleotide adjacent to at least one boundary of at least two interspersed adaptors, thereby identifying a nucleotide sequence of said target nucleic acid. - View Dependent Claims (62, 63, 64)
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65. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:
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a) providing a plurality of amplicons, wherein; i) each amplicon comprises multiple copies of a fragment of the target nucleic acid, ii) each amplicon comprises a plurality of interspersed adaptors at predetermined sites within the fragment, each adaptor comprising at least one anchor probe hybridization site, and iii) said plurality of amplicons comprise fragments that substantially cover the target nucleic acid; b) providing a random array of said amplicons fixed to a surface at a density such that at least a majority of said amplicons are optically resolvable; c) hybridizing one or more anchor probes to said random array; d) hybridizing one or more sequencing probes to said random array to form perfectly matched duplexes between said one or more sequencing probes and fragments of target nucleic acid; e) ligating the anchor probes to the sequencing probes; and f) identifying at least one nucleotide adjacent to at least one interspersed adaptor; and g) repeating steps (c) and (f) until a nucleotide sequence of said target nucleic acid is identified. - View Dependent Claims (66, 67, 68, 69)
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70. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:
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a) providing a plurality of amplicons, wherein; i) each amplicon comprises multiple copies of a fragment of the target nucleic acid, ii) each amplicon comprises a plurality of interspersed adaptors at predetermined sites within the fragment, each adaptor comprising at least one anchor probe hybridization site, and iii) said plurality of amplicons comprise fragments that substantially cover the target nucleic acid; b) providing a random array of said amplicons fixed to a surface at a density such that at least a majority of said amplicons are optically resolvable; c) hybridizing one or more anchor probes to said random array to form perfectly matched duplexes between said one or more anchor probes and anchor probe hybridization sites on said interspersed adaptors, d) identifying at least one nucleotide adjacent to at least one interspersed adaptor by extending said one or more anchor probes in a sequence specific reaction; and e) repeating steps (c) and (d) until a nucleotide sequence of said target nucleic acid is identified.
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71. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising:
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a) providing a random array of concatemers fixed to a planar surface, wherein; i) said surface has an array of optically resolvable discrete spaced apart regions, ii) each discrete spaced apart region has an area of less than 1 μ
m2,iii) substantially all of said discrete spaced apart regions have at most one of said concatemers attached, iv) each of said concatemers comprises multiple copies of a fragment of said target nucleic acid, v) each concatemer comprises a plurality of interspersed adaptors at predetermined sites within each fragment, and vi) said fragments of said concatemers substantially cover said target nucleic acid; b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers; c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers; d) ligating probes from said first and second sets which are hybridized to a concatemer at contiguous sites; e) identifying sequences of said ligated probes; thereby identifying a nucleotide sequence of said target nucleic acid. - View Dependent Claims (72)
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73. A method for identifying multiple nucleotides in a target nucleic acid, comprising the steps of:
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(a) providing a target nucleic acid comprising two or more adapters interspersed within said target nucleic acid, wherein each of said adaptors comprises at least one anchor site; (b) hybridizing anchor probes and sequencing probes to multiple anchor sites within said adaptors, (c) ligating anchor probes and sequencing probes that are hybridized to adjacent sites within the target nucleic acid to create anchor-probe ligations; (d) removing any probes that are not anchor-probe ligations; (e) identifying the probe-anchor ligation at each anchor site independently within the target nucleic acid; wherein each anchor-probe ligation is used to identity one or more nucleotides at a defined distance from each adaptor in the target nucleic acid. - View Dependent Claims (74, 75, 76)
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77. A method for identifying multiple nucleotides in target nucleic acid in a single ligation reaction, the method comprising:
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a) providing a target nucleic acid comprising a plurality of interspersed adaptors; b) hybridizing two or more labeled anchor probes to the target nucleic acid, wherein the different anchor probes, can be distinguished based on differential properties of the probes; c) ligating the labeled probes to identify a nucleotide position relative to each anchor d) identifying the labeled probes hybridized to the adaptors; e) removing one or more probes based on the differential properties of the probes; f) identifying the labeled probes remaining after removal of the probes having differential properties; g) identifying additional nucleotide positions in the target targets by comparison of the probes identified in steps (d) and (f), thereby providing identification of two or more nucleotides in a single ligation reaction. - View Dependent Claims (78, 79)
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80. A method for identifying multiple nucleotides in a target nucleic acid, comprising the steps of:
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(a) providing a target nucleic acid comprising two or more adapters interspersed within said target nucleic acid, wherein each of said adaptors comprises at least one anchor site; (b) hybridizing two-part anchor probes to the multiple anchor sites within said adaptors, wherein the first part of the anchor probe is substantially complementary to the adaptor, and wherein the second part of the anchor probes comprises one or more degenerate nucleotides at the ligation end of the probe; and (c) hybridizing sequencing probes to the target nucleic acid; (d) ligating anchor probes and sequencing probes that are hybridized to adjacent sites within the target nucleic acid to create anchor-probe ligations; (e) removing any hybridized probes that are not anchor-probe ligations; (f) identifying the probe-anchor ligation at each anchor site independently within the target nucleic acid; wherein each anchor-probe ligation is used to identity one or more nucleotides at a defined distance from each adaptor in the target nucleic acid.
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Specification