Molecular redundant sequencing
First Claim
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1. A method of sequencing nucleic acid fragments within a genetic target material, comprising:
- fractionating the genetic target material into separate fractions;
tagging each of said separate fractions with known exogenous registration sequences, wherein nucleic acid fragments in a single fraction are tagged with an identical known exogenous registration sequence and wherein nucleic acid fragments in said separate fractions are tagged with different known exogenous registration sequences; and
further wherein the exogenous registration sequences provide identification of which separate fraction a given nucleic acid fragment was derived from, thereby generating tagged fragments in each of said separate fractions;
circularizing the tagged fragments;
pooling the tagged fragments from all of said separate fractions;
sequencing the tagged fragments until the exogenous registration sequence has been sequenced at least twice, thereby generating sequence reads, each of which comprises at least two exogenous registration sequence portions and at least one tagged fragment sequence portion.
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Abstract
Methods, systems and compositions where a target nucleic acid includes a registration sequence disposed therein for identification of the number or relative position of determined sequence from the template sequence. Particularly preferred aspects include a registration sequence in a circular template nucleic acid sequence which is, in turn, used in sequence by incorporation processes that rely upon template dependent, polymerase mediated primer extension in the identification of the sequence of the template.
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Citations
20 Claims
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1. A method of sequencing nucleic acid fragments within a genetic target material, comprising:
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fractionating the genetic target material into separate fractions; tagging each of said separate fractions with known exogenous registration sequences, wherein nucleic acid fragments in a single fraction are tagged with an identical known exogenous registration sequence and wherein nucleic acid fragments in said separate fractions are tagged with different known exogenous registration sequences; and
further wherein the exogenous registration sequences provide identification of which separate fraction a given nucleic acid fragment was derived from, thereby generating tagged fragments in each of said separate fractions;circularizing the tagged fragments; pooling the tagged fragments from all of said separate fractions; sequencing the tagged fragments until the exogenous registration sequence has been sequenced at least twice, thereby generating sequence reads, each of which comprises at least two exogenous registration sequence portions and at least one tagged fragment sequence portion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of determining a nucleic acid sequence for a plurality of target nucleic acids, comprising:
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providing the plurality of target nucleic acids in separate fractions, wherein each of said separate fractions comprises a different subset of said target nucleic acids, wherein each different subset comprises target nucleic, acids from a genomic region of interest; tagging the target nucleic acids in the multiple separate fractions with exogenous registration sequences such that all of the target nucleic acids in a single fraction comprise the same exogenous registration sequence that is different from the exogenous registration sequences used to tag the target nucleic acids in other fractions, and further wherein said exogenous registration sequences tag said target nucleic acids at selected locations relative to a first sequence of nucleotides in said target nucleic acids; sequencing the target nucleic acids to generate sequence reads comprising both target sequence information and exogenous registration sequence information such that the first sequence of nucleotides and registration sequence are sequenced multiple times; aligning the sequence reads based at least in part upon the exogenous registration sequence information, thereby providing an aligned set of sequence reads for each of said separate fractions; and determining a consensus sequence from said aligned set of sequence reads to determine a nucleic acid sequence for each first sequence of nucleotides in each different subset of target nucleic acids. - View Dependent Claims (10, 11, 12, 13)
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14. A composition, comprising:
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a set of nucleic acid synthesis complexes comprising; a set of circular nucleic acid templates comprising different length fragments of a nucleic acid region of interest, each having a common 5′
-terminal sequence, wherein each of the circular nucleic acid templates comprises one of said different length fragments and one exogenous registration sequence, wherein the 5′
-end of said fragment is linked to a 3′
-end of the exogenous registration sequence, and the 3′
-end of said fragment is linked to a 5′
-end of the exogenous registration sequence;a primer sequence annealed to at least a portion of the exogenous registration sequence in each of said circular nucleic acid templates, and a nucleic acid polymerase bound to each of said circular nucleic acid templates at said primer sequence; and a plurality of types of nucleotides or nucleotide analogs. - View Dependent Claims (15)
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16. A method of sequencing a nucleic acid region of interest, the method comprising:
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providing a nucleic acid region of interest; fragmenting the nucleic acid region of interest to produce a nested set of fragments of the nucleic acid region of interest, wherein the nested set comprises fragments of varying lengths, and further wherein each fragment in the nested set comprises a common 5′
-terminal sequence and a variable 3′
-terminal sequence, such that each terminates at a different point in the nucleic acid region of interest depending on its length;circularizing each fragment in the nested set of fragments by linking a 3′
-end of an exogenous registration sequence to the 5′
-end of each fragment in the nested set and linking a 5′
-end of the exogenous registration sequence to the 3′
-end of each fragment in the nested set, thereby generating circular nucleic acid templates of varying sizes;sequencing each of the circular nucleic acid templates, thereby generating sequence reads comprising sequence information for the variable 3′
-terminal sequences; andassembling the sequence reads based upon the sequence information for the variable terminal sequences and the varying sizes of the circular nucleic acid templates. - View Dependent Claims (17, 18, 19, 20)
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Specification