Nucleic acid sequence analysis
First Claim
1. A method of generating redundant sequence information from a single template nucleic acid molecule using multiple sequencing enzymes, the method comprising:
- attaching the single template nucleic acid molecule to a solid surface;
sequencing a polynucleotide region of the single template nucleic acid molecule with a first single sequencing enzyme to obtain a first polynucleotide sequence read of the polynucleotide region;
exchanging the first single sequencing enzyme with a second single sequencing enzyme, wherein the first single sequencing enzyme is a first type of polymerase enzyme and the second single sequencing enzyme is a second type of polymerase enzyme that is different from the first type of polymerase enzyme; and
sequencing the polynucleotide region of the single template nucleic acid molecule with the second single sequencing enzyme to obtain a second polynucleotide sequence read of the polynucleotide region, wherein the first and second sequence reads comprise redundant sequence information for the polynucleotide region, thereby generating redundant sequence information from the single template nucleic acid molecule.
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Accused Products
Abstract
Provided are methods for sequencing a nucleic acid with a sequencing enzyme, e.g., a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. In certain embodiments, a template is fixed to a surface through a template localizing moiety. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Also provided are methods for using data from analytical reactions wherein two different enzymes are employed, e.g., at a same or different reaction regions.
47 Citations
16 Claims
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1. A method of generating redundant sequence information from a single template nucleic acid molecule using multiple sequencing enzymes, the method comprising:
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attaching the single template nucleic acid molecule to a solid surface; sequencing a polynucleotide region of the single template nucleic acid molecule with a first single sequencing enzyme to obtain a first polynucleotide sequence read of the polynucleotide region; exchanging the first single sequencing enzyme with a second single sequencing enzyme, wherein the first single sequencing enzyme is a first type of polymerase enzyme and the second single sequencing enzyme is a second type of polymerase enzyme that is different from the first type of polymerase enzyme; and sequencing the polynucleotide region of the single template nucleic acid molecule with the second single sequencing enzyme to obtain a second polynucleotide sequence read of the polynucleotide region, wherein the first and second sequence reads comprise redundant sequence information for the polynucleotide region, thereby generating redundant sequence information from the single template nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of sequencing a single template nucleic acid molecule, the method comprising:
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a) attaching the single template nucleic acid molecule to a solid surface; b) annealing an oligonucleotide primer to the template nucleic acid; c) using a first single polymerase to perform template-directed nascent strand synthesis using a polynucleotide region of the single template nucleic acid molecule to generate a first polynucleotide complement of the polynucleotide region, where the first single polymerase is a first type of polymerase that is not immobilized to the solid surface; d) detecting a first set of incorporations to generate a first temporal sequence of incorporations into the first polynucleotide complement, the first temporal sequence of incorporations comprising each nucleotide base incorporated during the template-directed nascent strand synthesis of step c; e) using a second single polymerase to perform template-directed nascent strand synthesis using the polynucleotide region of the single template nucleic acid molecule to generate a second polynucleotide complement of the polynucleotide region, where the second single polymerase is a second type of polymerase that is not immobilized to the solid surface; f) detecting a second set of incorporations to generate a second temporal sequence of incorporations into the second polynucleotide complement, the second temporal sequence of incorporations comprising each nucleotide base incorporated during the template-directed nascent strand synthesis of step e; and g) analyzing the first temporal sequence and the second temporal sequence to determine a consensus sequence for the polynucleotide region of the single template nucleic acid molecule. - View Dependent Claims (11, 12)
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13. A method of sequencing a plurality of identical template nucleic acids, the method comprising:
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attaching single molecules of the identical template nucleic acids to different single molecule reaction regions on a solid surface; exposing the solid surface to a reaction mixture comprising at least two different types of polymerases; forming a plurality of complexes at the single molecule reaction regions, the complexes comprising a single one of the molecules of identical template nucleic acids and a single polymerase selected from the at least two different types of polymerases; within each complex, using the single polymerase to synthesize a polynucleotide strand complementary to the single molecule of template nucleic acid; detecting a set of incorporations of nucleotide bases into the polynucleotide strand synthesized in each of the complexes, thereby generating a plurality of sequence reads for the identical template nucleic acids, wherein some of the sequence reads were generated by a first of the different types of polymerases and others of the sequence reads were generated by a second of the different types of polymerases; and subjecting the plurality of sequence reads to statistical analysis to determine a consensus sequence for the identical template nucleic acids. - View Dependent Claims (14, 15, 16)
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Specification