NUCLEIC ACID SEQUENCE ANALYSIS

• US 20120115736A1
• Filed: 10/12/2011
• Published: 05/10/2012
• Est. Priority Date: 09/19/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Bibillo, Arek, Christians, Fred, Travers, Kevin, Emig, Robin, Turner, Stephen
• Granted Patent: US 8,921,046 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2531/125 Rolling circle

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.

Claims

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 enzymes is a second 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 a single template nucleic acid molecule.

2. The method of claim 1, further comprising subjecting both the first polynucleotide sequence read and the second polynucleotide sequence read to statistical analysis to determine a consensus polynucleotide sequence of the polynucleotide region of the single template nucleic acid molecule.

4. The method of claim 1, wherein the first single sequencing enzyme and the second single sequencing enzyme are single polymerase enzymes.

6. The method of claim 4, wherein the first single sequencing enzyme has at least one characteristic that differs from the second single sequencing enzyme, wherein the characteristic is selected from the group consisting of fidelity, processivity, accuracy, activity, specificity, rate, retention time, sequence context response, and stability.

8. The method of claim 1, further comprising sequencing the polynucleotide region of the single template nucleic acid molecule multiple times with at least one of the first or second single sequencing enzyme.

9. The method of claim 1, wherein the single template nucleic acid molecule is a circular template nucleic acid molecule, 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.

10. The method of claim 9, further comprising sequencing the polynucleotide region of the single template nucleic acid molecule more than two times using both the first and second types of polymerase enzymes to generate a single nucleic acid strand comprising multiple copies of a polynucleotide complementary to the polynucleotide region.

11. The method of claim 1, wherein the first single sequencing enzyme and the second single sequencing enzyme are present in the same reaction mixture.

14. The method of claim 1, wherein the sequencing by the first single sequencing enzyme is performed under a first set of reaction conditions, and the sequencing by the second single sequencing enzyme is performed under a second set of reaction conditions, wherein the first set of reaction conditions is different from the second set of reaction conditions by at least one aspect selected from the group consisting of temperature, pH, types of nucleotides, types of labels, types of buffer, ion concentrations, illumination, and types of divalent or monovalent cations.

3. (canceled)

5. (canceled)

7. (canceled)

12-13. -13. (canceled)

15-32. -32. (canceled)

33. A reaction mixture, comprising:

a single template nucleic acid molecule attached to a solid surface in a single molecule reaction region;

a first type of polymerase;

a second type of polymerase;

a set of different nucleotide analogs of the invention, at least one nucleotide analog of which comprises a detectable moiety linked to a phosphate moiety that is released upon incorporation of said one nucleotide analog into a complementary copy of the single template nucleic acid molecule; and

multiple polynucleotide complements of the single template nucleic acid molecule, wherein some of the polynucleotide complements were synthesized by a single one of the first type of polymerase and others of the polynucleotide complements were synthesized by a single one of the second type of polymerase.

37. The reaction mixture of claim 33, wherein the single template nucleic acid is a circular template nucleic acid.

38. The reaction mixture of claim 37, wherein the multiple polynucleotide complements are within a linear concatemer molecule.

41. The reaction mixture of claim 33, wherein the first type of polymerase has at least one characteristic that differs from the second type of polymerase.

34-36. -36. (canceled)

39-40. -40. (canceled)

42. (canceled)

43. A method of sequencing a single template nucleic acid molecule, the method comprising:

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.

44. The method of claim 43, wherein the single template nucleic acid molecule is a circular nucleic acid.

47. The method of claim 43, wherein nucleotides used in the template-directed nascent strand synthesis comprise detectable labels that identify the base composition of the nucleotides.

45-46. -46. (canceled)

48-55. -55. (canceled)

56. A method of sequencing a plurality of identical template nucleic acids, the method comprising:

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.

58. The method of claim 56, wherein nucleotides used to synthesize the polynucleotide strand comprise detectable labels that identify the base composition of the nucleotides.

59. The method of claim 56, wherein the first of the different types of polymerases has at least one characteristic that differs from the second of the different types of polymerases, wherein the characteristic is selected from the group consisting of fidelity, processivity, accuracy, activity, specificity, rate, retention time, sequence context sensitivity, and stability.

61. The method of claim 56, further comprising performing the synthesis of the polynucleotide strand under at least two different reaction conditions.

57. (canceled)

60. (canceled)

62. (canceled)