阅读说明：本技术 用于核酸测序的系统和方法 (Systems and methods for nucleic acid sequencing ) 是由 H·埃斯范德亚珀 马里亚姆·尤兹 塞思·斯特恩 保罗·肯尼 于 2018-09-20 设计创作，主要内容包括：本公开内容提供了用于测序核酸分子的方法和系统。方法可以包括对双链核酸或单链核酸进行测序。测序可以包括使用偶联至静电部分的核苷酸。静电部分可以通过传感器阵列检测。静电部分可以是在掺入核苷酸后从核酸分子切割的可逆静电部分。静电部分可以是不可逆静电部分。可以将包含不可逆静电部分的核苷酸掺入核酸分子,由传感器阵列检测,并交换为不可检测的核苷酸。(The present disclosure provides methods and systems for sequencing nucleic acid molecules. The method may comprise sequencing the double-stranded nucleic acid or the single-stranded nucleic acid. Sequencing may include the use of nucleotides coupled to electrostatic moieties. The electrostatic part may be detected by a sensor array. The electrostatic moiety can be a reversible electrostatic moiety that is cleaved from the nucleic acid molecule upon incorporation of the nucleotide. The electrostatic moiety may be an irreversible electrostatic moiety. Nucleotides comprising an irreversible electrostatic moiety can be incorporated into a nucleic acid molecule, detected by a sensor array, and exchanged for an undetectable nucleotide.)

1. A method for detecting a nucleic acid molecule, comprising:

(a) providing a plurality of double-stranded nucleic acid molecules adjacent to a sensor array, wherein a given double-stranded nucleic acid molecule of the plurality of nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given double-stranded nucleic acid molecule comprises a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule having sequence complementarity to the first single-stranded nucleic acid molecule, and wherein the given sensor is electrically coupled to a charge bilayer comprising the given double-stranded nucleic acid molecule;

(b) subjecting at least a portion of the second single-stranded nucleic acid molecule to release from the first single-stranded nucleic acid molecule to provide a segment of the first single-stranded nucleic acid molecule that does not hybridize to the second single-stranded nucleic acid molecule;

(c) contacting the segment with an individual nucleotide such that the segment undergoes a nucleic acid incorporation reaction that generates a third single-stranded nucleic acid molecule from the individual nucleotide, wherein the third single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule; and

(d) detecting a signal indicative of the incorporation of the individual nucleotides into the third single-stranded nucleic acid molecule using the given sensor during or after the nucleic acid incorporation reaction is performed, thereby determining the sequence or length of the segment.

2. The method of claim 1, wherein releasing the at least a portion of the second single-stranded nucleic acid molecule forms a flap, and wherein the flap is cleaved from the second single-stranded nucleic acid molecule.

3. The method of claim 2, wherein the flap is cleaved upon detection of the signal indicative of incorporation of the individual nucleotide.

4. The method of claim 1, wherein the second single-stranded nucleic acid molecule is selected from a library of nucleic acid subunits.

5. The method of claim 4, wherein the library of nucleic acid subunits comprises random sequences.

6. The method of claim 4, wherein a given nucleic acid subunit in the library of nucleic acid subunits comprises at least five nucleotides.

7. The method of claim 4, wherein the library of nucleic acid subunits comprises peptide nucleic acids or locked nucleic acids.

8. The method of any one of claims 1-7, wherein the second single-stranded nucleic acid molecule comprises one or more detectable labels.

9. The method of claim 8, wherein releasing the second single-stranded nucleic acid molecule or portion thereof from the first single-stranded nucleic acid molecule generates the signal indicative of the incorporation of the individual nucleotide into the third single-stranded nucleic acid molecule.

10. The method of any one of claims 1-9, wherein the plurality of double-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the given double-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

11. The method of any one of claims 1-9, wherein the plurality of double-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein the given double-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

12. The method of any one of claims 1-11, wherein (c) further comprises providing a priming site adjacent to the segment and generating the third single-stranded nucleic acid molecule upon primer extension from the priming site.

13. The method of any one of claims 1-12, wherein the given sensor comprises at least two electrodes.

14. The method of any one of claims 1-13, wherein at least a subset of the individual nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule.

15. The method of claim 14, wherein the reversible terminator is removed after the individual nucleotide is incorporated into the third single-stranded nucleic acid molecule and before another individual nucleotide is incorporated into the third single-stranded nucleic acid molecule.

16. The method of any one of claims 1-15, wherein at least a subset of the individual nucleotides comprise a detectable label.

17. The method of claim 16, wherein the detectable label is an electrostatic moiety.

18. The method of claim 16, wherein the detectable label is coupled to nucleobases of the at least a subset of the individual nucleotides.

19. The method of claim 18, wherein the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a single type of detectable label.

20. The method of claim 16, wherein the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a different type of detectable label.

21. The method of any one of claims 19 and 20, wherein the detectable label is reversibly coupled to the different types of nucleotides by at least two coupling mechanisms.

22. The method of any one of claims 19 and 20, wherein the detectable label is reversibly coupled to the different type of nucleotide by a single coupling mechanism.

23. The method of any one of claims 21 and 22, further comprising removing the detectable label after detecting the signal indicative of incorporation of the individual nucleotide.

24. The method of any one of claims 1-23, wherein the individual nucleotides comprise different types of nucleotides, and wherein the segments are sequentially contacted with the different types of nucleotides.

25. The method of claim 24, wherein the segment is contacted with a first type of individual nucleotides at a given point in time during the nucleic acid incorporation reaction and the segment is contacted with a second type of individual nucleotides at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type.

26. The method of any one of claims 1-23, wherein the individual nucleotides comprise different types of nucleotides, and wherein the segments are contacted with the different types of nucleotides simultaneously.

27. The method of any one of claims 1-26, wherein the signal indicative of incorporation of the individual nucleotide is a steady state signal.

28. The method of claim 27, wherein the signal indicative of incorporation of a subject nucleotide is detected once after incorporation of the subject nucleotide.

29. The method of claim 27, wherein the signal indicative of incorporation of a subject nucleotide is detected at least twice after incorporation of the subject nucleotide.

30. The method of any one of claims 1-26, wherein the signal indicative of incorporation of the individual nucleotide is a transient signal.

31. The method of any one of claims 1-30, wherein the signal indicative of incorporation of the individual nucleotide is an electrical signal generated by impedance or impedance change in the charge bilayer.

32. The method of any one of claims 1-31, wherein the plurality of double-stranded nucleic acid molecules is a clonal population of the double-stranded nucleic acid molecules.

33. The method of any one of claims 1-32, wherein (b) - (d) are repeated until the sequence or length of the first single-stranded nucleic acid molecule is determined.

34. A method for detecting a nucleic acid molecule, comprising:

(a) providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, wherein a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule;

(b) contacting the first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides, wherein the second single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, wherein at least a subset of the individual nucleotides comprise a detectable label; and

(c) during or after performing the nucleic acid incorporation reaction, detecting a signal from the detectable label using the given sensor, the signal indicating incorporation of the individual nucleotide into the second single-stranded nucleic acid molecule, thereby determining the sequence or length of the first single-stranded nucleic acid molecule.

35. The method of claim 34, wherein the plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

36. The method of claim 34, wherein the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein the first single-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

37. The method of any one of claims 34-36, wherein (b) further comprises providing a priming site adjacent to the first single-stranded nucleic acid and generating the second single-stranded nucleic acid molecule upon primer extension from the priming site.

38. The method of claim 37, wherein the priming site is a self-priming loop.

39. The method of any one of claims 34-38, wherein the given sensor comprises at least two electrodes.

40. The method of any one of claims 34-39, wherein at least another subset of the individual nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule.

41. The method of claim 40, wherein the reversible terminator is removed after the individual nucleotide is incorporated into the second single-stranded nucleic acid molecule and before another individual nucleotide is incorporated into the second single-stranded nucleic acid molecule.

42. The method of any one of claims 34-41, wherein the detectable label is an electrostatic moiety.

43. The method of any one of claims 34-42, wherein the detectable label is coupled to nucleobases of the at least one subset of the individual nucleotides.

44. The method of any one of claims 34-43, wherein the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a single type of detectable label.

45. The method of any one of claims 34-43, wherein the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a different type of detectable label.

46. The method of any one of claims 44 and 45, wherein the detectable label is reversibly coupled to the different types of nucleotides by at least two coupling mechanisms.

47. The method of any one of claims 44 and 45, wherein the detectable label is reversibly coupled to the different type of nucleotide by a single coupling mechanism.

48. The method of any one of claims 46 and 47, wherein the detectable label is removed after detecting the signal indicative of incorporation of the individual nucleotide.

49. The method of any one of claims 34-48, wherein the individual nucleotides comprise different types of nucleotides, and wherein the first single-stranded nucleic acid molecule is sequentially contacted with the different types of nucleotides.

50. The method of claim 49, wherein the first single-stranded nucleic acid molecule is contacted with individual nucleotides of a first type at a given point in time during the nucleic acid incorporation reaction, and the first single-stranded nucleic acid molecule is contacted with individual nucleotides of a second type at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type.

51. The method of any one of claims 34-48, wherein the individual nucleotides comprise different types of nucleotides, and wherein the first single-stranded nucleic acid molecule is contacted with the different types of nucleotides simultaneously.

52. The method of any one of claims 34-51, wherein the signal indicative of incorporation of the individual nucleotide is a steady state signal.

53. The method of claim 52, wherein the signal indicative of incorporation of a subject nucleotide is detected once after incorporation of the subject nucleotide.

54. The method of claim 52, wherein the signal indicative of incorporation of a subject nucleotide is detected at least twice after incorporation of the subject nucleotide.

55. The method of any one of claims 34-51, wherein the signal indicative of incorporation of the individual nucleotide is a transient signal.

56. The method of any one of claims 34-55, wherein the signal indicative of incorporation of the individual nucleotide is an electrical signal generated by impedance or impedance change in the charge bilayer.

57. The method of any one of claims 34-56, wherein the plurality of single-stranded nucleic acid molecules is a clonal population of the first single-stranded nucleic acid molecule.

58. The method of any one of claims 34-57, wherein (b) - (c) are repeated until the sequence or the length of the first single-stranded nucleic acid molecule is determined.

59. A method for detecting a nucleic acid molecule, comprising:

(a) providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, wherein a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array;

(b) subjecting the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule that is a growing strand complementary to the first single-stranded nucleic acid molecule, wherein the nucleic acid incorporation reaction comprises alternately and sequentially (i) incorporating individual nucleotides in a first plurality of nucleotides that comprise a detectable label, and (ii) incorporating individual nucleotides in a second plurality of nucleotides that do not comprise a detectable label; and

(c) simultaneously with or after performing the nucleic acid incorporation reaction, detecting a signal indicative of a change in charge or conductivity from the bilayer comprising the detectable label using the given sensor, thereby determining the sequence or length of the first single-stranded nucleic acid molecule.

60. The method of claim 59, wherein the first plurality of nucleotides comprises a terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule.

61. The method of claim 59, wherein the first plurality of nucleotides comprises dideoxynucleotides.

62. The method of any one of claims 59-61, wherein the second plurality of nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid.

63. The method of claim 59, wherein the first plurality of nucleotides is exchanged with the second plurality of nucleotides.

64. The method of any one of claims 59-63, wherein the incorporation of the second plurality of nucleotides corrects for phase error by incorporating individual nucleotides from the second plurality of nucleotides at positions along the first single-stranded nucleic acid molecule where individual nucleotides from the first plurality of nucleotides are not incorporated.

65. The method of claim 64, further comprising continuing the nucleic acid incorporation reaction using the individual nucleotides from the first plurality of nucleotides.

66. The method of claim 62, wherein the reversible terminator is removed after incorporation of the individual nucleotide in the second plurality of nucleotides.

67. The method of any one of claims 59-66, wherein the detectable label is a non-removable electrostatic moiety.

68. The method of any one of claims 59-67, wherein the individual nucleotides in the first plurality of nucleotides comprise different types of nucleotides, each different type of nucleotide coupled to a single type of detectable label.

69. The method of any one of claims 59-67, wherein the individual nucleotides in the first plurality of nucleotides comprise different types of nucleotides, each different type of nucleotide coupled to a different type of detectable label.

70. The method of any one of claims 59-69, wherein the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule.

71. The method of claim 59, wherein the plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

72. The method of claim 59, wherein the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein the first single-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

73. The method of any one of claims 59-72, wherein (b) further comprises providing a priming site adjacent to the first single-stranded nucleic acid and generating the second single-stranded nucleic acid molecule upon primer extension from the priming site.

74. The method of claim 73, wherein the priming site is a self-priming loop.

75. The method of any one of claims 59-74, wherein the given sensor comprises at least two electrodes.

76. The method of any one of claims 59-75, wherein the individual nucleotides comprise different types of nucleotides, and wherein the first single-stranded nucleic acid molecule is sequentially contacted with the different types of nucleotides.

77. The method of claim 59, wherein the first single-stranded nucleic acid molecule is contacted with individual nucleotides of a first type at a given point in time during the nucleic acid incorporation reaction, and the segment is contacted with individual nucleotides of a second type at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type.

78. The method of any one of claims 59-77, wherein the individual nucleotides comprise different types of nucleotides, and wherein the first single-stranded nucleic acid molecule is contacted with the different types of nucleotides simultaneously.

79. The method of any one of claims 59-78, wherein the signal indicative of incorporation of the individual nucleotide is a steady state signal.

80. The method of claim 79, wherein the signal indicative of incorporation of a subject nucleotide is detected once after incorporation of the subject nucleotide.

81. The method of claim 79, wherein the signal indicative of incorporation of a subject nucleotide is detected at least twice after incorporation of the subject nucleotide.

82. The method of any one of claims 59-78, wherein the signal indicative of incorporation of the individual nucleotide is a transient signal.

83. The method of any one of claims 59-82, wherein the signal is an electrical signal generated by an impedance or impedance change in the charge bilayer.

84. The method of any one of claims 59-83, wherein the plurality of single-stranded nucleic acid molecules is a clonal population of the first single-stranded nucleic acid molecule.

85. The method of any one of claims 59-84, wherein (b) - (c) are repeated until the sequence or the length of the first single-stranded nucleic acid molecule is determined.

86. The method of claim 59, wherein said first single-stranded nucleic acid molecule is a portion of said plurality of single-stranded nucleic acid molecules adjacent to said given sensor, wherein individual single-stranded nucleic acid molecules of said plurality including said first single-stranded nucleic acid molecule have sequence homology to a template single-stranded nucleic acid molecule.

87. A system for detecting a nucleic acid molecule, comprising:

a sensor array comprising a plurality of sensors, wherein during use a given double-stranded nucleic acid molecule of a plurality of double-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given double-stranded nucleic acid molecule comprises a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule having sequence complementarity to the first single-stranded nucleic acid molecule, wherein the given sensor is electrically coupled to a charge bilayer comprising the given double-stranded nucleic acid molecule; and

one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact a segment of the first single-stranded nucleic acid molecule that is not hybridized to the second single-stranded nucleic acid molecule with an individual nucleotide to subject the segment to a nucleic acid incorporation reaction that generates the third single-stranded nucleic acid molecule from the individual nucleotide, wherein the third single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, and (ii) simultaneously with or subsequent to performing the nucleic acid incorporation reaction, detect a signal indicative of incorporation of the individual nucleotide into the third single-stranded nucleic acid molecule using the given sensor, thereby determining the sequence or length of the segment.

88. The system of claim 87, wherein, during use, the plurality of double-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the given double-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

89. The system of claim 87, wherein, during use, the plurality of double-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein the given double-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

90. The system of any one of claims 87-89, wherein the given sensor comprises at least two electrodes.

91. The system of any one of claims 87-90, wherein during use, the signal indicative of incorporation of the individual nucleotide is a steady state signal.

92. The system of any one of claims 87-90, wherein during use, the signal indicative of incorporation of the individual nucleotide is a transient signal.

93. The system of any one of claims 87-92, wherein, during use, the signal indicative of incorporation of the individual nucleotide is an electrical signal generated by impedance or impedance change in the charge bilayer.

94. A system for detecting a nucleic acid molecule, comprising:

a sensor array comprising a plurality of sensors, wherein during use a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule; and

one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact the first single-stranded nucleic acid molecule with an individual nucleotide, such that the first single-stranded nucleic acid molecule undergoes a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides, wherein the second single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, wherein at least a subset of the individual nucleotides comprise a detectable label, and (ii) a signal from the detectable label is detected using the given sensor, simultaneously with or subsequent to performing the nucleic acid incorporation reaction, the signal indicates that the individual nucleotide is incorporated into the second single-stranded nucleic acid molecule, thereby determining the sequence or length of the first single-stranded nucleic acid molecule.

95. The system of claim 94, wherein, during use, the plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

96. The system of claim 94, wherein, during use, the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein, during use, the first single-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

97. The system of any one of claims 94-96, wherein the given sensor comprises at least two electrodes.

98. The system of any one of claims 94-97, wherein, during use, the signal indicative of incorporation of the individual nucleotide is a steady state signal.

99. The system of any one of claims 94-97, wherein, during use, the signal indicative of incorporation of the individual nucleotide is a transient signal.

100. The system of any one of claims 94-99, wherein, during use, the signal indicative of incorporation of the individual nucleotide is an electrical signal generated by impedance or impedance change in the charge bilayer.

101. A system for detecting a nucleic acid molecule, comprising:

a sensor array comprising a plurality of sensors, wherein during use a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array; and

one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact the first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule, wherein the nucleic acid incorporation reaction comprises alternately and sequentially incorporating individual nucleotides of a first plurality of nucleotides comprising a detectable label and exchanging the individual nucleotides of the first plurality of nucleotides with individual nucleotides of a second plurality of nucleotides not comprising a detectable label, and (ii) detect a signal indicative of a change in charge or conductivity from a bilayer comprising the detectable label using the given sensor while or after performing the nucleic acid incorporation reaction, thereby determining the sequence or length of the first single-stranded nucleic acid molecule.

102. The system of claim 101, wherein during use, the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule.

103. The system of claim 102, wherein, during use, the plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads, wherein the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and wherein the charge bilayer is adjacent to a surface of the given bead.

104. The system of claim 102, wherein, during use, the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array, wherein, during use, the first single-stranded nucleic acid molecule is coupled to a surface of the given sensor, and wherein the charge bilayer is adjacent to the surface.

105. The system of any one of claims 101-104, wherein the given sensor comprises at least two electrodes.

106. The system of any one of claims 101-105, wherein during use, the signal indicative of incorporation of the individual nucleotide is a steady state signal.

107. The system of any one of claims 101-105, wherein during use, the signal indicative of incorporation of the individual nucleotide is a transient signal.

108. The system of any one of claims 101-107, wherein, during use, the signal indicative of incorporation of the individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

Background

The goal of elucidating the entire human genome has raised interest in rapid nucleic acid (e.g., DNA) sequencing technologies, whether for small-scale or large-scale applications. Important parameters are the sequencing speed, the length of sequence that can be read during a single sequencing run, and the amount of nucleic acid template required to generate sequencing information. Large-scale genomic projects are currently too expensive to be practically developed for a large number of subjects (e.g., patients). Furthermore, as the genetic basis of human disease is increasingly understood, the need for accurate, high-throughput DNA sequencing affordable for clinical use will continue to increase. Practical methods for determining the base pair sequence of a single nucleic acid molecule, including those with high speed and long read lengths, can provide measurement capability.

Nucleic acid sequencing is a process that can be used to provide sequence information for a nucleic acid sample. Such sequence information can aid in the diagnosis and/or treatment of a subject having a disorder. For example, a subject's nucleic acid sequence may be used to identify, diagnose, and possibly develop treatments for a genetic disorder. As another example, research into pathogens may lead to treatment of contagious diseases. Unfortunately, however, the sequencing techniques existing in the prior art are expensive and may not provide sequence information over a certain period of time and/or with a certain degree of accuracy, and thus may not be sufficient to diagnose and/or treat a subject with a disorder.

Disclosure of Invention

The present disclosure provides methods and systems for sample analysis or identification, such as nucleic acid sequencing. The present disclosure provides methods and systems that can achieve sample preparation and identification (e.g., sequencing) without the use of particles (e.g., beads). This may enable samples to be prepared and identified at significantly reduced cost and complexity compared to other systems and methods.

In one aspect, the present disclosure provides a method for detecting a nucleic acid molecule, comprising: providing a plurality of double-stranded nucleic acid molecules adjacent to a sensor array, wherein a given double-stranded nucleic acid molecule of the plurality of double-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given double-stranded nucleic acid molecule comprises a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule having sequence complementarity to the first single-stranded nucleic acid molecule, and wherein the given sensor is electrically coupled to a charge bilayer comprising the given double-stranded nucleic acid molecule; subjecting at least a portion of the second single-stranded nucleic acid molecule to release from the first single-stranded nucleic acid molecule to provide a segment of the first single-stranded nucleic acid molecule that is not hybridized to the second single-stranded nucleic acid molecule; contacting the segment with the individual nucleotides to subject the segment to a nucleic acid incorporation reaction that generates a third single-stranded nucleic acid molecule from the individual nucleotides, wherein the third single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule; and detecting a signal indicative of the incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule using a given sensor while performing the nucleic acid incorporation reaction, thereby determining the sequence and/or length of the segment.

In some embodiments, releasing at least a portion of the second single-stranded nucleic acid molecule forms a flap. In some embodiments, the flap is cleaved from the second single-stranded nucleic acid molecule. In some embodiments, the flap is cleaved upon detection of a signal indicative of incorporation of an individual nucleotide. In some embodiments, the flanks are cleaved by a flanking endonuclease. In some embodiments, the flanking endonucleases are mesophilic.

In some embodiments, the second single-stranded nucleic acid molecule is selected from a library of nucleic acid subunits. In some embodiments, the library of nucleic acid subunits comprises random sequences. In some embodiments, a given nucleic acid subunit in the library of nucleic acid subunits comprises at least five nucleotides. In some embodiments, a given nucleic acid subunit in the library of nucleic acid subunits has at least six nucleotides. In some embodiments, the library of nucleic acid subunits comprises peptide nucleic acids or locked nucleic acids.

In some embodiments, the second single-stranded nucleic acid molecule comprises one or more detectable labels. In some embodiments, release of the second single-stranded nucleic acid molecule or portion thereof from the first single-stranded nucleic acid molecule produces a detectable signal.

In some embodiments, a plurality of double-stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, the given double-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charge bilayer is adjacent to a surface of the given bead. In some embodiments, a plurality of double-stranded nucleic acid molecules are coupled to one or more surfaces of a sensor array. In some embodiments, a given double-stranded nucleic acid molecule is coupled to a surface of a given sensor, and a charge bilayer is adjacent to the surface.

In some embodiments, the method further comprises providing a priming site adjacent to the segment, and generating a third single-stranded nucleic acid molecule upon extension of the primer from the priming site. In some embodiments, the priming site is a primer sequence having sequence complementarity to the first single-stranded nucleic acid molecule. In some embodiments, the method further comprises incorporating the individual nucleotides using a polymerase. In some embodiments, a given sensor comprises at least two electrodes.

In some embodiments, at least a subset of the individual nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule. In some embodiments, the reversible terminator is removed after an individual nucleotide is incorporated into the third single-stranded nucleic acid molecule and before another individual nucleotide is incorporated into the third single-stranded nucleic acid molecule.

In some embodiments, at least a subset of the individual nucleotides comprise a detectable label. In some embodiments, the detectable label is an electrostatic moiety. In some embodiments, a detectable label is coupled to nucleobases of at least a subset of the individual nucleotides. In some embodiments, the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a single type of detectable label. In some embodiments, the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a different type of detectable label. In some embodiments, the detectable label is reversibly coupled to the different types of nucleotides by one or more coupling mechanisms. In some embodiments, the detectable label is reversibly coupled to the different types of nucleotides by a single coupling mechanism. In some embodiments, the detectable label is removed after detection of the signal indicative of incorporation of the individual nucleotide. In some embodiments, the individual nucleotides comprise different types of nucleotides, and the segments are sequentially contacted with the different types of nucleotides.

In some embodiments, a segment is contacted with a first type of individual nucleotide at a given point in time during a nucleic acid incorporation reaction, and a segment is contacted with a second type of individual nucleotide at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type. In some embodiments, the individual nucleotides comprise different types of nucleotides, and the segments are contacted with the different types of nucleotides simultaneously.

In some embodiments, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

In some embodiments, the plurality of double-stranded nucleic acid molecules is a clonal population of double-stranded nucleic acid molecules. In some embodiments, the method is repeated until the sequence of the first single-stranded nucleic acid molecule is determined.

In another aspect, the present disclosure provides a method for detecting a nucleic acid molecule, comprising: providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, wherein a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule; contacting the first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides, wherein the second single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, wherein at least a subset of the individual nucleotides comprise a detectable label; and detecting a signal from the detectable label, which signal indicates incorporation of the individual nucleotide into the second single-stranded nucleic acid molecule, using the given sensor, simultaneously with or after performing the nucleic acid incorporation reaction, thereby determining the sequence and/or length of the first single-stranded nucleic acid molecule.

In some embodiments, a plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charged bilayer is adjacent to a surface of the given bead. In some embodiments, the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array. In some embodiments, the first single-stranded nucleic acid molecule is coupled to a surface of a given sensor, and the charge bilayer is adjacent to the surface.

In some embodiments, the method further comprises providing a priming site adjacent to the first single-stranded nucleic acid and generating a second single-stranded nucleic acid molecule upon extension of the primer from the priming site. In some embodiments, the priming site is a primer sequence having sequence complementarity to the first single-stranded nucleic acid molecule. In some embodiments, the priming site is a self-priming loop. In some embodiments, the method further comprises incorporating the individual nucleotides using a polymerase. In some embodiments, a given sensor comprises at least two electrodes.

In some embodiments, at least another subset of the individual nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule. In some embodiments, the reversible terminator is removed after an individual nucleotide is incorporated into the second single-stranded nucleic acid molecule and before another individual nucleotide is incorporated into the second single-stranded nucleic acid molecule.

In some embodiments, the detectable label is an electrostatic moiety. In some embodiments, a detectable label is coupled to nucleobases of at least a subset of the individual nucleotides. In some embodiments, the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a single type of detectable label. In some embodiments, the individual nucleotides comprise different types of nucleotides, each different type of nucleotide being reversibly coupled to a different type of detectable label. In some embodiments, the detectable label is reversibly coupled to the different types of nucleotides by one or more coupling mechanisms. In some embodiments, the detectable label is reversibly coupled to the different types of nucleotides by a single coupling mechanism. In some embodiments, the detectable label is removed after detection of the signal indicative of incorporation of the individual nucleotide.

In some embodiments, the individual nucleotides comprise different types of nucleotides, and the first single-stranded nucleic acid molecule is sequentially contacted with the different types of nucleotides. In some embodiments, a first single-stranded nucleic acid molecule is contacted with a first type of individual nucleotides at a given point in time during a nucleic acid incorporation reaction, and the first single-stranded nucleic acid molecule is contacted with a second type of individual nucleotides at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type. In some embodiments, the individual nucleotides comprise different types of nucleotides, and the first single-stranded nucleic acid molecule is contacted with the different types of nucleotides simultaneously.

In some embodiments, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

In some embodiments, the plurality of single-stranded nucleic acid molecules is a clonal population of first single-stranded nucleic acid molecules. In some embodiments, the first single-stranded nucleic acid molecule comprises a self-priming loop. In some embodiments, the method is repeated until the sequence of the first single-stranded nucleic acid molecule is determined.

In another aspect, the present disclosure provides a method for detecting a nucleic acid molecule, comprising: providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, wherein a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array; subjecting the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule that is a growing strand complementary to the first single-stranded nucleic acid molecule, wherein the nucleic acid incorporation reaction comprises alternately and sequentially (i) incorporating individual nucleotides of a first plurality of nucleotides that comprise a detectable label, and (ii) incorporating individual nucleotides of a second plurality of nucleotides that do not comprise a detectable label; and detecting a signal indicative of a change in charge or conductivity from the bilayer comprising the detectable label using a given sensor, simultaneously with or after performing the nucleic acid incorporation reaction, thereby determining the sequence and/or length of the first single-stranded nucleic acid molecule.

In some embodiments, the first plurality of nucleotides comprises a terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule. In some embodiments, the first plurality of nucleotides comprises dideoxynucleotides. In some embodiments, the second plurality of nucleotides comprises a reversible terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid. In some embodiments, the reversible terminator is removed after individual nucleotides in the first plurality of nucleotides are exchanged with individual nucleotides in the second plurality of nucleotides.

In some embodiments, the first plurality of nucleotides is exchanged with the second plurality of nucleotides. In some embodiments, the incorporation of the second plurality of nucleotides corrects for phase error by incorporating individual nucleotides from the second plurality of nucleotides at positions along the first single-stranded nucleic acid molecule where individual nucleotides from the first plurality of nucleotides are not incorporated. In some embodiments, the method further comprises continuing the nucleic acid incorporation reaction using individual nucleotides from the first plurality of nucleotides.

In some embodiments, the detectable label is not removable. In some embodiments, the detectable label is an electrostatic moiety. In some embodiments, the detectable label is coupled to a nucleobase of an individual nucleotide of the first plurality of nucleotides. In some embodiments, individual nucleotides in the first plurality of nucleotides comprise different types of nucleotides, each different type of nucleotide coupled to a single type of detectable label. In some embodiments, individual nucleotides in the first plurality of nucleotides comprise different types of nucleotides, each different type of nucleotide coupled to a different type of detectable label.

In some embodiments, a given sensor is electrically coupled to a charge bilayer comprising a first single-stranded nucleic acid molecule. In some embodiments, a plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charged bilayer is adjacent to a surface of the given bead. In some embodiments, the plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array. In some embodiments, the first single-stranded nucleic acid molecule is coupled to a surface of a given sensor, and the charge bilayer is adjacent to the surface.

In some embodiments, the method further comprises providing a priming site adjacent to the first single-stranded nucleic acid and generating a second single-stranded nucleic acid molecule upon extension of the primer from the priming site. In some embodiments, the priming site is a primer sequence having sequence complementarity to the first single-stranded nucleic acid molecule. In some embodiments, the priming site is a self-priming loop. In some embodiments, the method further comprises incorporating the individual nucleotides using a polymerase.

In some embodiments, a given sensor comprises at least two electrodes. In some embodiments, the individual nucleotides comprise different types of nucleotides, and the first single-stranded nucleic acid molecule is sequentially contacted with the different types of nucleotides. In some embodiments, a first single-stranded nucleic acid molecule is contacted with a first type of individual nucleotides at a given point in time during a nucleic acid incorporation reaction, and a segment is contacted with a second type of individual nucleotides at a subsequent point in time during the nucleic acid incorporation reaction, wherein the first type is different from the second type. In some embodiments, the individual nucleotides comprise different types of nucleotides, and the first single-stranded nucleic acid molecule is contacted with the different types of nucleotides simultaneously.

In some embodiments, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

In some embodiments, the plurality of single-stranded nucleic acid molecules is a clonal population of first single-stranded nucleic acid molecules. In some embodiments, the method is repeated until the sequence of the first single-stranded nucleic acid molecule is determined. In some embodiments, the first single-stranded nucleic acid molecule is part of a plurality of single-stranded nucleic acid molecules adjacent to the given sensor, wherein individual single-stranded nucleic acid molecules of the plurality of single-stranded nucleic acid molecules, including the first single-stranded nucleic acid molecule, have sequence homology to the template single-stranded nucleic acid molecule.

In another aspect, the present disclosure provides a system for detecting a nucleic acid molecule, comprising: a sensor array comprising a plurality of sensors, wherein during use a given double-stranded nucleic acid molecule of the plurality of double-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given double-stranded nucleic acid molecule comprises a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule having sequence complementarity to the first single-stranded nucleic acid molecule, wherein the given sensor is electrically coupled to a charge bilayer comprising the given double-stranded nucleic acid molecule; and one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact a segment of the first single-stranded nucleic acid molecule that is not hybridized to the second single-stranded nucleic acid molecule with an individual nucleotide, such that the segment undergoes a nucleic acid incorporation reaction that generates a third single-stranded nucleic acid molecule from the individual nucleotide, wherein the third single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, and (ii) detect, with a given sensor, a signal indicative of incorporation of the individual nucleotide into the third single-stranded nucleic acid molecule, concurrent with or subsequent to the nucleic acid incorporation reaction, thereby determining the sequence and/or length of the segment.

In some embodiments, during use, a plurality of double stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, during use, a given double-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charge bilayer is adjacent to a surface of the given bead. In some embodiments, during use, a plurality of double-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array. In some embodiments, during use, a given double-stranded nucleic acid molecule is coupled to a surface of a given sensor, and a charge bilayer is adjacent to the surface. In some embodiments, a given sensor comprises at least two electrodes.

In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, individual nucleotides incorporate a detectable label. In some embodiments, the detectable label is an electrostatic moiety. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

In another aspect, the present disclosure provides a system for detecting a nucleic acid molecule, comprising: a sensor array comprising a plurality of sensors, wherein during use a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array, wherein the given sensor is electrically coupled to a charge bilayer comprising the first single-stranded nucleic acid molecule; and one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact the first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides, wherein the second single-stranded nucleic acid molecule has sequence complementarity with the first single-stranded nucleic acid molecule, wherein at least a subset of the individual nucleotides comprise a detectable label, and (ii) simultaneously with or subsequent to performing the nucleic acid incorporation reaction, detect a signal from the detectable label using a given sensor, the signal indicating incorporation of the individual nucleotide into the second single-stranded nucleic acid molecule, thereby determining the sequence and/or length of the first single-stranded nucleic acid molecule.

In some embodiments, during use, a plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, during use, the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charge bilayer is adjacent to a surface of the given bead. In some embodiments, during use, a plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array. In some embodiments, during use, the first single-stranded nucleic acid molecule is coupled to a surface of a given sensor, and the charge bilayer is adjacent to the surface. In some embodiments, a given sensor comprises at least two electrodes. In some embodiments, the detectable label is an electrostatic moiety.

In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

In another aspect, the present disclosure provides a system for detecting a nucleic acid molecule, comprising: a sensor array comprising a plurality of sensors, wherein during use a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules is positioned adjacent to a given sensor of the sensor array; and one or more computer processors operatively coupled to the sensor array, wherein the one or more computer processors are individually or collectively programmed to (i) contact the first single-stranded nucleic acid molecule with an individual nucleotide, such that the first single-stranded nucleic acid molecule undergoes a nucleic acid incorporation reaction to produce a second single-stranded nucleic acid molecule, wherein the nucleic acid incorporation reaction comprises alternately and sequentially incorporating individual nucleotides in a first plurality of nucleotides comprising a detectable label and exchanging individual nucleotides in the first plurality of nucleotides with individual nucleotides in a second plurality of nucleotides not comprising a detectable label, and (ii) detecting a signal indicative of a change in charge or conductivity from the bilayer comprising the detectable label using a given sensor, simultaneously with or subsequent to performing the nucleic acid incorporation reaction, thereby determining the sequence and/or length of the first single-stranded nucleic acid molecule.

In some embodiments, during use, a given sensor is electrically coupled to a charge bilayer comprising a first single-stranded nucleic acid molecule. In some embodiments, during use, a plurality of single-stranded nucleic acid molecules are coupled to a plurality of beads. In some embodiments, during use, the first single-stranded nucleic acid molecule is coupled to a given bead of the plurality of beads, and the charge bilayer is adjacent to a surface of the given bead. In some embodiments, during use, a plurality of single-stranded nucleic acid molecules are coupled to one or more surfaces of the sensor array. In some embodiments, during use, the first single-stranded nucleic acid molecule is coupled to a surface of a given sensor, and the charge bilayer is adjacent to the surface. In some embodiments, a given sensor comprises at least two electrodes. In some embodiments, the detectable label is an electrostatic moiety.

In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a steady state signal. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected once after incorporation of the individual nucleotide. In some embodiments, the signal indicative of incorporation of an individual nucleotide is detected at least twice after incorporation of the individual nucleotide. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is a transient signal. In some embodiments, during use, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in the charge bilayer.

Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the disclosure is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"); in the drawings:

FIG. 1 shows a schematic representation of an unstructured template nucleic acid molecule coupled to a bead;

FIG. 2 shows a schematic representation of a structured template nucleic acid molecule coupled to a bead;

FIG. 3 shows an example process flow for relaxed template sequencing;

FIGS. 4A-4D show examples of double-stranded sequencing methods and sequencing results using labeled and unlabeled nucleotides; FIG. 4A shows an exemplary comparison between single-stranded sequencing results and double-stranded sequencing results; FIG. 4B shows exemplary sequencing results of double-stranded sequencing using polyanionic electrostatic moieties; FIG. 4C shows exemplary sequencing results of double-stranded sequencing using polycationic electrostatic moieties; FIG. 4D shows example sequencing results of double-stranded sequencing using both a polyanionic electrostatic moiety and a polycationic electrostatic moiety;

FIG. 5 shows an exemplary method of double-strand sequencing;

FIG. 6 shows an example method of double strand sequencing using random hexamers;

FIGS. 7A and 7B illustrate an exemplary method of sequencing a duplex with a reversible terminator; FIG. 7A shows an example method of double-stranded sequencing using a reversible terminator and a flanking endonuclease; FIG. 7B shows an example method of double-stranded sequencing using a reversible terminator and a nucleic acid subunit;

FIG. 8 shows an example sequencing method using different types of electrostatic moieties for each type of nucleotide;

FIG. 9 shows an example sequencing method using a single type of electrostatic moiety for each type of nucleotide;

FIG. 10 shows an example method of sequencing using an electrostatic moiety and a reversible terminator;

FIGS. 11A and 11B illustrate an example method of double-stranded sequencing using a detectable label on a second single-stranded nucleic acid molecule; FIG. 11A shows an exemplary sequencing method using a detectable label cleaved by a flanking endonuclease; FIG. 11B shows an exemplary sequencing method using a detectable label and a reversible terminator;

FIGS. 12A and 12B illustrate an example method of double-stranded sequencing using a detectable label and a flanking endonuclease; FIG. 12A shows an example method of double-stranded sequencing using a detectable label and a mesophilic flanking endonuclease; FIG. 12B shows an example method of double-stranded sequencing using a detectable label and a thermostable flanking endonuclease;

FIGS. 13A and 13B illustrate an example method of double-stranded sequencing using a detectable label, a flanking endonuclease, and a reversible terminator; FIG. 13A shows an exemplary method of double-strand sequencing using a detectable label, a mesophilic flanking endonuclease, and a reversible terminator; FIG. 13B shows an example method of double-strand sequencing using a detectable label, a thermostable flanking endonuclease, and a reversible terminator;

FIG. 14A shows an example method of double-stranded sequencing using a detectable label and nucleic acid subunits;

FIG. 14B shows an example method of double-strand sequencing using a detectable label, a nucleic acid subunit, and a reversible terminator;

FIG. 15 shows an example method for pyrophosphorolysis-mediated terminator exchange sequencing;

FIG. 16 illustrates a computer system programmed or otherwise configured to implement the methods provided herein;

figure 17 shows an example of a modified nucleotide comprising a detectable label or effector coupled to a nucleobase by a linker;

18A-18C show examples of detectable labels; figure 18A shows an example of a polycationic electrostatic moiety having lysine residues; fig. 18B shows an example of a polyanion electrostatic moiety having a carboxylic acid group; fig. 18C shows an example of a switch label comprising a histidine imidazole residue that can switch between a neutral state and a positive charge state in response to the pH of a buffer;

FIG. 19 shows the activity of the polymerase at different salt concentrations and in the presence or absence of polyethylene glycol (PEG);

FIG. 20 illustrates a method for correcting phase errors during a sequencing reaction;

FIG. 21 is an example of a method for correcting phase errors; and is

Fig. 22 is another example of a method for correcting a phase error.

Detailed Description

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term "adjacent" as used herein generally refers to being immediately adjacent, proximate to, or within a sensing or electronic range (or distance). For example, a first object that is adjacent to a second object may be in contact with the second object, or may not be in contact with the second object but may be proximate to the second object. In some examples, the first to second objects are within about 0 microns ("millionths of a meter"), 0.001 microns, 0.01 microns, 0.1 microns, 0.2 microns, 0.3 microns, 0.4 microns, 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 10 microns, or 100 microns of the second object.

As used herein, the term "nucleic acid" generally refers to a molecule comprising one or more nucleic acid subunits. The nucleic acid may comprise one or more subunits selected from adenosine (a), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. Nucleotides may include A, C, G, T or U or variants thereof. The nucleotide may comprise any subunit that can be incorporated into a growing nucleic acid strand. Such a subunit may be A, C, G, T or U, or any other subunit specific for one or more complementary A, C, G, T or U, or complementary to a purine (i.e., a or G or variant thereof) or pyrimidine (i.e., C, T or U or variant thereof). In some examples, the nucleic acid may be single-stranded or double-stranded, in some cases the nucleic acid molecule is circular.

As used herein, the terms "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" generally refer to a polymeric form of nucleotides that can be of various lengths, i.e., Deoxyribonucleotides (DNA) or Ribonucleotides (RNA) or analogs thereof. Oligonucleotides are generally composed of a specific sequence of the following four nucleotide bases: adenine (a); cytosine (C); guanine (G); and thymine (T) (in the case of RNA, uracil (U) replaces thymine (T)). Thus, the term "oligonucleotide sequence" is an alphabetical representation of a polynucleotide molecule; alternatively, the term may apply to the polynucleotide molecule itself. The letter representation can be entered into a database of a computer having a central processor unit and used for bioinformatics applications such as functional genomics and homology searches. The oligonucleotide may comprise one or more non-standard nucleotides, nucleotide analogs, and/or modified nucleotides. In some cases, an oligonucleotide may refer to a shorter single-stranded nucleic acid sequence having at most 300 base pairs (bp), at most 200bp, at most 100bp, at most 90bp, at most 80bp, at most 70bp, at most 60bp, at most 50bp, at most 40bp, at most 30bp, at most 20bp, at most 10bp, or fewer base pairs. In some cases, the oligonucleotide may have-C6-NH at its 3 'or 5' end suitable for conjugation₂A functional group.

Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-galactosyltetraoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylstevioside, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine (wybutoxosine), pseudouracil, stevioside, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2, 6-diaminopurine, and the like. Nucleic acid molecules can also be modified at the base moiety (e.g., at one or more atoms that can typically form hydrogen bonds with a complementary nucleotide and/or at one or more atoms that cannot typically form hydrogen bonds with a complementary nucleotide), the sugar moiety, or the phosphate backbone. The nucleic acid molecule may also contain amine-modifying groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP), to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). In the oligonucleotides of the present disclosure, alternatives to standard DNA base pairs or RNA base pairs may provide higher density (bits per cubic millimeter), higher safety (resistance to accidental or purposeful synthesis of natural toxins), easier differentiation in a photo-programmed polymerase, or less secondary structure. Alternative base pairs compatible with natural and mutant polymerases for de novo synthesis and/or amplification synthesis are described in Betz K, Malyshiev D A, Lavergne T, Welte W, Diederich K, Dwyer T J, Ordoukhanian P, Romesberg F E, Marx A (2012).

As used herein, the term "nucleotide" generally refers to an organic molecule that functions as a monomer or subunit of a nucleic acid molecule, such as a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. In some embodiments, the nucleotides may also be Peptide Nucleic Acid (PNA) nucleotides, Locked Nucleic Acid (LNA) nucleotides, or dideoxynucleotides.

As used herein, the term "primer" generally refers to a nucleic acid strand that serves as a starting point for nucleic acid synthesis, such as Polymerase Chain Reaction (PCR). In one example, during replication of a DNA sample, an enzyme that catalyzes replication begins to replicate at the 3' end of the primer attached to the DNA sample and replicates the opposite strand.

As used herein, the term "polymerization enzyme" generally refers to any enzyme capable of catalyzing a polymerization reaction. Examples of polymerases include, but are not limited to, nucleic acid polymerases. The polymerase may be naturally occurring or synthetic. An exemplary polymerase is Φ 29 polymerase or a derivative thereof. The polymerase may be a polymerization enzyme. In some cases, a transcriptase or ligase (i.e., an enzyme that catalyzes the formation of a bond) is used. Examples of polymerases include DNA and RNA polymerases, thermostable polymerases, wild-type polymerases, modified polymerases, E.coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase Φ 29(phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, Ex-Taq polymerase, LA-Taw polymerase, Sso polymerase Poc polymerase, Pab polymerase, mth polymerase ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment polymerase with 3 'to 5' exonuclease activity, or variants, modifications, and derivatives thereof. In some embodiments, the polymerase is a single subunit polymerase. The polymerase may have a high persistence, i.e., the ability of the polymerase to persist nucleotides into the nucleic acid template without releasing the nucleic acid template.

The term "detectable label" as used herein generally refers to any detectable moiety coupled to the molecule to be detected. Non-limiting examples of detectable labels may include electrostatic moieties, fluorescent moieties, chemiluminescent moieties, radioactive moieties, colorimetric moieties, or any combination thereof. The detectable label may be reversibly or irreversibly coupled to the molecule to be detected. Such a portion may be a mark. Examples of electrostatic moieties include charge labels. A detectable label may be coupled to the nucleobase at the C5 or C7 position. For example, a reversible electrostatic moiety may be coupled to a nucleotide incorporated into a nucleic acid molecule.

The detectable label may be coupled to the nucleobase via a linker. The linker may be coupled to the nucleobase at the C5 or C7 position. The linker may be a non-nucleotide molecule. The linker may be acid labile, photolabile or contain a disulfide bond. The linker may hold the detectable label at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and the enzyme. In some examples, the detectable linker is at a distance of at least about 1 nanometer (nm), 2nm, 3nm, 4nm, 5nm, 10nm, 20nm, 30nm, 40nm, 50nm, 100nm, 200nm, 300nm, 400nm, 500nm, or more from the nucleotide. Figure 17 shows an example of a modified nucleotide with a detectable label coupled to the nucleotide by a linker. In this example, the detectable label may also be referred to as an effector molecule, since it may affect the charge distribution around the nucleotide.

As used herein, the term "electrostatic moiety" generally refers to a detectable label comprising a net positive or negative charge, or to a moiety attached to a chemical or biological unit to render the chemical or biological unit detectable. For example, the electrostatic moiety may comprise a charged functional group, a portion of a functional group having a charge, a charge label, or a charged molecule that is a detectable label. The electrostatic moiety may be monovalent (e.g., having a charge of +1 or-1) or multivalent (e.g., having a charge of +2, +3, +4, +5, +6, etc., or-1, -2, -3, -4, -5, -6, etc.). The electrostatic moiety may have a net positive or negative charge. The electrostatic moiety may have one or more anionic or cationic charge groups. In one example, the electrostatic moiety has both an anionically charged group and a cationically charged group and a net positive or negative charge. In another example, the electrostatic moiety is not a zwitterion. The electrostatic moiety may have a constant net charge or may change charge. In one example, the electrostatic moiety switches or changes charge depending on solution conditions (e.g., pH, temperature, etc.).

As used herein, the term "clone" generally means that at least some, substantially all, or all of a population of sensor regions have the same nucleic acid sequence. There may be two populations associated with a single sample nucleic acid fragment that can be used for "pairing", "paired ends", or other similar methods; the populations may be present in substantially similar numbers within the sensor area and may be randomly distributed across the sensor area.

As used herein, the term "phase error" generally refers to the error or difference between a given polynucleotide sequence (e.g., a second or third single-stranded nucleic acid molecule) and the template nucleic acid molecule from which the given polynucleotide sequence was derived. A given polynucleotide sequence may be part of a clonal population, and a given nucleotide sequence may have a longer or shorter sequence than the consensus state (e.g., a reference sequence) of the clonal population. The phase error may be a leading or lagging phase error. Lead phase error may include additional nucleotide bases not present in the consensus (e.g., reference) sequence. The lag phase error may include fewer nucleotide bases relative to a consensus (e.g., reference) sequence. The phase error may be a product of misincorporation of nucleotide bases by the polymerase or insufficient nucleotide base incorporation. Phase errors may limit the read length of the sequencing system.

As used herein, the term "flanking" generally refers to the portion of a single-stranded nucleic acid molecule that does not hybridize or associate with another single-stranded nucleic acid molecule where a portion of the single-stranded nucleic acid molecule hybridizes or associates with the other single-stranded nucleic acid molecule. The flap can be at least about 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 30, 40, 50, or more nucleotide bases in length.

Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first of a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

Whenever the term "no more than," "less than," or "less than or equal to" precedes the first of a series of two or more values, the term "no more than," "less than," or "less than or equal to" applies to each value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

Method for nucleic acid sequencing

In one aspect, the present disclosure provides a method for nucleic acid sequencing. The method can include providing a plurality of double stranded nucleic acid molecules adjacent to a sensor array. A given or individual double stranded nucleic acid molecule can be placed adjacent to a given or individual sensor of a sensor array. The double-stranded nucleic acid molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule. The first and second single stranded nucleic acid molecules can have sequence complementarity with each other. The sensor can be electrically coupled to a charge bilayer (e.g., within the debye length) of the double-stranded nucleic acid molecule. A portion of the second single-stranded nucleic acid molecule can be released from the first single-stranded nucleic acid molecule to provide a segment of the first single-stranded nucleic acid molecule that does not hybridize to the second single-stranded nucleic acid molecule. The segment can be contacted with the individual nucleotide. Individual nucleotides can be subjected to a nucleic acid incorporation reaction that generates a third single-stranded nucleic acid molecule. The third single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. During the nucleic acid incorporation reaction, the sensor can be used to detect a signal indicative of the incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule, thereby determining the sequence or length of the non-hybridizing segment.

In another aspect, the present disclosure can provide a method for detecting a nucleic acid molecule. The method may include providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, contacting a first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides, and, while or after performing the nucleic acid incorporation reaction, detecting a signal from a detectable label with a given sensor, the signal indicating incorporation of the individual nucleotides into the second single-stranded nucleic acid molecule, thereby determining the sequence and/or length of the first single-stranded nucleic acid molecule. A first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. A given sensor may be electrically coupled to a charge bilayer (e.g., within the debye length) of a first single-stranded nucleic acid molecule. The second single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. At least a subset of the individual nucleotides may comprise a detectable label.

In another aspect, the present disclosure may provide a method for nucleic acid sequencing. The method can include providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, subjecting a first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule as a growing strand complementary to the first single-stranded nucleic acid molecule, and, while or after performing the nucleic acid incorporation reaction, detecting a signal from a detectable label with a given sensor, the signal indicating incorporation of individual nucleotides of the first plurality of nucleotides into the second single-stranded nucleic acid molecule, thereby determining the sequence or length of the first single-stranded nucleic acid molecule. A first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. The nucleic acid incorporation reaction can include alternately and sequentially (i) incorporation of individual nucleotides in a first plurality of nucleotides comprising a detectable label, and (ii) incorporation of individual nucleotides in a second plurality of nucleotides that do not comprise a detectable label.

The systems and methods described herein can be used to detect biomolecules and reactions. For example, the systems and methods described can be used to detect the presence or absence of binding events, reactions, and reaction products and/or biomolecules. In one example, the systems and methods can be used to determine the sequence of a nucleic acid molecule. In another example, the systems and methods can be used to determine the length (e.g., number of nucleotides) of a nucleic acid molecule. In one example, the systems and methods can be used to determine the sequence and length of a target nucleic acid molecule. The systems and methods can be used to detect nucleic acid polymorphisms such as, but not limited to, misincorporated nucleotides, altered fragment sizes, repeated nucleotide sequences, and/or deleted nucleotide sequences. Determining the length of a nucleic acid molecule can have applications for healthcare, such as for diagnostics (e.g., cancer detection). For example, the system and method may detect microsatellite instability by detecting increases in segment length.

Sequencing or determining the length of a nucleic acid molecule may utilize nucleic acid templates that are free in solution or coupled to a support. The support may comprise beads, flat surfaces, wells or any other structure capable of coupling to nucleic acid molecules. The support may be positioned adjacent to the sensors of the sensor array. Alternatively or additionally, the support may be part of a sensor (e.g., an electrode, a passivation layer, a dielectric layer, etc.) in a sensor array. Nucleic acid templates coupled to a support may be unstructured (e.g., extend linearly from the surface of the support) or may be structured (e.g., form loops, hairpins, and/or other secondary structures). FIG. 1 shows an example of an unstructured template nucleic acid coupled to a bead. The beads may be coupled to a single nucleic acid template or multiple nucleic acid templates. The unstructured templates may not interact with each other around the surface of the bead. Alternatively or additionally, the unstructured nucleic acid templates may interact with each other around the surface of the bead. Non-interacting nucleic acid templates can produce a monotonic signal during the sequencing process (e.g., each nucleotide incorporated generates a constant signal). FIG. 2 shows an example of a structured template nucleic acid coupled to a bead. The nucleic acid template may interact with itself to form loops, hairpins, and/or other secondary structures. The bead may have a single nucleic acid template or multiple nucleic acid templates coupled thereto. In one example, the bead is coupled to a plurality of nucleic acid templates, and the nucleic acid templates can interact with each other. Nucleic acid templates that interact with each other can generate non-monotonic signals during sequencing (e.g., each nucleotide incorporated generates a different non-linear signal).

The structured nucleic acid template may be unstructured or relaxed prior to sequencing to generate a monotonic signal. The template structure may be relaxed prior to nucleotide incorporation (e.g., primer extension reaction) or prior to reading or detecting the incorporation event. Figure 3 shows an example method for relaxed template sequencing. The structured template may include random coil, secondary structure, and/or hairpin. In one example, the template includes a self-initiating loop. The self-priming loop may be a hairpin structure that allows extension of a single-stranded nucleic acid structure without the need for a separate primer sequence. In the structured state, self-priming loops can be arranged by loop-mediated amplification (LAMP) to facilitate the primer extension reaction. The self priming loop facilitates incorporation of nucleotides into the 3' end of the nucleic acid template. Alternatively or additionally, the self-priming loop may incorporate nucleotides into the 5' end of the nucleic acid template. After incorporation of the nucleotide, the structure of the nucleic acid template may be relaxed. The template structure may be relaxed by changing the solution conditions, including but not limited to applying heat, changing the pH, changing the ionic strength, and/or introducing one or more organic solvents (e.g., formamide or urea) into the solution. The relaxed nucleic acid template can then be read to detect nucleotide incorporation. The detected signal may be a linear or monotonic signal.

Signal linearity can be increased using double-stranded sequencing. Double-stranded sequencing can include double-stranded nucleic acid templates free in solution or coupled to a support. The double-stranded nucleic acid template may have a secondary structure, such as a double-helical structure. The duplex structure may reduce or prevent interaction between double stranded nucleic acid templates coupled to the same support. Reducing or preventing interaction between double-stranded nucleic acid templates can increase the linearity of the signal detected during sequencing. Furthermore, combining double-stranded sequencing with nucleotides comprising a detectable label can increase both linearity and signal-to-noise ratio. FIGS. 4A-4D show an example of a double-stranded sequencing method and an example of sequencing results using labeled and unlabeled nucleotides; fig. 4A shows an example comparison between single stranded 401 and double stranded sequencing results 402. The example of single strand sequencing 401 shows signals that are both positive and negative with respect to the y-axis of the graph and that do not vary monotonically. The example of double-stranded sequencing 402 shows a signal that is positive with respect to the y-axis of the graph and varies monotonically with the number of incorporated nucleotides. Fig. 4B shows example sequencing results of double-stranded sequencing using polyanionic electrostatic moieties. The polyanionic electrostatic moiety can include one or more of phosphate, phosphonate, sulfate, sulfonate, borate, or carboxylate groups. In this example, the detected signal is positive and monotonic with respect to the y-axis. Further, the detected signal may be outside of the detectable signal noise (e.g., have a high signal-to-noise ratio). Figure 4C shows example sequencing results of double-stranded sequencing using polycationic electrostatic moieties. The polycationic electrostatic moiety can include one or more of pyridinium, imidazolium, guanidinium, iminium, primary amine, secondary amine, tertiary amine, or quaternary ammonium. Like polyanionic electrostatic moieties, polycationic electrostatic moieties can generate signals in addition to detectable signal noise. However, the polycationic electrostatic moiety may generate a negative signal or a signal that is opposite to the signal generated by the polyanionic electrostatic moiety. Fig. 4D shows example sequencing results of double-stranded sequencing using both polyanionic and polycationic electrostatic moieties. The polyanionic electrostatic moiety and polycationic electrostatic moiety can generate detectable signals that are in addition to the detectable signal noise and that are both positive and negative (e.g., have opposite signal directions from each other).

Polycationic electrostatic moieties can be used to improve signal to noise ratio, such as during sequencing. Detectable labels, such as polycationic or polyanionic electrostatic moieties, can be used to generate a monotonic signal, i.e., a linear signal, as compared to the signal from an unmodified nucleotide. The linear signal may be due to a structural transition of the nucleic acid molecule caused by the detectable label. This structural transformation can result in a change in the distribution of ions around the nucleic acid molecule, thereby producing a signal with the same amplitude as the signal generated by the incorporation of a single nucleotide.

The polycationic electrostatic moiety may comprise amine groups or amino acid residues, such as lysine, histidine, arginine, or any combination thereof. The polycationic electrostatic moiety can displace or repel other polycations, such as magnesium ions (Mg), from the vicinity of the nucleic acid molecule^{2 +}). Displacement of other polycations may result in a lower conduction current that can be detected by the sensor as a negative signal. Polyanionic electrostatic moieties such as carboxylic acid groups can attract or concentrate polycations, such as Mg, around nucleic acid molecules²⁺. The detectable label may comprise a charged group. The detectable label may be monovalent (e.g., having a single positive or negative charge, such as, for example, +1 or-1) or multivalent (e.g., having multiplePositive or negative charge, such as +2 or-2, for example). A detectable label, such as a polycationic or polyanionic detectable label, may have a positive or negative charge of about one to about fifty or more. In some cases, a detectable label can have greater than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, or more charge groups. The detectable label may comprise about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50 charge groups. In one example, the detectable label can be a polycationic electrostatic moiety comprising three lysine residues, six lysine residues, or more than six lysine residues. In another example, the detectable label can be a polyanionic electrostatic moiety comprising three carboxylic acid groups, six carboxylic acid groups, or more than six carboxylic acid groups. Higher concentrations of polycationic electrostatic moieties can result in higher conduction currents, which can be detected by the sensor as a positive signal. The number of polycations or polyanions in the detectable label can be correlated to the intensity of the signal detected by the sensor. For example, a detectable label with six lysine residues (e.g., a K6 label) may produce a stronger negative signal than a detectable label with three lysine groups (i.e., a K3 label). Similarly, six carboxylic acid groups can produce a stronger positive signal than three carboxylic acid groups in the detectable label. The higher charge state of the detectable label may result in stronger non-specific binding to a surface such as glassware. For example, the K6 label may have a higher charge state than the K3 label, and thus, the K6 label may have stronger non-specific binding than the K3 label. An example of a polycationic electrostatic moiety with lysine residues is shown in fig. 18A. An example of a polyanionic electrostatic moiety having a carboxylic acid group is shown in fig. 18B.

The detectable label can be switched between a charged state and a neutral state or between one charged state and another charged state (e.g., positive to negative or negative to positive). The detectable label may switch charge states in response to solution conditions (such as buffer conditions, e.g., such as the pH or ionic strength of the buffer). The switchable detectable label may be in a charged state during the nucleic acid incorporation reaction (e.g., during signal detection), and may be in a neutral state for the remainder of the time. In one example, nucleotide incorporation is detected during an incorporation event, and the detectable label can be charged during incorporation. In another example, nucleotide incorporation can be detected after nucleotide incorporation, and the detectable label can be uncharged during incorporation, but can be switched such that the detectable label is charged during detection. In another example, the detectable label has one charge (e.g., positive, negative, or neutral) during incorporation and converts to have another charge (e.g., negative or positive) during detection. The switch label can be used to reduce non-specific binding compared to a detectable label that remains charged throughout the process (e.g., a K6 label). An example of a histidine tag is shown in fig. 18C. As shown in fig. 18C, the switch label can comprise a histidine imidazole residue that can switch between a neutral state and a positive charge state in response to the pH of the buffer. Exemplary switch labels can include detectable labels having greater than or equal to 1, 2, 3, 4, 5, 6, 8, 10, 12, or more histidine groups. In one example, the detectable label has three histidine groups (e.g., H3), six histidine groups (e.g., H6), or more than six histidine groups. When the pH is equal to or greater than 7, the transition mark may be in a neutral state. When the pH is equal to or less than 5, the transition label may be in a positive charge state. When the switch label is in a neutral state, the label cannot bind non-specifically to the surface and may have greater mobility than the label in a positive charge state. The transition mark may be kept in a positive charge state during signal detection by the sensor in order to fix the mark. The switch label can remain in a neutral state when the nucleotide is directed toward and/or away from the target nucleic acid molecule (e.g., when the nucleotide is mobile within the system).

Non-specific binding of the detectable label can be reduced by changing the reaction conditions. For example, the K6 label can be used with high concentrations of low affinity peptides (such as the K3 label). In this case, the K6 label may exhibit reduced non-specific binding due to competition from the low affinity peptide for the binding surface. In some cases, non-specific binding can be reduced by using a buffer of high ionic strength. For example, a buffer with 200mM potassium chloride (KCl) can reduce non-specific binding of the K6 label, thereby making the K6 label mobile to keep the K6 label in solution.

The polymerase may be kinetically active under altered reaction conditions for use with a switch label and/or with a nucleotide comprising a detectable label. In some cases, the polymerase may be selected based on kinetic activity and/or compatibility with a detectable label. For example, a type B polymerase with a large binding pocket (such as 9 ° N, RB69, KOD polymerase) can be used with a large detectable label. In some cases, a high ionic strength buffer tolerant polymerase (such as type B polymerase, thermoator IX, Bst 3.0, Φ 29, Taq polymerase) may be used with a high salt buffer and a polycationic electrostatic moiety (such as the K6 label). Tolerance of the polymerase can be improved by the addition of a volume exclusion agent such as, for example, polyethylene glycol (PEG), dextran, or similar compounds. As shown in fig. 19, the polymerase may exhibit improved salt tolerance during the nucleic acid incorporation reaction in the presence of PEG. The template may be coupled to a bead. The primer may be complementary to the 3' end of the template strand. The primers may be fluorescently labeled with a 6-FAM fluorophore and extended by incorporation of individual nucleotides. The primer extension reaction can be detected by a sensor.

The primer extension reaction may be facilitated by a polymerase (such as, for example, a thermostable polymerase). Examples of polymerases that can be used for the extension reaction include, but are not limited to, Thermus thermophilus (Thermus thermophilus) HB8, Thermus ohydralis (Thermus osimai) mutants, Thermus nigricans (Thermus scotoductus); thermus thermophilus 1B21, Thermus thermophilus GK24, Thermus aquaticus polymerase(s) ((II))FS or Taq (G46D, F667Y), Taq (G46D, F667Y, E6811) and Taq (G46D, F667Y, T664N, R660G), Pyrococcus furiosus (Pyrococcus furiosus) polymerase, Pyrococcus serpentis (Thermococcus gorgonnarius) polymerase, Pyrococcus GB-D polymerase, Pyrococcus (9 ℃ N-7 strain) polymerase, Bacillus stearothermophilus (Bacillus stearothermophilus) polymerase (Bst), Bacillus caldotena (Bacillus caldotenax) DNA polymerase (Bca) Tsp polymerase, ThermalAce (ThermalAce) Tsp polymerase^TMPolymerase (Invitrogen), Thermus flavus polymerase, Thermus litoralis polymerase, Thermus Z05 polymerase, Z05 polymerase (e.g., Z05 Gold DNA polymerase), Sulfolobus (Sulfolobus) DNA polymerase IV, or mutants, variants, or derivatives thereof. Other examples of polymerases that can be used for the primer extension reaction are non-thermostable polymerases, including but not limited to DNA polymerase I, DNA polymerase I mutants, including but not limited to Klenow and Klenow fragments (no 3 'to 5' exonuclease activity), T4 DNA polymerase, T4 DNA polymerase mutant, T7 DNA polymerase, T7 DNA polymerase mutant, phi29 DNA polymerase, and phi29 DNA polymerase mutant.

In some examples, the primer extension reaction can be performed at various salt concentrations, such as three salt concentrations (about 0mM, 100mM, and 200mM) and in the presence or absence of PEG. For example, one set of experiments may be performed with PEG, and another set of experiments may be performed without PEG. FIG. 19 shows exemplary results of primer extension reactions with different types of polymerizers at various salt and PEG concentrations. When the buffer lacks KCl (e.g., with 0mM KCl), bst2.0 polymerase can incorporate nucleotides, whether PEG is present (e.g., + PEG) or absent (e.g., -PEG), as indicated by the peaks that are present in both the case of + PEG and-PEG. When the buffer lacks KCl (e.g., with 0mM KCl), the TIX polymerase cannot incorporate nucleotides, either in the presence or absence of PEG, as shown by the absence of peaks in the case of + PEG and-PEG. When the buffer contains 100mM KCl, both polymerases can incorporate nucleotides, whether in the presence or absence of PEG, as shown by the peaks that are present in both the case of + PEG and-PEG. When the buffer contained 200mM KCl, both polymerases could incorporate nucleotides in the presence of PEG, as shown by the peak in + PEG, but could not incorporate nucleotides in the absence of PEG.

In some cases, polycations (e.g., Mg) can be improved by inclusion²⁺，Ca²⁺，Zn²⁺) The resulting molecules that conduct current improve the signal-to-noise ratio. Such molecules may associate with polycations that can lead to an increase in conduction current. Non-limiting examples of molecules that can improve conduction current include, but are not limited to, phosphodiester backbones of nucleic acid molecules (e.g., dT3, dT6, dT12, etc.), carboxyglutamic acid Gla (e.g., gamma-carboxyglutamic acid Gla3, Gla6, Gla12, etc.), specific peptides (e.g., peptides having the sequences DIETDIET, fdgdfdfdgd, and/or stllpp), or small molecules (e.g., pyridine, NTA, IDA, or phosphine).

The target nucleic acid molecule can be sequenced and/or the length of the target nucleic acid molecule can be determined. The target nucleic acid molecule can be a fragmented nucleic acid molecule or can be a non-fragmented nucleic acid molecule. The target nucleic acid molecule can be amplified prior to detection. The target nucleic acid molecule can be amplified in solution and/or on a support. The target nucleic acid molecules amplified on the support can be immobilized on the support prior to amplification. The target nucleic acid molecule can be amplified by bridge amplification, wild fire (wild fire) amplification, recombinase polymerase amplification, isothermal amplification, or using any other amplification technique. Sequencing or determining the length of a target nucleic acid molecule can include providing a plurality of double stranded nucleic acid molecules adjacent to a sensor array. A given or individual double stranded nucleic acid molecule can be placed adjacent to a given or individual sensor of the sensor array. The double-stranded nucleic acid molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule. The first and second single stranded nucleic acid molecules can have sequence complementarity with each other. The sensor can be electrically coupled to a charge bilayer (e.g., within the debye length) of the double-stranded nucleic acid molecule. A portion of the second single-stranded nucleic acid molecule can be released from the first single-stranded nucleic acid molecule to provide a segment of the first single-stranded nucleic acid molecule that does not hybridize to the second single-stranded nucleic acid molecule. The segment can be contacted with the individual nucleotide. Individual nucleotides can be subjected to a nucleic acid incorporation reaction that generates a third single-stranded nucleic acid molecule. The third single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. During the nucleic acid incorporation reaction, the sensor can be used to detect a signal indicative of the incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule, thereby determining the sequence of the non-hybridizing segment.

The double stranded nucleic acid molecule may be coupled to a support. The support may be a bead or one or more surfaces of a sensor array. Multiple double-stranded nucleic acid molecules can be coupled to multiple beads or multiple locations on the surface of the sensor array. Each bead of the plurality of beads may be positioned adjacent to a given sensor. The charge bilayer (e.g., debye length) may be adjacent to the surface of the bead. Alternatively or additionally, a plurality of double stranded nucleic acid molecules may be coupled to one or more surfaces of the sensor array. A given double stranded nucleic acid molecule can be coupled to the surface of a given sensor. The charge bilayer (e.g., debye length) may be adjacent to the surface of a given sensor. Double-stranded nucleic acid molecules coupled to the support can be clonally amplified prior to sequencing to couple the support surface to a clonal population of double-stranded nucleic acid molecules.

A given sensor may comprise at least one, at least two, at least three or at least four electrodes or more electrodes. In one example, a given sensor contains at least two electrodes. In another example, a given sensor contains two electrodes. The electrode may be exposed to a solution in which a primer extension reaction takes place. Alternatively or additionally, the electrodes may be buried within the sensor array and may therefore not be exposed to the solution in which the primer extension reaction takes place. The electrodes of a given sensor can detect a signal indicative of the incorporation of an individual nucleotide into a double-stranded nucleic acid molecule. The signal indicative of an incorporation event may comprise a change in impedance, conductance, or charge in the electronic bilayer. In one example, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in a charge bilayer. The signal indicative of incorporation of an individual nucleotide may be a steady state signal, a transient signal, or a combination of steady state and transient signals. The signal may be detected instantaneously or under steady state conditions. In the transient signal detection mode, detection occurs during or immediately following nucleotide incorporation. In steady state detection, the reading of the sensor may occur after the incorporation event is complete. The steady state change in the signal may be constant until a change is introduced to the environment surrounding the sensor.

FIG. 5 shows an example method for double-stranded sequencing. The double-stranded nucleic acid template can have a uniform structure that produces a linear, substantially linear, or semi-linear response to charge changes due to nucleotide incorporation. The double-stranded nucleic acid can comprise a priming site adjacent to the 3' end of the first single-stranded nucleic acid (e.g., the nucleic acid template to be sequenced). Primer 503 may have complementarity to the 3 'end of the first single-stranded nucleic acid molecule and may hybridize to the 3' end of the first single-stranded nucleic acid molecule. Alternatively or additionally, the second double-stranded nucleic acid may be nicked to provide a primer and a strand to be displaced (e.g., a displaced strand). The second single-stranded nucleic acid may comprise a uracil nucleotide. The second single-stranded nucleic acid molecule may be nicked at the uracil nucleotide. The second single-stranded nucleic acid molecule can be nicked by any enzyme capable of cleaving uracil (e.g., uracil DNA glycosidase). The polymerase 502 can bind to double-stranded nucleic acids and facilitate the primer extension reaction. In one example, the polymerase 502 is a polymerase, such as Bst DNA polymerase. The primer extension reaction can displace the end of the second single-stranded nucleic acid and produce the single-stranded flap 505 and a segment of the first single-stranded nucleic acid molecule that does not hybridize to the second single-stranded nucleic acid molecule. A segment can be a portion of a first single-stranded nucleic acid molecule that does not hybridize to a second or third single-stranded nucleic acid molecule. The segment may not comprise the entire first single-stranded nucleic acid molecule. The length of the segment may be a single nucleotide or may be multiple nucleotides. The flap 505 may be a nucleotide coupled to the second single-stranded nucleic acid molecule but not hybridized to the first single-stranded nucleic acid molecule. Flap 505 may induce the slip of the polymerase 502 (stutter) and result in phasing during sequencing (phasing). The flap 505 may be recognized and cleaved by a Flap Endonuclease (FEN) 501. FEN 501 may be thermostable or mesophilic. The thermostable FEN can remain associated with the nucleic acid after cleavage of the flap 505 and during subsequent nucleic acid incorporation reactions. Mesophilic FEN can be inactivated during the primer extension reaction and can be replenished into the system after each incorporation and detection cycle. The flap may be cleaved after detection of a signal indicative of nucleotide 504 incorporation and prior to subsequent nucleotide incorporation. Incorporation of nucleotide 504 can result in an increase in the negative charge of the double-stranded nucleic acid molecule. Cleavage of flap 505 can result in a loss of negative charge of the double-stranded nucleic acid. Thus, incorporation of nucleotides with subsequent cleavage of the flap can produce a net neutral change in charge, resulting in little or no detectable signal.

The second single-stranded nucleic acid of the double-stranded nucleic acid may comprise a subunit. FIG. 6 shows an exemplary method for double-strand sequencing using nucleic acid subunits 601. The nucleic acid subunits 601 may be selected from a library of nucleic acid subunits 601. The library of nucleic acid subunits may comprise random sequences. The nucleic acid subunit 601 can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more nucleotides. In one example, the nucleic acid subunit 601 comprises at least 5 nucleotides. In one example, the nucleic acid subunit 601 comprises at least 6 nucleotides. The nucleic acid subunits 601 may all be the same length or may be different lengths. The library of nucleic acid subunits may comprise DNA subunits, Peptide Nucleic Acid (PNA) subunits, RNA subunits, or Locked Nucleic Acid (LNA) subunits. The association between the nucleic acid subunit 601 and the first single-stranded nucleic acid (e.g., a nucleic acid template molecule) can generate a double-stranded nucleic acid molecule and linearize the nucleic acid template. Nucleotide 504 incorporation (e.g., by a primer extension reaction) can displace the subunits and provide a segment of non-hybridized single-stranded nucleic acid template. In one example, the nucleic acid subunits are uncharged, and thus, displacement of the nucleic acid subunit 601 does not alter the charge state of the double stranded nucleic acid molecule. In one example, the nucleic acid subunits are charged and displacement of subunit 601 changes the charge state of the double stranded nucleic acid molecule. The use of nucleic acid subunits may facilitate double-stranded sequencing without the use of FEN.

Individual nucleotides may comprise a reversible terminator. The reversible terminator may prevent the addition of subsequent nucleotides to the third single-stranded nucleic acid molecule. Alternatively or additionally, the reversible terminator may prevent stable hybridization of the additional nucleotide to the first single-stranded nucleic acid molecule. Reversible terminators can reduce homopolymer formation and/or incorporation of more than one nucleotide during the incorporation cycle. The reversible terminator may be coupled to the oxygen atom of the 3 'hydroxyl group of the nucleotide pentose (e.g., a 3' -O-blocked reversible terminator). Alternatively or additionally, a reversible terminator may be coupled to the nucleobase of the nucleotide (e.g., a 3' -unblocked reversible terminator). The reversible terminator may comprise a detectable label. The reversible terminator may comprise an allyl, hydroxylamine, acetic acid, benzoic acid, phosphoric acid, azidomethyl, or amide group. The reversible terminators may be removed by treatment with a reducing agent, an acid or base, an organic solvent, an ionic surfactant, photons (photolysis), or any combination thereof. Removal of the reversible terminator of the 3' -O-blocked reversible terminator may return the hydroxyl group to the pentose sugar of the nucleotide and allow the subsequent nucleotide to be incorporated into the third single-stranded nucleic acid molecule.

Fig. 7A and 7B illustrate an example method of double-strand sequencing using a reversible terminator 701. Figure 7A shows an example method for double-stranded sequencing using a reversible terminator and FEN 501. The second single-stranded nucleic acid of the double-stranded nucleic acid may comprise a uracil nucleotide that is nicked by uracil-DNA glycosidase. Alternatively or additionally, the second single-stranded nucleic acid molecule may comprise a replacement strand and a primer. The polymerase 502 may be bound to the primer 503. The polymerase may be an enzyme that allows for efficient and fidelity of incorporation. The polymerase may be, but is not limited to, Bst polymerase, reverse transcriptase, type A polymerase, type B polymerase, or type C polymerase. The polymerizer enzyme may incorporate individual nucleotides that include reversible terminator 701. Incorporation of nucleotide 701 can generate a flank. The incorporated nucleotides 701 can be detected and, after detection, the flanks can be cleaved by FEN 501. FEN 501 may be mesophilic. After detection of the incorporated nucleotides, mesophilic FEN 501 may be contacted with the flank and may be removed before the next cycle of incorporation. FEN 501 can be supplemented with each nucleotide incorporation cycle. The reversible terminator may be reversed during or after the flanking cleavage. The reversible terminator can be reversed by introducing a reducing agent into the solution. In one example, the reducing agent is Dithiothreitol (DTT). After reversible terminator reversal, the cycle of nucleotide incorporation, detection, flanking cleavage, and terminator reversal is repeated until a portion or all of the first single-stranded nucleic acid is sequenced.

FIG. 7B shows an example method of double-stranded sequencing using a reversible terminator and a nucleic acid subunit. The second single-stranded nucleic acid molecule can comprise a random nucleic acid subunit. The second single-stranded nucleic acid molecule may additionally comprise a primer 503. The polymerase 502 can bind to the primer and incorporate the individual nucleotides 701 into the third single-stranded nucleic acid molecule. Individual nucleotides may replace some or all of the nucleic acid subunits. The incorporated nucleotide may be detected after incorporation into the third single-stranded nucleic acid molecule. Individual nucleotides may comprise a reversible terminator. The reversible terminator may be reversed upon detection of the incorporated nucleotide. The reversible terminator can be reversed by introducing a reducing agent into the solution. In one example, the reductant is DTT. After reversible terminator reversal, the cycle of nucleotide incorporation, detection, and reversal of the terminator can be repeated until a portion or all of the first single-stranded nucleic acid is sequenced.

The double-stranded nucleic acid molecule can comprise a detectable label. The detectable label may be an electrostatic moiety, a fluorescent label, a colorimetric label, a chemiluminescent label, a radioactive label, or any combination thereof. The detectable label may be coupled to the second single-stranded nucleic acid molecule, the nucleotide to be incorporated into the third single-stranded nucleic acid, or any combination thereof. The detectable label may be coupled to the phosphate of the nucleotide, the nucleobase of the nucleotide or to a reversible terminator coupled to the nucleotide. In one example, the detectable label is coupled to a nucleobase of a nucleotide. The detectable label may be reversibly or irreversibly coupled to the nucleotide. The detectable label can generate a signal indicating incorporation of a nucleotide into the third single-stranded nucleic acid molecule or cleavage of a nucleotide from the second single-stranded nucleic acid molecule. The signal from the detectable label may be detected by a sensor array.

In one example, each different type of nucleotide can be coupled to a different detectable label. Each type of detectable label may be indicative of the nucleotide base to which it is bound. For example, each of guanine, cytosine, adenine, thymine, and uracil may have a different detectable label that is distinguishable from one another. Fig. 8 shows example nucleotides each having a different electrostatic moiety and reversible terminator 801. The electrostatic moiety may be a polyanionic or polycationic electrostatic moiety. One or more individual nucleotides may not have an electrostatic moiety. One or more individual nucleotides may have a polycationic electrostatic moiety. The polycationic electrostatic moieties may have different degrees of charge. One or more individual nucleotides may have a polyanionic electrostatic moiety. The polyanionic electrostatic moiety can have different degrees of charge. The presence of excess charge on double-stranded nucleic acid molecules can reduce the efficiency of the polymerase. The polymerizing enzyme may be an enzyme capable of efficiently and faithfully incorporating a nucleotide with a detectable label. The polymerase may be, but is not limited to, Bst polymerase, reverse transcriptase, type A polymerase, type B polymerase, or type C polymerase. The electrostatic moiety may be reversibly or irreversibly coupled to the nucleotide. The electrostatic portion of a nucleotide may be coupled to the second single-stranded nucleic acid molecule or to a medium nucleotide incorporated into a third single-stranded nucleic acid. In one example, the electrostatic moiety is coupled to an individual nucleotide incorporated into the third single-stranded nucleic acid molecule. Individual nucleotides can be directed to contact double-stranded nucleic acids individually and sequentially (e.g., contact with a, then T, then C, then G, etc.), and the sensor can detect nucleotide incorporation between each addition. Alternatively or additionally, the nucleotides can be directed to be contacted with the double-stranded nucleic acid molecule simultaneously (e.g., contacted with a solution comprising all G, A, T and C at one time), and the sensor can detect the change in charge upon nucleotide incorporation. Different electrostatic moieties coupled to different types of individual nucleotides can allow each type of individual nucleotide incorporated into the third single-stranded nucleic acid molecule to be detected and distinguished from each other. Individual nucleotides can be resolved using a single read per cycle of incorporation. Upon detection of nucleotide incorporation, the detectable label may be cleaved from the nucleotide. The cleavage reaction may include a reduction reaction, acid or base cleavage, cleavage in an organic solvent (e.g., formamide or urea), cleavage by an ionic surfactant, or a combination thereof.

In one example, each different type of nucleotide can have the same detectable label. Fig. 9 shows exemplary nucleotides each having the same electrostatic moiety and a different reversible coupling mechanism. The coupling mechanism may be uncoupled or cleaved by reduction, acid or base cleavage, cleavage in organic solvents, cleavage by ionic surfactants, or a combination thereof. In addition, the electrostatic moiety can be coupled to the individual nucleotide by a variety of coupling mechanisms including, but not limited to, covalent bonds, association-dissociation interactions, ligand and binding pair interactions (e.g., streptavidin-biotin interactions), hybridization interactions, or any combination thereof. Individual nucleotides can be directed to contact double-stranded nucleic acids individually and sequentially (e.g., contact with a, then T, then C, then T, etc.), and the sensor can detect nucleotide incorporation between each addition. Alternatively or additionally, the nucleotides can be directed to be contacted with the double-stranded nucleic acid simultaneously (e.g., contacted with a solution having all A, T, C and G at one time), and the sensor can detect the change in charge after nucleotide incorporation. The change in charge can be used to determine the length of the nucleic acid target molecule. In one example (see fig. 9), both nucleotides G, A and T have the same polyanionic electrostatic moiety cleaved by condition one. Nucleotide C may not have an electrostatic moiety. The electrostatic moiety of a may further comprise a second coupling mechanism that cleaves under condition two. The electrostatic portion of T may not be present initially during nucleotide incorporation, but may be coupled to the nucleotide using a third coupling mechanism (e.g., streptavidin-biotin). Double-stranded nucleic acid molecules can be contacted with all four nucleotides at once. One or more nucleotides can be incorporated into a plurality of third single-stranded nucleic acid molecules adjacent to the sensor array. Following the incorporation reaction, nucleotide incorporation can be read or detected. In this example, nucleotides G and a may have polyanionic electrostatic moieties during initial reading (e.g., detection cycles), and both G and a may generate detectable signals. Nucleotides T and C may not initially contain electrostatic moieties or may not generate detectable signals. The electrostatic moiety of a can be removed by contacting the nucleotide with a second cleavage condition, and T can be obtained by introducing an electrostatic moiety comprising a third coupling mechanism (e.g., a streptavidin group). A second read (e.g., a detection cycle) may be performed and both G and T may generate detectable signals, while both a and C may not generate detectable signals. The incorporated nucleotides can then be resolved and distinguished by combining the signals from the first and second reads and matching the signals to the corresponding nucleotides. After the first and second reading, the electrostatic part may be cut using cutting condition 1.

FIG. 10 shows an example method of sequencing using an electrostatic moiety and a reversible terminator. A nucleic acid template to be sequenced can undergo a nucleic acid incorporation reaction (e.g., a primer extension reaction). The incorporated nucleotides can have a cleavable electrostatic moiety and a reversible terminator. The reversible terminator may prevent the incorporation of subsequently added nucleotides into the extended primer. Following incorporation of the nucleotide, the incorporated nucleotide can be read or detected. After reading, the electrostatic moiety can be cleaved and the reversible terminator can be removed. The electrostatic portion may be cleaved and the terminator may be removed using the same chemical method. Exemplary chemical methods include treatment with reducing agents such as Dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP). Alternatively or additionally, the electrostatic portion may be cleaved, and the terminator may be removed using different cleavage and removal chemistries.

The detectable label may be coupled to a nucleotide incorporated during a primer extension reaction, or may be coupled to a nucleotide of the second single-stranded nucleic acid molecule. In one example, the second single-stranded nucleic acid (e.g., displacement strand) can comprise one or more detectable labels. Fig. 11A and 11B illustrate an example method of double-stranded sequencing using a detectable label coupled to a second single-stranded nucleic acid molecule. FIG. 11A shows an example sequencing method using a detectable label cleaved by a flanking endonuclease 501. The second single-stranded nucleic acid molecule of the double-stranded nucleic acid molecule may comprise a detectable label. The detectable label may be an electrostatic moiety. Each nucleotide of the second single stranded nucleic acid molecule can be coupled to an electrostatic moiety. Each different type of nucleotide in the second single-stranded nucleic acid molecule can be coupled to a different electrostatic moiety or to the same electrostatic moiety. The polymerase 502 can bind to the primer 503 adjacent to the displaced strand end. Polymerase 503 can incorporate nucleotide 504 into the third single-stranded nucleic acid molecule. The incorporated nucleotides may or may not have an electrostatic moiety. Incorporation of nucleotides can generate flanks. The flap may be cut through the FEN 501. FEN 501 may be a thermostable or mesophilic FEN. The flap cleavage by FEN 501 removes the electrostatic moiety from the displaced strand, thereby changing the charge state of the double-stranded nucleic acid molecule. The change in charge state can be detected by a sensor array to generate a sequence of the first single-stranded nucleic acid molecule. The cycle of nucleotide incorporation, flanking cleavage, and charge change detection can be repeated until the sequence of the first single-stranded nucleic acid molecule is determined.

Fig. 11B illustrates an example double-stranded sequencing method using both a detectable label coupled to a second single-stranded nucleic acid molecule and a reversible terminator. The detectable label may be an electrostatic moiety. Each nucleotide of the second single stranded nucleic acid molecule can be coupled to an electrostatic moiety. Each different type of nucleotide in the second single-stranded nucleic acid molecule can be coupled to a different electrostatic moiety or to the same electrostatic moiety. The polymerase 502 can bind to the primer 503 adjacent to the displaced strand end. The polymerase 502 may incorporate individual nucleotides 701 into the third single-stranded nucleic acid molecule. The incorporated nucleotide may or may not have an electrostatic moiety and a reversible terminator. The flap may be cut by FEN. The FEN may be thermostable or mesophilic. The flanking cleavage by FEN removes the electrostatic moiety from the displaced strand, thereby altering the charge state of the double stranded nucleic acid molecule. The change in charge state can be detected to generate a sequence of the first single-stranded nucleic acid molecule. After detecting nucleotide incorporation, the newly incorporated nucleotide can undergo a cleavage reaction to remove the reversible terminator. Removal of the reversible terminator may allow for subsequent incorporation of the nucleotide into the third single-stranded nucleic acid molecule. The cycle of nucleotide incorporation, cleavage of the resulting flap, detection of the change in charge, and removal of the reversible terminator may be repeated until the sequence of the first single-stranded nucleic acid molecule is determined. The method can include performing greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection cycles. Nucleotide incorporation and detection may be performed for a set number of cycles, or may be performed until the primer extension reaction is complete.

The detectable label may be coupled to individual nucleotides incorporated into the third single-stranded nucleic acid molecule. Fig. 12A shows an example method of double-stranded sequencing using individual nucleotide-coupled electrostatic moieties 1201 and mesophilic FEN 501. The polymerase may bind to a primer of the second single-stranded nucleic acid molecule to facilitate incorporation of the individual nucleotide 1201. The individual nucleotides 1201 may comprise an electrostatic moiety bound to the nucleobase of the nucleotide. Incorporation of individual nucleotides can generate flanks. The flap may be cut by FEN. The FEN may be mesophilic FEN and may be regenerated after each incorporation cycle. The flap may be cleaved upon detection of a nucleotide incorporation event. The electrostatic moiety can be reversibly coupled to the nucleotide. The electrostatic portion may be cut at the same time or after the flap is cut. The cycle of nucleotide incorporation, detection of nucleotide incorporation, cleavage of the resulting flap, and cleavage of the electrostatic moiety can be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined.

Figure 12B shows an example method of double-stranded sequencing using electrostatic moieties and thermostable FEN. The polymerase 502 may bind to a primer 503 of the second single-stranded nucleic acid molecule to facilitate incorporation of the individual nucleotides 1201. The individual nucleotides 1201 may comprise an electrostatic moiety bound to the nucleobase of the nucleotide. Incorporation of individual nucleotides can generate flanks. The flap may be cut through the FEN 501. FEN 501 can be a thermostable FEN and can remain associated with the double stranded nucleic acid molecule after cleaving the wings. After each incorporation cycle, the thermally stable FEN may not be regenerated. The flanks can be cleaved prior to detection of nucleotide incorporation events. The electrostatic moiety can be reversibly coupled to the nucleotide and can be cleaved upon detection of a nucleotide incorporation event. The cycle of nucleotide incorporation, cleavage of the generated flap, detection of nucleotide incorporation, and removal of the electrostatic moiety can be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined.

Individual nucleotides may comprise both a detectable label and a reversible terminator. Fig. 13A shows an example method of double-strand sequencing using electrostatic moieties, reversible terminators, and mesophilic FEN. The polymerase 502 may bind to the primer 503 of the second single-stranded nucleic acid molecule to facilitate incorporation of the individual nucleotides 1301. The individual nucleotides 1301 can include an electrostatic moiety bound to the nucleobase of the nucleotide and a reversible terminator bound to the 3' side of the pentose sugar. Reversible terminators may reduce homopolymer formation and/or incorporation of multiple nucleotides per cycle. Incorporation of individual nucleotides can generate flanks. The flap may be cut through the FEN 501. FEN 501 may be mesophilic FEN and may be regenerated after each incorporation cycle. The flap may be cleaved upon detection of a nucleotide incorporation event. The electrostatic moiety can be reversibly coupled to the nucleotide. The electrostatic portion may be cut at the same time or after the flap is cut. The reversible terminator may be reversed before, simultaneously with, or after cleavage of the electrostatic moiety. In one example, the electrostatic moiety is cleaved and the reversible terminator is reversed by a reducing agent (such as DTT or TCEP). The cycle of nucleotide incorporation, detection of nucleotide incorporation, cleavage of the resulting flank, cleavage of the electrostatic moiety, and reversal of the reversible terminator may be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection. Nucleotide incorporation and detection may be performed for a set number of cycles, or may be performed until the primer extension reaction is complete.

Figure 13B shows an example method of double-stranded sequencing using electrostatic moieties, thermostable FEN, and reversible terminators. The polymerase 502 may bind to the primer 503 of the second single-stranded nucleic acid molecule to facilitate incorporation of the individual nucleotides 1301. Individual nucleotides 1301 can include an electrostatic portion of a nucleobase bound to the nucleotide and a reversible terminator on the 3' side. Reversible terminators may reduce homopolymer formation and/or incorporation of multiple nucleotides per cycle. Incorporation of individual nucleotides can generate flanks. The flap may be cut through the FEN 501. The FEN 501 may be a thermostable FEN that may remain associated with the double stranded nucleic acid molecule after cleaving the wings. After each incorporation cycle, the thermally stable FEN may not be regenerated. The flanks can be cleaved prior to detection of nucleotide incorporation events. The electrostatic moiety can be reversibly coupled and can be cleaved upon detection of a nucleotide incorporation event. The reversible terminator may be reversed at the same time as or after cleavage of the electrostatic moiety. In one example, the electrostatic moiety is cleaved and the reversible terminator is reversed by a reducing agent (such as DTT or TCEP). The cycle of nucleotide incorporation, cleavage of the generated flap, detection of nucleotide incorporation, cleavage of the electrostatic moiety, and removal of the reversible terminator may be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection. Nucleotide incorporation and detection may be performed for a set number of cycles, or may be performed until the primer extension reaction is complete.

FIG. 14A illustrates an example method of double-stranded sequencing using a detectable label and a nucleic acid subunit. The detectable label may be an electrostatic moiety. The detectable label may be reversibly coupled to the individual nucleotide. The second single-stranded nucleic acid can comprise a random nucleic acid subunit and a primer 503. The polymerase 502 may be bound to the primer 503. The polymerase 502 can incorporate individual nucleotides having electrostatic moieties into the third single-stranded nucleic acid molecule. Incorporation of individual nucleotides 1201 may displace a portion or all of the random nucleic acid subunits. The sensor array may detect a signal indicative of an incorporation event after incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule. Upon detection of the incorporation event, the reversible electrostatic moiety can be cleaved. In one example, the reversible electrostatic moiety is cleaved with a reducing agent (such as DTT or TCEP). The cycle of nucleotide incorporation, nucleic acid subunit substitution, individual nucleotide detection, and electrostatic partial cleavage can be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection. Nucleotide incorporation and detection may be performed for a set number of cycles, or may be performed until the primer extension reaction is complete.

FIG. 14B illustrates an example method of double-strand sequencing using a detectable label, a nucleic acid subunit, and a reversible terminator. The detectable label may be an electrostatic moiety. A detectable label may be reversibly coupled to individual nucleotides 1301. Individual nucleotides may comprise a reversible terminator on the 3' side. The second single-stranded nucleic acid can comprise a random nucleic acid subunit and a primer 503. The polymerase 502 may be bound to the primer 503. The polymerase 502 can incorporate individual nucleotides 1301 having an electrostatic moiety and a reversible terminator into the third single-stranded nucleic acid molecule. Incorporation of individual nucleotides may replace some or all of the random nucleic acid subunits. The sensor array may detect a signal indicative of an incorporation event after incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule. Upon detection of the incorporation event, the reversible electrostatic moiety can be cleaved. The reversible terminator may be removed simultaneously with or after cleavage of the electrostatic moiety. In one example, the reversible electrostatic moiety and the reversible terminator are cleaved simultaneously with a reducing agent (such as DTT or TCEP). Cycles of nucleotide incorporation, nucleic acid subunit substitution, individual nucleotide detection, electrostatic partial cleavage, and reversible terminator removal can be repeated until the sequence of at least a portion of the first single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection. Nucleotide incorporation and detection may be performed for a set number of cycles, or may be performed until the primer extension reaction is complete.

The target nucleic acid molecule can be sequenced and/or the length of the target nucleic acid molecule can be determined. The target nucleic acid molecule can be a fragmented nucleic acid molecule or can be a non-fragmented nucleic acid molecule. The target nucleic acid molecule can be amplified prior to detection. The target nucleic acid molecule can be amplified in solution and/or on a support. The target nucleic acid molecules amplified on the support can be immobilized on the support prior to amplification. The target nucleic acid molecule can be amplified by bridge amplification, wildfire amplification, recombinase polymerase amplification, isothermal amplification, or using any other amplification technique. Sequencing or determining the length of a target nucleic acid molecule can include providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array. A first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. A given sensor may be electrically coupled to a charge bilayer (e.g., within the debye length) containing a first single-stranded nucleic acid molecule. The first single-stranded nucleic acid molecule can be contacted with an individual nucleotide to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction. A nucleic acid incorporation reaction (e.g., a primer extension reaction) can generate a second single-stranded nucleic acid molecule from the individual nucleotides. The second single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. At least a subset of the individual nucleotides may comprise a detectable label. A given sensor in the sensor array can be used to detect a signal from the detectable label indicative of incorporation of an individual nucleotide during or after a nucleic acid incorporation reaction is performed. The detected signal can be used to determine the sequence of the first single-stranded nucleic acid molecule.

Multiple single-stranded nucleic acid molecules can be coupled to multiple supports. The plurality of supports may be a plurality of beads or a plurality of surfaces on the sensor array. In one example, a plurality of single-stranded nucleic acid molecules may be coupled to a plurality of beads, and a given single-stranded nucleic acid molecule may be coupled to a given bead. The charge bilayer may be adjacent to the surface of a given bead. Single-stranded nucleic acid molecules can be amplified on the surface of the beads. The amplification products may be coupled to the surface of the beads. The amplification products can form a clonal population of single-stranded nucleic acid molecules on the surface of the beads. Clonal populations of single-stranded nucleic acid molecules can be sequenced.

In one example, a plurality of single-stranded nucleic acid molecules can be coupled to a plurality of surfaces on a sensor array, and a given single-stranded nucleic acid molecule is coupled to a surface of a given sensor. The charge bilayer may be adjacent to the surface of a given sensor. Single-stranded nucleic acid molecules can be amplified on the surface of the sensor. The amplification product may be coupled to the surface of the sensor. The amplification products may form a clonal population of single-stranded nucleic acid molecules on the surface of the sensor. Clonal populations of single-stranded nucleic acid molecules can be sequenced.

A given sensor in the sensor array may comprise at least one, at least two, at least three, at least four, or more electrodes. In one example, a given sensor contains at least two electrodes. In another example, a given sensor contains two electrodes. The electrode may be exposed to a solution in which a primer extension reaction takes place. Alternatively or additionally, the electrodes may be buried within the sensor array and may therefore not be exposed to the solution in which the primer extension reaction takes place. The sensor can detect a signal indicative of a nucleotide incorporation event. The sensor can detect a detectable label coupled to the individual nucleotide. The sensor may detect the detectable label during transient or steady state conditions. Nucleotide incorporation can be detected once, twice, three times, four times, or more than four times per incorporation cycle during steady state conditions. In one example, nucleotide incorporation can be detected at least twice per cycle of incorporation during steady state conditions. The sensor array may detect electrical signals during transient or steady state conditions. The electrical signal may include, but is not limited to, a change in the charge state of the molecule, a change in the conductivity of the surrounding solution, an impedance signal, or a change in an impedance signal. The sensor may detect a change in charge and/or conductivity or a change in impedance. The sensor can detect a change in charge and/or conductivity or impedance within a charge bilayer (e.g., debye length) of the sensor, support, or nucleic acid molecule (e.g., sample). Detectable labels coupled to individual nucleotides can alter the electrical environment surrounding the single-stranded nucleic acid molecule, and a given sensor can detect an electrical change.

The second single-stranded nucleic acid molecule can comprise a priming site adjacent to the first single-stranded nucleic acid molecule. The priming site may be a primer having sequence complementarity to the first single-stranded nucleic acid molecule. The second single-stranded nucleic acid molecule can be generated by initiating a primer extension reaction from the primer. In one example, the primer is a self-priming loop. The self-priming loop may be in a structural or circular configuration during the primer extension reaction. Upon incorporation of individual nucleotides, the structure of the self-priming loop can relax to form a linear nucleic acid molecule. The incorporation of individual nucleotides can be detected during the relaxed unstructured state. The self-priming loop can be relaxed by increasing the reaction temperature, changing the solution pH, changing the solution ionic strength, introducing formamide into the solution, or by any other method that denatures the nucleic acid structure.

Individual nucleotides of different types may be sequentially contacted with a single-stranded nucleic acid molecule (e.g., a single nucleotide at a time). After each type of individual nucleotide is contacted with the single-stranded nucleic acid molecule, a signal indicative of nucleotide incorporation can be detected. In one example, a single-stranded nucleic acid molecule can be contacted with an a nucleotide, followed by detection of a signal indicative of nucleotide incorporation. The single-stranded nucleotides can then be contacted with T nucleotides, followed by signal detection. The single-stranded nucleotides can then be contacted with the G nucleotides, followed by signal detection. Single-stranded nucleotides can be contacted with C nucleotides and then signal detected. This cycle may be repeated until all or part of the sequence of the single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection.

Single-stranded nucleic acid molecules can be contacted with different types of nucleotides simultaneously. For example, a single-stranded nucleic acid molecule can be contacted with all A, T, C and G at once. Alternatively or additionally, the single stranded nucleic acid molecule may be contacted with any combination of A, T, C and G at once. For example, single stranded nucleic acid molecules can be contacted with a and T, C and G, A and C, A and G, T and C or T and G at one time. In one example, a single stranded nucleic acid molecule is contacted with all A, T, C and G simultaneously, followed by signal detection to determine the sequence length or sequence of the nucleic acid molecule. This cycle can be repeated until all or part of the sequence of the single-stranded nucleic acid molecule is determined.

Individual nucleotides may comprise a detectable label. The detectable label may be reversibly or irreversibly coupled to the individual nucleotide. The detectable label may be coupled to the nucleobase of the individual nucleotide. Individual nucleotides may comprise different types of nucleotides. In one example, the detectable label may be an electrostatic moiety. Each different type of individual nucleotide may be coupled to the same or a single type of detectable label. Each different type of individual nucleotide may be coupled to the same type of detectable label by a different coupling mechanism. Detectable labels can be selectively coupled to or cleaved from individual nucleotides. For example, individual nucleotides may comprise a detectable label when contacted with a single-stranded nucleic acid molecule. After incorporation, the signal of the individual nucleotide can be detected and a positive signal generated (e.g., a signal detected). The detectable label may be removed under given cleavage conditions. Upon removal of the detectable label, the incorporated nucleotide can generate a null signal (e.g., no signal is detected). In examples using a dual read sequencing method (see fig. 9), individual nucleotides may have a positive signal/null signal, a positive signal/positive signal, a null signal/null signal, or a null signal/positive signal. In one example, a single-stranded nucleic acid molecule can be contacted with four different types of nucleotides simultaneously, and a polymerase can facilitate nucleotide incorporation. Each nucleotide may have the same or different detectable label. In one example, each type of nucleotide has a different detectable label and the sequence of the nucleic acid molecule is detected. In another example, each type of nucleotide has the same detectable label and the length of the nucleic acid molecule is detected. After nucleotide incorporation, a signal indicative of nucleotide incorporation can be measured. The incorporated nucleotides can generate a variety of positive and null signals. Single stranded nucleic acid molecules can be treated with cleavage and/or coupling conditions. After treatment with cleavage and/or coupling conditions, the signal indicative of nucleotide incorporation can be measured again. The incorporated nucleotides can generate a variety of positive and null signals. The pattern of positive and empty signals can be used to determine which type of nucleotide is incorporated into the second single-stranded nucleic acid molecule. After the second detection cycle, the electrostatic portion may be removed using a second cutting condition.

In one example, each different individual nucleotide may be coupled to a different detectable label. The detectable label may be an electrostatic moiety. The electrostatic moiety may comprise a polyanion, polycation or neutral electrostatic moiety. Single-stranded nucleic acid molecules can be contacted with different nucleotides sequentially or simultaneously. In one example, a single-stranded nucleic acid molecule can be contacted with different individual nucleotides simultaneously, and each of the different individual nucleotides can be coupled to a different electrostatic moiety. The polymerase can facilitate incorporation of the individual nucleotides into the second single-stranded nucleic acid molecule. After incorporation of individual nucleotides, the sensor array can detect signals indicative of the incorporation of individual nucleotides. Different individual nucleotides can generate signals representing different charge groups and signals representing different charge magnitudes corresponding to the electrostatic moiety to which they are coupled. After detection of the signal, the detectable label may be removed.

Individual nucleotides can comprise a reversible terminator, a detectable label, and both a reversible terminator and a detectable label. Reversible terminators may be coupled to the 3' side of individual nucleotides. The reversible terminator may prevent stable hybridization of the additional nucleotide to the first single-stranded nucleic acid molecule. The reversible terminator may be removed upon detection of a signal indicative of nucleotide incorporation. Removal of the reversible terminator may allow for subsequent nucleotide incorporation. In one example, a single-stranded nucleic acid molecule can be contacted simultaneously with different individual nucleotides comprising different detectable moieties and a reversible terminator. The polymerase can facilitate the incorporation of a single individual nucleotide into the second single-stranded nucleic acid molecule. The sensor can detect a signal indicative of nucleotide incorporation. Following signal detection, the detectable label and reversible terminator may be removed simultaneously or sequentially. This cycle may be repeated until all or part of the sequence of the first single-stranded nucleic acid molecule is determined.

Cleavage of the detectable label can leave a scar on the individual nucleotide after cleavage. The scar may include a portion of the detectable mark that is not completely removed during cutting of the mark. In one example, the scar can reduce the efficiency of the polymerase. In one example, the scar can inhibit a polymerase. Pyrophosphorolysis-mediated terminator exchange (PMTE) sequencing can reduce the amount of scar that accumulates during sequencing.

The target nucleic acid molecule can be sequenced and/or the length of the target nucleic acid molecule can be determined. The target nucleic acid molecule can be a fragmented nucleic acid molecule or can be a non-fragmented nucleic acid molecule. The target nucleic acid molecule can be amplified prior to detection. The target nucleic acid molecule can be amplified in solution and/or on a support. The target nucleic acid molecules amplified on the support can be immobilized on the support prior to amplification. The target nucleic acid molecule can be amplified by bridge amplification, wildfire amplification, recombinase polymerase amplification, isothermal amplification, or using any other amplification technique. Sequencing or determining the length of a target nucleic acid molecule can include providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array. A first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. The first single-stranded nucleic acid molecule can be subjected to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule. The nucleic acid incorporation reaction can include the alternating and sequential incorporation of individual nucleotides in a first plurality of nucleotides comprising a detectable label followed by the incorporation of a second plurality of individual nucleotides without a detectable label. The nucleic acid incorporation reaction can include alternately and sequentially incorporating individual nucleotides in a first plurality of nucleotides comprising a detectable label and exchanging the first plurality of individual nucleotides with individual nucleotides in a second plurality of individual nucleotides without the detectable label (e.g., removing the first nucleotide). The first plurality of nucleotides can be covalently incorporated into the growing nucleic acid strand or can be transiently bound (e.g., not covalently bound) to the growing nucleic acid strand. The second plurality of individual nucleotides may not comprise a detectable label. A given sensor can detect a signal from a detectable label simultaneously with or after a nucleic acid incorporation reaction is performed. The detected signal may be generated from the detectable label and may indicate that the first plurality of individual nucleotides is incorporated into the second single-stranded nucleic acid molecule, thereby determining the sequence of the first single-stranded nucleic acid molecule.

Multiple single-stranded nucleic acid molecules can be coupled to multiple supports. The plurality of supports can be a plurality of beads. In one example, a plurality of single-stranded nucleic acid molecules may be coupled to a plurality of beads, and a given single-stranded nucleic acid molecule may be coupled to a given bead. A given sensor may be electrically coupled to a charge bilayer comprising a first single-stranded nucleic acid molecule. The charge bilayer may be adjacent to the surface of a given bead. Single-stranded nucleic acid molecules can be amplified on the surface of the beads. The amplification products may be coupled to the surface of the beads. The amplification products can form a clonal population of single-stranded nucleic acid molecules on the surface of the beads. Clonal populations of single-stranded nucleic acid molecules can be sequenced.

In one example, a plurality of single-stranded nucleic acid molecules can be coupled to a plurality of surfaces on a sensor array, and a given single-stranded nucleic acid molecule is coupled to a surface of a given sensor. A given sensor may be electrically coupled to a charge bilayer comprising a first single-stranded nucleic acid molecule. The charge bilayer may be adjacent to the surface of a given sensor. Single-stranded nucleic acid molecules can be amplified on the surface of the sensor. The amplification product may be coupled to the surface of the sensor. The amplification products may form a clonal population of single-stranded nucleic acid molecules on the surface of the sensor. Clonal populations of single-stranded nucleic acid molecules can be sequenced and/or the length of the single-stranded nucleic acid can be determined.

A given sensor in the sensor array may comprise at least one, at least two, at least three, at least four, or more electrodes. In one example, a given sensor contains at least two electrodes. In another example, a given sensor contains two electrodes. The electrode may be exposed to a solution in which a primer extension reaction takes place. Alternatively or additionally, the electrodes may be buried within the sensor array and may therefore not be exposed to the solution in which the primer extension reaction takes place. The sensor can detect a signal indicative of a nucleotide incorporation event. The sensor can detect a detectable label coupled to the individual nucleotide. The sensor may detect the detectable label during transient or steady state conditions. Nucleotide incorporation can be detected once, twice, three times, four times, or more than four times per incorporation cycle during steady state conditions. In one example, nucleotide incorporation can be detected at least twice per cycle of incorporation during steady state conditions. The sensor array may detect electrical signals during transient or steady state conditions. The electrical signal may include, but is not limited to, a change in the charge state of the molecule, a change in the conductivity of the surrounding solution, an impedance signal, or a change in an impedance signal. The sensor may detect a change in charge and/or conductivity or a change in impedance. The sensor can detect a change in charge and/or conductivity or impedance within a charge bilayer (e.g., debye length) of the sensor, support, or nucleic acid molecule (e.g., sample). Detectable labels coupled to individual nucleotides can alter the electrical environment surrounding the single-stranded nucleic acid molecule, and a given sensor can detect an electrical change.

The second single-stranded nucleic acid molecule can comprise a priming site adjacent to the first single-stranded nucleic acid molecule. The priming site may be a primer having sequence complementarity to the first single-stranded nucleic acid molecule. The second single-stranded nucleic acid molecule can be generated by initiating a primer extension reaction from the primer. In one example, the primer is a self-priming loop. The self-priming loop may be in a structural or circular configuration during the primer extension reaction. Upon incorporation of individual nucleotides, the structure of the self-priming loop can relax to form a linear nucleic acid molecule. The incorporation of individual nucleotides can be detected during the relaxed unstructured state. The self-priming loop can be relaxed by increasing the reaction temperature, changing the solution pH, changing the solution ionic strength, introducing formamide into the solution, or by any other method that denatures the nucleic acid structure.

The first plurality of nucleotides can comprise a terminator that prevents stable hybridization of additional nucleotides to the first single-stranded nucleic acid molecule. The terminator may be a reversible terminator or an irreversible terminator. In one example, the terminator is an irreversible terminator. The terminator may reduce the occurrence of homopolymer and/or the incorporation of multiple individual nucleotides per cycle of incorporation. The first plurality of individual nucleotides may comprise dideoxynucleotides (ddNTPs) or 3-fluorodeoxynucleotides. ddntps can be chain-extension inhibitors. The first plurality of nucleotides can comprise a detectable label. The detectable label may not be removed after detection of nucleotide incorporation. The detectable label may be an electrostatic moiety, a fluorescent label, a chemiluminescent label, a colorimetric label, a radioactive label, or any other detectable label. In one example, the detectable label is an electrostatic moiety. A detectable label may be coupled to the nucleobase of the first plurality of nucleotides.

The first plurality of nucleotides can comprise different types of nucleotides. In one example, different types of nucleotides can be coupled to different types of detectable labels. Each individual type of nucleotide may be coupled to an individual type of detectable label. The first single-stranded nucleic acid molecule can be contacted with all of the different types of nucleotides simultaneously. The sensor array can then detect different detectable electrostatic moieties coupled to different individual nucleotides. Alternatively or additionally, each type of nucleotide may have the same detectable label, and the sensor may detect the addition of nucleotides without resolving different nucleotides (e.g., determining sequence length). In this example, a single read may be used per doping cycle. In one example, each type of individual nucleotide may be coupled to the same detectable label. The first single-stranded nucleic acid molecule can be contacted with each type of nucleotide in sequence (e.g., contacted with one type of nucleotide, followed by contact with another type of nucleotide). After incorporation of one type of nucleotide, the sensor array can detect a signal indicative of nucleotide incorporation. The first single-stranded nucleic acid molecule can then be contacted with a different type of nucleotide. The detectable label may not be cleaved from the first plurality of nucleotides (e.g., the detectable label may be irreversible).

The first plurality of individual nucleotides may be exchanged for a second plurality of individual nucleotides. The exchange reaction can be accomplished by back-driving the polymerization reaction with an excess of pyrophosphate, triphosphate or tetraphosphate. The second plurality of individual nucleotides may not comprise a detectable label. Exchanging the first plurality of individual nucleotides for the second plurality of individual nucleotides can reduce scar formation. The second plurality of individual nucleotides can comprise a reversible terminator. The reversible terminator may be reversed by contact with a reducing agent, by changing the solution pH, by changing the solution ionic strength, by contact with an ionic surfactant, or by any other terminator removal method.

Figure 15 shows an example PMTE sequencing method. The first single stranded nucleic acid molecule can be coupled to a bead. The first single-stranded nucleic acid molecule can have a priming site. The priming site may be complementary to a portion of the first single-stranded nucleic acid molecule. A first single stranded nucleic acid molecule can be contacted with the first plurality of individual nucleotides 1501. The first plurality of individual nucleotides 1501 may comprise a single type of nucleotide. The first plurality of individual nucleotides 1501 may comprise an irreversible terminator and an irreversibly detectable electrostatic moiety. The irreversibly detectable electrostatic moiety may be the same for each different type of nucleotide. The polymerase 502 may facilitate the incorporation of the first bulk nucleotide 1501 into the second single-stranded nucleic acid molecule. A given sensor can detect the presence or absence of nucleotide incorporation by the presence or absence of a detectable label. The first plurality of individual nucleotides 1501 may then be exchanged for the second plurality of individual nucleotides 701. The second plurality of individual nucleotides 701 may be the same type of nucleotide as the first plurality of individual nucleotides 1501. The second plurality of individual nucleotides 701 can have no detectable label and can have a reversible terminator. The reversible terminator may be removed or reversed after the second plurality of individual nucleotides is incorporated into the second single-stranded nucleic acid molecule. The terminator may be reversed by the reducing agent. This cycle may be repeated until all or part of the sequence of the first single-stranded nucleic acid molecule is determined. The method can include performing cycles of greater than or equal to 1, 2, 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or more nucleotide incorporation and detection.

In one example (see fig. 15), a first single-stranded nucleic acid molecule can be coupled to a bead. The first single-stranded nucleic acid molecule can have a priming site coupled to the primer 502. The priming site may be complementary to a portion of the first single-stranded nucleic acid molecule. A first single-stranded nucleic acid molecule can be contacted with a first plurality of individual nucleotides. The first plurality of individual nucleotides 1501 can comprise a plurality of different types of nucleotides. The first plurality of individual nucleotides 1501 may comprise an irreversible terminator and an irreversibly detectable electrostatic moiety. The irreversibly detectable electrostatic moiety may be different for each type of individual nucleotide. The polymerase 502 may facilitate the incorporation of the first bulk nucleotide 1501 into the second single-stranded nucleic acid molecule. A given sensor can detect the type of nucleotide incorporated into the second single-stranded nucleic acid molecule by the type of detectable label present on the sensor. The first plurality of individual nucleotides 1501 may then be exchanged for the second plurality of individual nucleotides 701. The second plurality of individual nucleotides 701 can include the same type of nucleotide as the first plurality of individual nucleotides. The second plurality of individual nucleotides 701 can have no detectable label and can have a reversible terminator. The reversible terminator may be removed or reversed after the second plurality of individual nucleotides is incorporated into the second single-stranded nucleic acid molecule. The terminator may be reversed by the reducing agent. This cycle may be repeated until all or part of the sequence of the first single-stranded nucleic acid molecule is determined.

The PMTE sequencing method described above can be used in combination with a double-stranded sequencing method. For example, a double-stranded nucleic acid molecule comprising a first and a second single-stranded nucleic acid molecule can be contacted with a polymerizer enzyme. The polymerase can incorporate individual nucleotides comprising an irreversible terminator and a detectable label into the third single-stranded nucleic acid molecule. Incorporation of individual nucleotides can generate flanks. The flap may be cleaved before or after detecting the nucleotide incorporation of the individual. Double-stranded nucleic acid molecules can be contacted with different types of individual nucleotides simultaneously or sequentially. Different types of individual nucleotides may comprise the same or different detectable labels. After incorporation of individual nucleotides, the incorporation event can be detected by a sensor array. After detection, individual nucleotides comprising a detectable label and an irreversible terminator can be exchanged for individual nucleotides comprising a reversible terminator. The reversible terminator can then be reversed to allow for subsequent incorporation of the individual nucleotide.

The method may further comprise monitoring and/or correcting for phase errors. A nucleic acid molecule with phase error may be more or less extended than the consensus state (e.g., reference sequence) of the clonal population of which it is a member or the template nucleic acid molecule of which it is a copy or representative sequence. For fragments that include erroneously incorporated bases (e.g., extra bases or incorrect bases added to the growing strand), the phase error can be considered to be a lead. For other nucleic acid molecules that have bases that are not incorporated into the growing strand relative to the consensus sequence, the polynucleotide can be considered to be lagging. Since the polymerase may be deficient, some phase errors may occur in the colony with long extension reactions as part of the colony-based sequencing process. Phase errors can limit the read length of commercial clonal sequencing systems.

Phase errors can lead to sequencing-look-ahead incorporation errors. A look-ahead sequencing error can refer to a sequence that is longer than the main sequence due to incorrect or excessive (e.g., homopolymer) addition of nucleotides. Incorrect or excessive addition may be caused by polymerase error, especially when high concentrations of dNTP are used in non-competitive reactions. Alternatively or additionally, the sequencing-ahead incorporation error may be due to insufficient washing or non-specific binding of dntps that can subsequently be released and incorporated. The sequencing-advance incorporation errors may be due to nucleotide incorporation without a valid 3' terminator, thus allowing one more cycle of incorporation events. For example, a pre-sequencing incorporation error may be due to the presence of trace amounts of unprotected or unblocked 3' -OH nucleotides in the nucleic acid incorporation event. Unprotected 3' -OH nucleotides may be produced during manufacture or possibly during storage and handling of reagents.

Phase errors can lead to lagging sequencing errors. Late sequencing incorporation errors can refer to sequences that are shorter than the main sequence due to the absence of the addition of the correct nucleotide. Lagged sequencing errors can occur due to non-optimal reaction conditions, steric hindrance, secondary structure, or other sources of polymerase inhibition. Non-limiting examples of processes that can cause hysteretic sequencing errors include: reversible terminators, detectable labels, flanks derived from the second single-stranded nucleic acid molecule, incomplete removal of modified nucleotides and/or linkers. Longer cycle times may allow the polymerase more opportunity to incorporate the wrong nucleotide. Similarly, nucleic acid molecules (e.g., DNA) that are difficult to access can result in an insufficient chance of incorporating the correct nucleotide. It is contemplated that the temperature, step time, polymerase choice, nucleotide concentration, salt concentration, and buffer choice can be optimized to minimize incorporation errors.

For example, a nucleic acid (e.g., DNA) sample can have a TGTTC sequence in a first region following the region complementary to the primer. The fluid cycle may first introduce deoxycytidine triphosphate (dCTP), second introduce deoxythymidine triphosphate (dTTP), third introduce deoxyadenosine triphosphate (dATP), fourth introduce deoxyguanosine triphosphate (dGTP), and insert wash steps. In the first part of the fluid cycle, dCTP molecules that are in-flowing as part of the first cycle may not be properly washed out and away from the nucleic acid template. In the second part of the fluid cycle, dTTP molecules that are in-flowing as part of the second cycle may not be properly washed out of the pore structure. Dntps may not be incorporated during the first and second portions of the first fluidic cycle. During the third part of the fluidic cycle, dATP can be introduced and incorporated as it is complementary to the first base T of the sample. Any non-specifically bound dCTP molecules can also terminate non-specific binding and be incorporated during this third portion of the fluid cycle. These unbound dCTP molecules can be incorporated after incorporation of the dATP molecules. After incorporation of the dCTP molecule, two more dATP molecules can be incorporated, which can result in some molecules of the monoclonal beads having a leading sequencing phase error. Thus, some molecules of the monoclonal beads may become out of phase.

Phase errors can be detected by comparing the sequences of multiple double-stranded or single-stranded molecules from a clonal population and/or by comparison to a reference sequence. For example, mismodulation (miscalls) in the sequence, such as substitution-type or insertion-deletion-type mismodulation, may be analyzed. Misregulation can be detected by measuring signal intensity during a nucleic acid incorporation reaction using a single stranded molecule as a template for generating a complementary single stranded molecule. A double-stranded or single-stranded molecule may comprise a clonal population. In one example, a portion of the clonal population can have a significantly lower detectable signal intensity, such as less than a threshold, than the remaining clonal population. This may indicate that the nucleotide incorporation may be at fewer than all available positions and may lead to indel-type mismodulation. Indel-type mismodulation may be due to incomplete extension of single-stranded molecules and may lead to lag sequencing errors. In another example, a portion of a clonal population can have a substituted base when compared to a reference sequence. Substitutional misadjustment can be caused by a leading sequencing error resulting from the incorporation of additional nucleotides in the nucleic acid incorporation reaction. The additional nucleotides may be different from the nucleotides in the reference sequence.

Phase errors can be reduced by performing the nucleic acid incorporation reaction under competitive conditions. For example, the concentration of nucleotides can be reduced to mitigate sequencing errors ahead. In another example, the cycle time and/or number of cycles per read can be reduced to avoid erroneous incorporation of nucleotides resulting in a leading sequencing error. In some cases, phase error can be reduced by using nucleotide-based polymerizers. For example, when incorporating unmodified nucleotides, a type a polymerase, such as Bst polymerase, can be used to reduce phase error. When modified nucleotides are incorporated, B-type polymerases, such as Therminator IX, can be used^TM(NEB) to reduce phase errors.

In one aspect, phase error can be reduced by incorporating unmodified nucleotides and modified nucleotides sequentially or simultaneously in a nucleic acid incorporation reaction.

A method of nucleic acid sequencing can include providing a plurality of double-stranded or single-stranded nucleic acid molecules adjacent to a sensor array. A first double-stranded or single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array.

The first single-stranded nucleic acid molecule can be subjected to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule as a growing strand complementary to the first single-stranded nucleic acid molecule. The nucleic acid incorporation reaction can include alternately and sequentially (i) incorporation of individual nucleotides in a first plurality of nucleotides comprising a detectable label, and (ii) incorporation of individual nucleotides in a second plurality of nucleotides that do not comprise a detectable label. A given sensor may be used to detect a signal from the detectable label that may indicate that an individual nucleotide of the first plurality of nucleotides is incorporated into the second single-stranded nucleic acid molecule, thereby determining the sequence of the first single-stranded nucleic acid molecule. The first plurality of nucleotides can be exchanged for the second plurality of nucleotides. Incorporation of a second plurality of nucleotides can correct for phase errors. Incorporation of the second plurality of nucleotides may correct for phase error by incorporating individual nucleotides from the second plurality of nucleotides at positions along the first single-stranded nucleic acid molecule where individual nucleotides from the first plurality of nucleotides are not incorporated. The nucleic acid incorporation reaction can be continued by using individual nucleotides from the first plurality of nucleotides. The second single-stranded nucleic acid molecule may have sequence homology with the template single-stranded nucleic acid molecule.

An example of a method for nucleic acid sequencing to correct for phase error is shown in figure 20. A plurality of single stranded nucleic acid molecules may be coupled to the bead. The plurality of single-stranded nucleic acid molecules can comprise a clonal population of a given single-stranded nucleic acid molecule. The first single-stranded nucleic acid molecule can have a priming site coupled to an individual primer. The priming site may be complementary to a portion of the first single stranded molecule. The first single-stranded molecule can be contacted with a first plurality of individual nucleotides. The first plurality of individual nucleotides can comprise a plurality of different types of nucleotides. The first plurality of individual nucleotides may comprise a single type of nucleotide. The first plurality of individual nucleotides may be modified nucleotides. The first plurality of individual nucleotides can comprise an irreversible terminator and an irreversibly detectable electrostatic moiety. The irreversibly detectable electrostatic moiety may be different for each type of individual nucleotide. The polymerase can facilitate incorporation of the first individual nucleotide into the second single-stranded nucleic acid molecule. A given sensor can detect the type of nucleotide incorporated into the second single-stranded nucleic acid molecule by the type of detectable label present on the sensor. The first plurality of individual nucleotides can then be exchanged for a second plurality of individual nucleotides. The second plurality of individual nucleotides may comprise the same type of nucleotide as the first plurality. The second plurality of individual nucleotides may have no detectable label and may have a reversible terminator. The addition of the second plurality of nucleotides may correct for phase error by incorporating individual nucleotides from the second plurality of nucleotides at positions along the first single-stranded nucleic acid molecule where individual nucleotides from the first plurality of nucleotides are not incorporated.

As shown in fig. 20, the phase error may be a leading sequencing error, as indicated by the addition of an additional nucleotide (n +1) in the leading second single-stranded molecule in read 1. Individual nucleotides from the second plurality of nucleotides can be incorporated into the lagging second single-stranded molecule such that the lagging molecule can be synchronized with the leading molecule, as indicated by "n + 1" in the next cycle before read 2. The reversible terminator may be removed or reversed after the second plurality of individual nucleotides is incorporated into the second single-stranded nucleic acid molecule. The terminator may be reversed by the reducing agent. Once two molecules can be synchronized to have the same number of incorporated nucleotides of (n +1), the nucleic acid incorporation reaction can be continued using individual nucleotides from the first plurality of nucleotides. In the next cycle read 2, the detectable label in the first plurality of nucleotides can be cleaved for detection by the sensor. The detectable label may be cleaved by using a phosphate reagent such as tris (hydroxypropyl) phosphine (THPP). Cleavage of the detectable label can leave a scar on the individual nucleotide after cleavage. The scar may include a portion of the detectable mark that is not completely removed during cutting of the mark. The first single-stranded nucleic acid molecule can be alternately provided with a first plurality of individual nucleotides and a second plurality of individual nucleotides to generate a second single-stranded nucleic acid molecule until the sequence of all or a portion of the first single-stranded nucleic acid molecule is determined.

Fig. 21 and 22 show example results of reducing phase error during nucleic acid sequencing using this method. To generate the second single-stranded molecule, the first single-stranded molecule can be contacted with a first plurality of nucleotides having a detectable label, such as three lysine amino acid residues. The first plurality of nucleotides can be exchanged for the second plurality of nucleotides. Incorporation of the first plurality of nucleotides can cause a leading phase error (n +1) in the second single-stranded molecule in read 1. The phase error can be corrected by incorporating a second plurality of nucleotides into the lag molecule so that the lag molecule can be synchronized with the lead molecule. As shown in FIG. 21, the X-axis indicates the number of flows (flow number) corresponding to the number of incorporated nucleotides, and the Y-axis indicates the signal derived from the cleavage of the detectable label. In read 1, a detectable label can be coupled to the first plurality of nucleotides, which can result in a negative signal on the Y-axis. The negative signal may be due to the substitution of a detectably labeled lysine residue for a cation (such as Mg)²⁺). In read 2, the detectable label can be cleaved from the first plurality of nucleotides, thereby generating a positive signal on the Y-axis derived from the scar nucleotide. In thatUpon removal of the detectable label comprising a lysine residue, a positive signal may be derived from a cation (such as Mg)²⁺) The concentration of (c). In reads 1 and 2, the second plurality of individual nucleotides may be free of detectable label, which may result in a signal on the Y-axis approaching zero. The change in signal during sequencing is shown in FIG. 22. The presence of a detectable label (three lysine residues) in the first multiple of nucleotides can result in a net negative signal on the Y-axis, as shown by Δ K3 in read 1. Addition of unmodified nucleotides can result in a neutral signal, as shown by Δ Chase in read 2, approaching zero on the Y-axis. Cleavage of the detectable label by THPP can result in a sharp increase in net positive signal, as shown by Δ THPP in read 3. Upon cleavage of the detectable label, the neutral signal due to the unmodified nucleotide can be converted to a positive signal, as indicated by the arrow during Δ chase. The positive signal can be attributed to the negative phosphate group in the nucleotide which in turn can concentrate Mg²⁺Cations thus produce a net positive signal.

System for nucleic acid sequencing

The present disclosure provides a system for nucleic acid sequencing that can include multiple components. The system can be used for a variety of applications, such as sequencing nucleic acid samples from living subjects. For example, a sensor array having sites occupied by beads or sites directly occupied by a plurality of nucleic acid templates comprising a clonal population can be contacted with a fluid comprising primers that hybridize to the clonal nucleic acids. The sensor array may then be washed and contacted with a fluid containing one or more types of nucleotides, polymerizers, and/or any cofactors in a suitable buffer. The array can then be washed and the incorporated nucleotides can be detected. The cycle of incorporation, washing, detection may be repeated until the sample nucleic acid bound to the beads or to the sensor surface has been sequenced.

The sensor array may be incorporated into an integrated sequencing platform. The integrated sequencing platform can include one or more of a nucleic acid (e.g., DNA) extraction module, a library construction module, an amplification module, an extraction module, and a sequencing module. In some embodiments, the systems may be stand alone and/or in modular form. In some embodiments, the integrated sequencing platform may comprise one, two, three, four, or all five of these systems. In some cases, the modules may be integrated in a single unit (e.g., a microfluidic device), a single array (e.g., a reusable sensor array), or even a single device. Examples of integrated sequencing platforms can be found in PCT patent application No. PCT/US2011/054769, PCT patent application No. PCT/US2012/039880, PCT patent application No. PCT/US2012/067645, PCT patent application No. PCT/US2014/027544, PCT patent application No. PCT/US2014/069624, and PCT patent application No. PCT/US2015/020130, each of which is incorporated by reference herein in its entirety.

In another aspect, the present disclosure provides a system for nucleic acid sequencing. The system may include a sensor array including a plurality of individual sensors. During use, a given double-stranded nucleic acid molecule of the plurality of double-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. A given double-stranded nucleic acid molecule can comprise a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule. A given sensor may be electrically coupled to a charge bilayer (e.g., within the debye length) of a given double-stranded nucleic acid molecule. The system may further comprise one or more computer processors operatively coupled to the sensor array. The one or more computer processors can be programmed to contact the non-hybridizing segment of the first single-stranded nucleic acid molecule with an individual nucleotide to subject the non-hybridizing segment to a nucleic acid incorporation reaction to generate a third single-stranded nucleic acid molecule of the individual nucleotide. The third single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. During or after the nucleic acid incorporation reaction, a given sensor can detect a signal indicative of the incorporation of an individual nucleotide into the third single-stranded nucleic acid molecule, thereby determining the sequence of the non-hybridizing segment.

The double stranded nucleic acid molecule may be coupled to a support. The support may be a bead, or the surface of a sensor array. Multiple double-stranded nucleic acid molecules can be coupled to multiple beads or multiple locations on the surface of the sensor array. Each bead of the plurality of beads may be positioned adjacent to a given sensor. The plurality of beads may be magnetic or non-magnetic beads. The beads may have a surface coating that facilitates coupling of double-stranded nucleic acid molecules to the beads. The charge bilayer (e.g., debye length) may be adjacent to the surface of the bead. Alternatively or additionally, a plurality of double stranded nucleic acid molecules may be coupled to one or more surfaces of the sensor array. A given double stranded nucleic acid molecule can be coupled to the surface of a given sensor. The charge bilayer (e.g., debye length) may be adjacent to the surface of a given sensor. Double-stranded nucleic acid molecules coupled to the surface of a bead or sensor array may be clonally amplified prior to sequencing such that each bead is coupled to a clonal population of double-stranded nucleic acid molecules, or such that each surface of a given sensor is coupled to a clonal population of double-stranded nucleic acid molecules.

A given sensor may comprise at least one, at least two, at least three or at least four electrodes. In one example, a given sensor contains at least two electrodes. The electrodes of a given sensor can detect a signal indicative of the incorporation of an individual nucleotide into a double-stranded nucleic acid molecule. The signal indicative of an incorporation event may comprise a change in impedance, conductance, or charge in the electronic bilayer. In one example, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in a charge bilayer. The signal indicative of incorporation of an individual nucleotide may be a steady state signal, a transient signal, or a combination of steady state and transient signals. The signal may be detected instantaneously or under steady state conditions. In the transient signal detection mode, detection occurs during or immediately following nucleotide incorporation. In steady state detection, the reading of the sensor may occur after the incorporation event is "complete". Steady state changes in the signal may be maintained until a change is introduced to the environment surrounding the sensor.

One or more computer processors may be programmed to direct fluid flow through the sensor array. During fluid flow conditions, double-stranded nucleic acid molecules can be stably coupled to one or more surfaces. The double stranded nucleic acid molecule can be stably coupled to a plurality of beads. The beads may be stably placed adjacent to the sensor array. The beads may be held adjacent to the sensor array by a magnetic or electric field. The fluid flow does not damage or dislodge the beads. The fluid directed through the sensor array may include nucleic acid molecules, primers, polymerizers, individual nucleotides, cofactors for nucleotide incorporation reactions (e.g., primer extension reactions), and/or buffers. The fluid may be a wash fluid comprising a buffer. In one example, a fluid may be directed to and incubated with a sensor array. The fluid may be incubated with the sensor array for the duration of a single cycle of the nucleotide incorporation reaction. Between incubation cycles, the sensor array may be washed with a washing fluid.

In another aspect, the present disclosure provides a system for nucleic acid sequencing. The system may include a sensor array including a plurality of sensors. During use, a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. A given sensor may be electrically coupled to a charge bilayer (e.g., within the debye length) of a first single-stranded nucleic acid molecule. The system may include one or more computer processors coupled to the sensor array. The one or more computer processors may be programmed to contact the first single-stranded nucleic acid molecule with individual nucleotides to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that generates a second single-stranded nucleic acid molecule from the individual nucleotides. The second single-stranded nucleic acid molecule can have sequence complementarity with the first single-stranded nucleic acid molecule. At least a subset of the individual nucleotides may comprise a detectable label. A given sensor can detect a signal from a detectable label during or after a nucleic acid incorporation reaction. The signal may indicate that an individual nucleotide is incorporated into the second single-stranded nucleic acid molecule. This signal can be used to determine the sequence of the first single-stranded nucleic acid molecule.

The single stranded nucleic acid molecule may be coupled to a support. The support may be a bead, or the surface of a sensor array. A plurality of single-stranded nucleic acid molecules may be coupled to a plurality of beads or a plurality of locations on the surface of a sensor array. Each bead of the plurality of beads may be positioned adjacent to a given sensor. The plurality of beads may be magnetic or non-magnetic beads. The beads may have a surface coating that facilitates coupling of the single stranded nucleic acid molecules to the beads. The charge bilayer (e.g., debye length) may be adjacent to the surface of the bead. Alternatively or additionally, a plurality of double stranded nucleic acid molecules may be coupled to one or more surfaces of the sensor array. A given single-stranded nucleic acid molecule can be coupled to the surface of a given sensor. The charge bilayer (e.g., debye length) may be adjacent to the surface of a given sensor. Single-stranded nucleic acid molecules coupled to the surface of a bead or sensor array may be clonally amplified prior to sequencing such that each bead is coupled to a clonal population of single-stranded nucleic acid molecules, or such that each surface of a given sensor is coupled to a clonal population of single-stranded nucleic acid molecules.

A given sensor may comprise at least one, at least two, at least three or at least four electrodes. In one example, a given sensor contains at least two electrodes. In another example, a given sensor contains two electrodes. The electrode may be exposed to a solution in which a primer extension reaction takes place. Alternatively or additionally, the electrodes may be buried within the sensor array and may therefore not be exposed to the solution in which the primer extension reaction takes place. The electrodes of a given sensor can detect a signal indicative of the incorporation of an individual nucleotide into a single-stranded nucleic acid molecule. The signal indicative of an incorporation event may comprise a change in impedance, conductance, or charge in the electronic bilayer. In one example, the signal indicative of incorporation of an individual nucleotide is an electrical signal generated by an impedance or impedance change in a charge bilayer. The signal indicative of incorporation of an individual nucleotide may be a steady state signal, a transient signal, or a combination of steady state and transient signals. The signal may be detected instantaneously or under steady state conditions. In the transient signal detection mode, detection occurs during or immediately following nucleotide incorporation. In steady state detection, the reading of the sensor may occur after the incorporation event is complete. Steady state changes in the signal may be maintained until a change is introduced to the environment surrounding the sensor. The sensor can detect an incorporation event (e.g., count incorporation events) or can resolve incorporated nucleotides individually (e.g., determine which nucleotide was incorporated).

One or more computer processors may be programmed to direct fluid flow through the sensor array. During fluid flow conditions, single-stranded nucleic acid molecules can be stably coupled to one or more surfaces. Single-stranded nucleic acid molecules can be stably coupled to a plurality of beads. The beads may be stably placed adjacent to the sensor array. The beads may be held adjacent to the sensor array by a magnetic or electric field. The fluid flow does not damage or dislodge the beads. The fluid directed through the sensor array may include nucleic acid molecules, primers, polymerizers, individual nucleotides, cofactors for nucleotide incorporation reactions (e.g., primer extension reactions), and/or buffers. The fluid may be a wash fluid comprising a buffer. In one example, a fluid may be directed to and incubated with a sensor array. The fluid may be incubated with the sensor array for the duration of a single cycle of the nucleotide incorporation reaction. Between incubation cycles, the sensor array may be washed with a washing fluid.

In another aspect, the present disclosure provides a system for nucleic acid sequencing. The system may include a sensor array including a plurality of sensors. During use, a first single-stranded nucleic acid molecule of the plurality of single-stranded nucleic acid molecules can be positioned adjacent to a given sensor of the sensor array. The system may include one or more computer processors operatively coupled to the sensor array. The one or more computer processors can be programmed to subject the first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction that includes alternating and sequential incorporation of individual nucleotides in a first plurality of nucleotides that include a detectable label and exchange of individual nucleotides in the first plurality of nucleotides with individual nucleotides in a second plurality of nucleotides that do not include a detectable label. A given sensor can detect a signal from a detectable label during or after a nucleic acid incorporation reaction. The signal may indicate that an individual nucleotide is incorporated into the second single-stranded nucleic acid molecule. This signal can be used to determine the sequence of the first single-stranded nucleic acid molecule.

Multiple single-stranded nucleic acid molecules can be coupled to multiple supports. The plurality of supports may be a plurality of beads or a plurality of surfaces on the sensor array. In one example, a plurality of single-stranded nucleic acid molecules may be coupled to a plurality of beads, and a given single-stranded nucleic acid molecule may be coupled to a given bead. A given sensor may be electrically coupled to a charge bilayer comprising a first single-stranded nucleic acid molecule. The charge bilayer may be adjacent to the surface of a given bead or on the surface of a given sensor. Single-stranded nucleic acid molecules can be amplified on the surface of a support. The amplification products may be coupled to the surface of the support. The amplification products may form a clonal population of single-stranded nucleic acid molecules on the surface of the support. Clonal populations of single-stranded nucleic acid molecules can be sequenced.

A given sensor in the sensor array may comprise at least one, at least two, at least three, at least four, or more electrodes. In one example, a given sensor contains at least two electrodes. In another example, a given sensor contains two electrodes. The electrode may be exposed to a solution in which a primer extension reaction takes place. Alternatively or additionally, the electrodes may be buried within the sensor array and may therefore not be exposed to the solution in which the primer extension reaction takes place. The sensor can detect a signal indicative of a nucleotide incorporation event. The sensor can detect a detectable label coupled to the individual nucleotide. The sensor may detect the detectable label during transient or steady state conditions. Nucleotide incorporation can be detected once, twice, three times, four times, or more than four times per incorporation cycle during steady state conditions. In one example, nucleotide incorporation can be detected at least twice per cycle of incorporation during steady state conditions. The sensor array may detect electrical signals during transient or steady state conditions. The electrical signal may include, but is not limited to, a change in the charge state of the molecule, a change in the conductivity of the surrounding solution, an impedance signal, or a change in an impedance signal. The sensor may detect a change in charge and/or conductivity or a change in impedance. The sensor can detect a change in charge and/or conductivity or impedance within a charge bilayer (e.g., debye length) of the sensor, support, or nucleic acid molecule (e.g., sample). Detectable labels coupled to individual nucleotides can alter the electrical environment surrounding the single-stranded nucleic acid molecule, and a given sensor can detect an electrical change. The sensor can detect an incorporation event (e.g., count incorporation events) or can resolve incorporated nucleotides individually (e.g., determine which nucleotide was incorporated).

One or more computer processors may be programmed to direct fluid flow through the sensor array. During fluid flow conditions, single-stranded nucleic acid molecules can be stably coupled to one or more surfaces. Single-stranded nucleic acid molecules can be stably coupled to a plurality of beads. The beads may be stably placed adjacent to the sensor array. The beads may be held adjacent to the sensor array by a magnetic or electric field. The fluid flow does not damage or dislodge the beads. The fluid directed through the sensor array may include nucleic acid molecules, primers, polymerizers, individual nucleotides, cofactors for nucleotide incorporation reactions (e.g., primer extension reactions), and/or buffers. The fluid may be a wash fluid comprising a buffer. In one example, a fluid may be directed to and incubated with a sensor array. The fluid may be incubated with the sensor array for the duration of a single cycle of the nucleotide incorporation reaction. Between incubation cycles, the sensor array may be washed with a washing fluid.

Computer system

The present disclosure provides a computer system programmed to implement the methods of the present disclosure. Fig. 16 illustrates a computer system programmed or otherwise configured to sequence a nucleic acid molecule. Computer system 1601 can regulate various aspects of the sequencing system of the present disclosure, such as, for example, controlling flow of nucleic acid templates to a sensor array, controlling flow of individual nucleotides to a sensor array, and controlling incorporation reaction conditions. The computer system 1601 can be a user's electronic device or a computer system that is remotely located with respect to the electronic device. The electronic device may be a mobile electronic device.

The computer system 1601 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1605, which may be a single or multi-core processor, or multiple processors for parallel processing. The computer system 1601 also includes a memory or memory location 1610 (e.g., random access memory, read only memory, flash memory), an electronic storage unit 1615 (e.g., hard disk), a communication interface 1620 for communicating with one or more other systems (e.g., a network adapter), and peripheral devices 1625 such as a cache, other memory, data storage, and/or an electronic display adapter. The memory 1610, storage unit 1615, interface 1620, and peripheral devices 1625 communicate with the CPU 1605 through a communication bus (solid line) such as a motherboard. The storage unit 1615 may be a data storage unit (or data store) for storing data. Computer system 1601 can be operatively coupled to a computer network ("network") 1630 by way of a communication interface 1620. Network 1630 may be the internet, an internet and/or an extranet, or an intranet and/or extranet in communication with the internet. In some cases, network 1630 is a telecommunications and/or data network. Network 1630 may include one or more computer servers, which may implement distributed computing, such as cloud computing. In some cases, network 1630 may implement a peer-to-peer network with computer system 1601, which may enable devices coupled to computer system 1601 to function as clients or servers.

CPU 1605 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 1610. The instructions may be directed to CPU 1605, which may then program CPU 1605 or otherwise configure CPU 1605 to implement the methods of the present disclosure. Examples of operations performed by CPU 1605 may include reading, decoding, executing, and writing back.

CPU 1605 may be part of a circuit such as an integrated circuit. One or more other components of system 1601 may be included in a circuit. In some cases, the circuit is an Application Specific Integrated Circuit (ASIC).

The storage unit 1615 may store files such as drivers, libraries, and saved programs. The storage unit 1615 may store user data, such as user preferences and user programs. In some cases, computer system 1601 can include one or more additional data storage units that are external to computer system 1601, such as on a remote server that communicates with computer system 1601 over an intranet or the internet.

Computer system 1601 canAnd communicates with one or more remote computer systems over the network 1630. For example, the computer system 1601 may communicate with a remote computer system of a user (e.g., the user's laptop or cell phone). Examples of remote computer systems include personal computers (e.g., laptop PCs), tablet or tablet computers (e.g.,iPad、galaxy Tab), telephone, smartphone (e.g.,iPhone, Android enabled device,) Or a personal digital assistant. A user may access computer system 1601 via network 1630.

The methods described herein may be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location (e.g., on memory 1610 or electronic storage unit 1615) of computer system 1601. The machine executable or machine readable code can be provided in the form of software. In use, the code may be executed by processor 1605. In some cases, the code may be retrieved from the storage unit 1615 and stored in the memory 1610 for ready access by the processor 1605. In some cases, the electronic storage unit 1615 may be eliminated, and the machine executable instructions stored on the memory 1610.

The code may be precompiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled at runtime. The code can be provided in a programming language that is selected to enable the code to be executed in a pre-compiled or just-in-time manner.

Aspects of the systems and methods provided herein, such as computer system 1601, may be embodied in programming. Various aspects of the technology may be considered as an "article of manufacture" or an "article of manufacture" in the form of machine (or processor) executable code and/or associated data that is typically carried or embodied in some type of machine-readable medium. The machine executable code may be stored on an electronic storage unit, such as a memory (e.g., read only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory, processors, etc. of a computer, or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., that may provide non-transitory storage for software programming at any time. All or portions of the software may at times communicate via the internet or various other telecommunications networks. Such communication may, for example, enable loading of software from one computer or processor to another computer or processor, e.g., from a management server or host to the computer platform of an application server. Thus, another type of media that can carry software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, over wired and optical land-line networks, and over various air links. The physical elements that carry these waves, such as wired or wireless links, optical links, etc., may also be considered to be media that carry software. As used herein, unless limited to a non-transitory tangible "storage" medium, terms such as a computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

Thus, a machine-readable medium, such as computer executable code, may take many forms, including but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any storage device such as in any computer, such as may be used to implement the databases shown in the figures, and so forth. Volatile storage media includes dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

Computer system 1601 can include or be in communication with an electronic display 1635 that includes a User Interface (UI)1640 for providing, for example, current operating conditions of the system or sequencing results. Examples of UIs include, but are not limited to, Graphical User Interfaces (GUIs) and web-based user interfaces.

The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented in software as the central processing unit 1605 executes. The algorithm may, for example, convert a signal indicative of nucleotide incorporation into a nucleic acid sequence.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited to the specific examples provided in the specification. While the invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Further, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.