U.S. patent application number 17/383716 was filed with the patent office on 2022-06-16 for systems and methods for nucleic acid sequencing.
The applicant listed for this patent is GenapSys, Inc.. Invention is credited to Hesaam Esfandyarpour, Maryam Jouzi, Paul Kenney, Seth Stern.
Application Number | 20220186303 17/383716 |
Document ID | / |
Family ID | 1000006181819 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220186303 |
Kind Code |
A1 |
Esfandyarpour; Hesaam ; et
al. |
June 16, 2022 |
SYSTEMS AND METHODS FOR NUCLEIC ACID SEQUENCING
Abstract
The present disclosure provides methods and systems for
sequencing nucleic acid molecules. Methods may include sequencing
double-stranded nucleic acids or single-stranded nucleic acids.
Sequencing may include the use of nucleotides coupled to
electrostatic moieties. The electrostatic moieties may be detected
by a sensor array. The electrostatic moieties may be reversible
electrostatic moieties that are cleaved from the nucleic acid
molecule after incorporation of the nucleotide. The electrostatic
moieties may be irreversible electrostatic moieties. Nucleotides
comprising irreversible electrostatic moieties may be incorporated
into the nucleic acid molecule, detected by the sensor array, and
exchanged for non-detectable nucleotides.
Inventors: |
Esfandyarpour; Hesaam;
(Redwood City, CA) ; Jouzi; Maryam; (Redwood City,
CA) ; Stern; Seth; (Menlo Park, CA) ; Kenney;
Paul; (Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GenapSys, Inc. |
Redwood City |
CA |
US |
|
|
Family ID: |
1000006181819 |
Appl. No.: |
17/383716 |
Filed: |
July 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17122049 |
Dec 15, 2020 |
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17383716 |
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16141215 |
Sep 25, 2018 |
10900075 |
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17122049 |
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PCT/US2018/052072 |
Sep 20, 2018 |
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16141215 |
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62561358 |
Sep 21, 2017 |
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62655083 |
Apr 9, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6825 20130101; C12Q 2565/30 20130101; C12Q 2563/107
20130101; C12Q 1/6837 20130101 |
International
Class: |
C12Q 1/6869 20060101
C12Q001/6869; C12Q 1/6825 20060101 C12Q001/6825 |
Claims
1-58. (canceled)
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 said plurality of single-stranded nucleic acid
molecules is disposed adjacent to a given sensor of said sensor
array; (b) subjecting said 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 said first single-stranded nucleic acid molecule,
wherein said nucleic acid incorporation reaction comprises
alternately and sequentially (i) incorporating individual
nucleotides of a first plurality of nucleotides comprising
detectable labels, and (ii) incorporating individual nucleotides of
a second plurality of nucleotides that do not comprise detectable
labels; and (c) while or subsequent to conducting said nucleic acid
incorporation reaction, using said given sensor to detect signals
indicative of a change in charge or conductivity from a double
layer comprising said detectable labels, thereby determining a
sequence or length of said first single-stranded nucleic acid
molecule.
60. The method of claim 59, wherein said first plurality of
nucleotides comprises a terminator that prevents an additional
nucleotide from stably hybridizing to said first single-stranded
nucleic acid molecule.
61. The method of claim 59, wherein said first plurality of
nucleotides comprises dideoxynucleotides.
62. The method of any of claims 59-61, wherein said second
plurality of nucleotides comprises a reversible terminator that
prevents an additional nucleotide from stably hybridizing to said
first single-stranded nucleic acid.
63. The method of claim 59, wherein said first plurality of
nucleotides is exchanged with said second plurality of
nucleotides.
64. The method of any of claims 59-63, wherein said incorporation
of said second plurality of nucleotides corrects phase error by
incorporating an individual nucleotide from said second plurality
of nucleotides at a location along said first single-stranded
nucleic acid molecule in which an individual nucleotide from said
first plurality of nucleotides has not been incorporated.
65. The method of claim 64, further comprising continuing said
nucleic acid incorporation reaction using said individual
nucleotides from said first plurality of nucleotides.
66. The method of claim 62, wherein said reversible terminator is
removed subsequent to incorporating said individual nucleotides of
said second plurality of nucleotides.
67. The method of any of claims 59-66, wherein said detectable
labels are electrostatic moieties that are not removable.
68. The method of any of claims 59-67, wherein said individual
nucleotides of said first plurality of nucleotides include
different types of nucleotides, each of which different types of
nucleotides is coupled to a single type of detectable label.
69. The method of any of claims 59-67, wherein said individual
nucleotides of said first plurality of nucleotides include
different types of nucleotides, each of which different types of
nucleotides is coupled to a different type of detectable label.
70. The method of claims 59-69, wherein said given sensor is
electrically coupled to a charge double layer comprising said first
single-stranded nucleic acid molecule.
71. The method of claim 59, wherein said plurality of
single-stranded nucleic acid molecules is coupled to a plurality of
beads, wherein said first single-stranded nucleic acid molecule is
coupled to a given bead of said plurality of beads, and wherein
said charge double layer is adjacent to a surface of said given
bead.
72. The method of claim 59, wherein said plurality of
single-stranded nucleic acid molecules is coupled to one or more
surfaces of said sensor array, wherein said first single-stranded
nucleic acid molecule is coupled to a surface of said given sensor,
and wherein said charge double layer is adjacent to said
surface.
73. The method of any of claims 59-72, wherein (b) further
comprises providing a priming site adjacent to said first
single-stranded nucleic acid and generating said second
single-stranded nucleic acid molecule upon primer extension from
said priming site.
74. The method of claim 73, wherein said priming site is a
self-priming loop.
75. The method of any of claims 59-74, wherein said given sensor
comprises at least two electrodes.
76. The method of any of claims 59-75, wherein said individual
nucleotides include different types of nucleotides, and wherein
said first single-stranded nucleic acid molecule is brought in
contact with said different types of nucleotides sequentially.
77. The method of claim 59, wherein at a given time point during
said nucleic acid incorporation reaction, said first
single-stranded nucleic acid molecule is brought in contact with
individual nucleotides of a first type, and at a subsequent time
point during said nucleic acid incorporation reaction, said segment
is brought in contact with individual nucleotides of a second type,
wherein said first type is different than said second type.
78. The method of any of claims 59-77, wherein said individual
nucleotides include different types of nucleotides, and wherein
said first single-stranded nucleic acid molecule is brought in
contact with said different types of nucleotides
simultaneously.
79.-108. (canceled)
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 17/122,049, filed Dec. 15, 2020, which is a continuation of
U.S. application Ser. No. 16/141,215, filed Sep. 25, 2018, now U.S.
Pat. No. 10,900,075, which is a continuation of International
Patent Application No. PCT/US2018/52072, filed Sep. 20, 2018, which
claims the benefit of U.S. Provisional Patent Application No.
62/561,358, filed Sep. 21, 2017, and U.S. Provisional Patent
Application No. 62/655,083, filed Apr. 9, 2018, each of which is
entirely incorporated herein by reference.
BACKGROUND
[0002] The goal to elucidate the entire human genome has created
interest in technologies for rapid nucleic acid (e.g., DNA)
sequencing, both for small and large scale applications. Important
parameters are sequencing speed, length of sequence that can be
read during a single sequencing run, and amount of nucleic acid
template required to generate sequencing information. Large scale
genome projects are currently too expensive to realistically be
carried out for a large number of subjects (e.g., patients).
Furthermore, as knowledge of the genetic basis for human diseases
increases, there will be an ever-increasing demand for accurate,
high-throughput DNA sequencing that is affordable for clinical
applications. Practical methods for determining the base pair
sequences of single molecules of nucleic acids, including those
with high speed and long read lengths, may provide measurement
capability.
[0003] Nucleic acid sequencing is a process that can be used to
provide sequence information for a nucleic acid sample. Such
sequence information may be helpful in diagnosing and/or treating a
subject with a condition. For example, the nucleic acid sequence of
a subject may be used to identify, diagnose and potentially develop
treatments for genetic diseases. As another example, research into
pathogens may lead to treatment for contagious diseases.
Unfortunately, though, existing sequencing technology of the status
quo is expensive and may not provide sequence information within a
time period and/or at an accuracy that may be sufficient to
diagnose and/or treat a subject with a condition.
SUMMARY
[0004] 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 may enable
sample preparation and identification (e.g., sequencing) without
the use of particles, such as beads. This may enable a sample to be
prepared and identified at substantially reduced cost and
complexity as compared to other systems and methods.
[0005] In an aspect, the present disclosure provides methods 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 nucleic acid molecules is disposed 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 with the first
single-stranded nucleic acid molecule, and wherein the given sensor
is electrically coupled to a charge double layer 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; bringing the segment in contact with
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 while conducting the nucleic acid incorporation
reaction, using the given sensor to detect signals indicative of
incorporation of the individual nucleotides into the third
single-stranded nucleic acid molecule, thereby determining a
sequence and/or a length of the segment.
[0006] In some embodiments, releasing the 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
after detecting the signals indicative of incorporation of the
individual nucleotides. In some embodiments, the flap is cleaved by
a flap endonuclease. In some embodiments, the flap endonuclease is
mesophilic.
[0007] 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
of the library of nucleic acid subunits comprises at least five
nucleotides. In some embodiments, the given nucleic acid subunit of
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.
[0008] 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 a portion thereof from the first single-stranded
nucleic molecule generates a detectable signal.
[0009] In some embodiments, the plurality of double-stranded
nucleic acid molecules is 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
double layer is adjacent to a surface of the given bead. In some
embodiments, the plurality of double-stranded nucleic acid
molecules is coupled to one or more surfaces of the sensor array.
In some embodiments, the given double-stranded nucleic acid
molecule is coupled to a surface of the given sensor and the charge
double layer is adjacent to the surface.
[0010] In some embodiments, the method 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. In some embodiments, the priming site is a primer
sequence having sequence complementarity with the first
single-stranded nucleic acid molecule. In some embodiments, the
method further comprises using a polymerizing enzyme to incorporate
the individual nucleotides. In some embodiments, the given sensor
comprises at least two electrodes.
[0011] In some embodiments, at least a subset of the individual
nucleotides comprises a reversible terminator that prevents an
additional nucleotide from stably hybridizing to the first
single-stranded nucleic acid molecule. In some embodiments, the
reversible terminator is removed after incorporation of the
individual nucleotide into the third single-stranded nucleic acid
molecule and prior to incorporation of another individual
nucleotide into the third single-stranded nucleic acid
molecule.
[0012] In some embodiments, at least a subset of the individual
nucleotides includes detectable labels. In some embodiments, the
detectable labels are electrostatic moieties. In some embodiments,
the detectable labels are coupled to nucleobases of the at least a
subset of the individual nucleotides. In some embodiments, the
individual nucleotides include different types of nucleotides, each
of which different types of nucleotides is reversibly coupled to a
single type of detectable label. In some embodiments, the
individual nucleotides include different types of nucleotides, each
of which different types of nucleotides is reversibly coupled to a
different type of detectable label. In some embodiments, the
detectable labels are reversibly coupled to the different types of
nucleotides by one or more coupling mechanisms. In some
embodiments, the detectable labels are reversibly coupled to the
different types of nucleotides by a single coupling mechanism. In
some embodiments, the detectable labels are removed after detection
of the signals indicative of incorporation of the individual
nucleotides. In some embodiments, the individual nucleotides
include different types of nucleotides and the segment is
sequentially brought in contact with the different types of
nucleotides.
[0013] In some embodiments, at a given time point during the
nucleic acid incorporation reaction, the segment is brought in
contact with individual nucleotides of a first type, and at a
subsequent time point during the nucleic acid incorporation
reaction, the segment is brought in contact with individual
nucleotides of a second type, wherein the first type is different
than the second type. In some embodiments, the individual
nucleotides include different types of nucleotides and the segment
is simultaneously brought in contact with the different types of
nucleotides.
[0014] In some embodiments, the signals indicative of incorporation
of the individual nucleotides are steady state signals. In some
embodiments, the signals indicative of incorporation of the
individual nucleotides are detected once after incorporation of an
individual nucleotide. In some embodiments, the signals indicative
of incorporation of the individual nucleotides are detected at
least twice after incorporation of an individual nucleotide. In
some embodiments, the signals indicative of incorporation of the
individual nucleotides are transient signals. In some embodiments,
the signals indicative of incorporation of the individual
nucleotides are electrical signals generated by an impedance or
impedance change in the charge double layer.
[0015] In some embodiments, the plurality of double-stranded
nucleic acid molecules is a clonal population of the
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.
[0016] In another aspect, the present disclosure provides methods
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
disposed adjacent to a given sensor of the sensor array, wherein
the given sensor is electrically coupled to a charge double layer
comprising the first single-stranded nucleic acid molecule;
bringing the first single-stranded nucleic acid molecule in contact
with individual nucleotides to subject the first single-stranded
nucleic acid molecule to a nucleic acid incorporation reaction
which 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 comprises detectable labels; and while
or subsequent to conducting the nucleic acid incorporation
reaction, using the given sensor to detect signals from the
detectable labels indicative of incorporation of the individual
nucleotides into the second single-stranded nucleic acid molecule,
thereby determining a sequence and/or a length of the first
single-stranded nucleic acid molecule.
[0017] In some embodiments, the plurality of single-stranded
nucleic acid molecules is 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 charge
double layer is adjacent to a surface of the given bead. In some
embodiments, the plurality of single-stranded nucleic acid
molecules is 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 the given sensor and the charge
double layer is adjacent to the surface.
[0018] In some embodiments, the method 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. In some embodiments,
the priming site is a primer sequence having sequence
complementarity with 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 using a
polymerizing enzyme to incorporate the individual nucleotides. In
some embodiments, the given sensor comprises at least two
electrodes.
[0019] In some embodiments, at least another subset of the
individual nucleotides comprises a reversible terminator that
prevents an additional nucleotide from stably hybridizing to the
first single-stranded nucleic acid molecule. In some embodiments,
the reversible terminator is removed after incorporation of the
individual nucleotide into the second single-stranded nucleic acid
molecule and prior to incorporation of another individual
nucleotide into the second single-stranded nucleic acid
molecule.
[0020] In some embodiments, the detectable labels are electrostatic
moieties. In some embodiments, the detectable labels are coupled to
nucleobases of the at least a subset of the individual nucleotides.
In some embodiments, the individual nucleotides include different
types of nucleotides, each of which different types of nucleotides
is reversibly coupled to a single type of detectable label. In some
embodiments, the individual nucleotides include different types of
nucleotides, each of which different types of nucleotides is
reversibly coupled to a different type of detectable label. In some
embodiments, the detectable labels are reversibly coupled to the
different types of nucleotides by one or more coupling mechanisms.
In some embodiments, the detectable labels are reversibly coupled
to the different types of nucleotides by a single coupling
mechanism. In some embodiments, the detectable labels are removed
after detection of the signals indicative of incorporation of the
individual nucleotides.
[0021] In some embodiments, the individual nucleotides include
different types of nucleotides and the first single-stranded
nucleic acid molecule is brought in contact with the different
types of nucleotides sequentially. In some embodiments, at a given
time point during the nucleic acid incorporation reaction, the
first single-stranded nucleic acid molecule is brought in contact
with individual nucleotides of a first type, and at a subsequent
time point during the nucleic acid incorporation reaction, the
first single-stranded nucleic acid molecule is brought in contact
with individual nucleotides of a second type, wherein the first
type is different than the second type. In some embodiments, the
individual nucleotides include different types of nucleotides and
the first single-stranded nucleic acid molecule is brought in
contact with the different types of nucleotides simultaneously.
[0022] In some embodiments, the signals indicative of incorporation
of the individual nucleotides are steady state signals. In some
embodiments, the signals indicative of incorporation of the
individual nucleotides are detected once after incorporation of an
individual nucleotide. In some embodiments, the signals indicative
of incorporation of the individual nucleotides are detected at
least twice after incorporation of an individual nucleotide. In
some embodiments, the signals indicative of incorporation of the
individual nucleotides are transient signals. In some embodiments,
the signals indicative of incorporation of the individual
nucleotides are electrical signals generated by an impedance or
impedance change in the charge double layer.
[0023] In some embodiments, the plurality of single-stranded
nucleic acid molecules is a clonal population of the 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.
[0024] In another aspect, the present disclosure provides methods
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
disposed 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 as 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 comprising detectable labels, and (ii) incorporating
individual nucleotides of a second plurality of nucleotides that do
not comprise detectable labels; and while or subsequent to
conducting the nucleic acid incorporation reaction, using the given
sensor to detect signals indicative of a change in charge or
conductivity from a double layer comprising the detectable labels,
thereby determining a sequence and/or a length of the first
single-stranded nucleic acid molecule.
[0025] In some embodiments, the first plurality of nucleotides
comprises a terminator that prevents an additional nucleotide from
stably hybridizing 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 an additional nucleotide from stably hybridizing to the
first single-stranded nucleic acid. In some embodiments, the
reversible terminator is removed after exchanging the individual
nucleotides of the first plurality of nucleotides with the
individual nucleotides of the second plurality of nucleotides.
[0026] 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 phase error by incorporating an individual
nucleotide from the second plurality of nucleotides at a location
along the first single-stranded nucleic acid molecule in which an
individual nucleotide from the first plurality of nucleotides has
not been incorporated. In some embodiments, the method further
comprises continuing the nucleic acid incorporation reaction using
the individual nucleotides from the first plurality of
nucleotides.
[0027] In some embodiments, the detectable labels are not
removable. In some embodiments, the detectable labels are
electrostatic moieties. In some embodiments, the detectable labels
are coupled to nucleobases of the individual nucleotides of the
first plurality of nucleotides. In some embodiments, the individual
nucleotides of the first plurality of nucleotides include different
types of nucleotides, each of which different types of nucleotides
is coupled to a single type of detectable label. In some
embodiments, the individual nucleotides of the first plurality of
nucleotides include different types of nucleotides, each of which
different types of nucleotides is coupled to a different type of
detectable label.
[0028] In some embodiments, the given sensor is electrically
coupled to a charge double layer comprising the first
single-stranded nucleic acid molecule. In some embodiments, the
plurality of single-stranded nucleic acid molecules is 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 charge double layer is adjacent to a surface of
the given bead. In some embodiments, the plurality of
single-stranded nucleic acid molecules is 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
the given sensor and the charge double layer is adjacent to the
surface.
[0029] In some embodiments, the method 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. In some embodiments,
the priming site is a primer sequence having sequence
complementarity with 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 using a
polymerizing enzyme to incorporate the individual nucleotides.
[0030] In some embodiments, the given sensor comprises at least two
electrodes. In some embodiments, the individual nucleotides include
different types of nucleotides and the first single-stranded
nucleic acid molecule is brought in contact with the different
types of nucleotides sequentially. In some embodiments, at a given
time point during the nucleic acid incorporation reaction, the
first single-stranded nucleic acid molecule is brought in contact
with individual nucleotides of a first type, and at a subsequent
time point during the nucleic acid incorporation reaction, the
segment is brought in contact with individual nucleotides of a
second type, wherein the first type is different than the second
type. In some embodiments, the individual nucleotides include
different types of nucleotides and the first single-stranded
nucleic acid molecule is brought in contact with the different
types of nucleotides simultaneously.
[0031] In some embodiments, the signals indicative of incorporation
of the individual nucleotides are steady state signals. In some
embodiments, the signals indicative of incorporation of the
individual nucleotides are detected once after incorporation of an
individual nucleotide. In some embodiments, the signals indicative
of incorporation of the individual nucleotides are detected at
least twice after incorporation of an individual nucleotide. In
some embodiments, the signals indicative of incorporation of the
individual nucleotides are transient signals. In some embodiments,
the signals indicative of incorporation of the individual
nucleotides are electrical signals generated by an impedance or
impedance change in the charge double layer.
[0032] In some embodiments, the plurality of single-stranded
nucleic acid molecules is a clonal population of the 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 the 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 a template single-stranded nucleic acid
molecule.
[0033] In another aspect, the present disclosure provides systems
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 disposed 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 with the first single-stranded
nucleic acid molecule, wherein the given sensor is electrically
coupled to a charge double layer 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) bring a segment of the first-single stranded
nucleic acid molecule that is not hybridized to the second
single-stranded nucleic acid molecule in contact with individual
nucleotides to subject the segment to a nucleic acid incorporation
reaction that generates the 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 (ii)
while or subsequent to conducting the nucleic acid incorporation
reaction, use the given sensor to detect signals indicative of
incorporation of the individual nucleotides into the third
single-stranded nucleic acid molecule, thereby determining a
sequence and/or a length of the segment.
[0034] In some embodiments, during use, the plurality of
double-stranded nucleic acid molecules is coupled to a plurality of
beads. In some embodiments, during use, the given double-stranded
nucleic acid molecule is coupled to a given bead of the plurality
of beads and the charge double layer is adjacent to a surface of
the given bead. In some embodiments, during use, the plurality of
double-stranded nucleic acid molecules is coupled to one or more
surfaces of the sensor array. In some embodiments, during use, the
given double-stranded nucleic acid molecule is coupled to a surface
of the given sensor and the charge double layer is adjacent to the
surface. In some embodiments, the given sensor comprises at least
two electrodes.
[0035] In some embodiments, during use, the signals indicative of
incorporation of the individual nucleotides are steady state
signals. In some embodiments, the signals indicative of
incorporation of the individual nucleotides are detected once after
incorporation of an individual nucleotide. In some embodiments, the
individual nucleotide incorporates detectable labels. In some
embodiments, the detectable labels are electrostatic moieties. In
some embodiments, the signals indicative of incorporation of the
individual nucleotides are detected at least twice after
incorporation of an individual nucleotide. In some embodiments,
during use, the signals indicative of incorporation of the
individual nucleotides are transient signals. In some embodiments,
during use, the signals indicative of incorporation of the
individual nucleotides are electrical signals generated by an
impedance or impedance change in the charge double layer.
[0036] In another aspect, the present disclosure provides systems
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 a plurality of
single-stranded nucleic acid molecules is disposed adjacent to a
given sensor of the sensor array, wherein the given sensor is
electrically coupled to a charge double layer 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) bring the first single-stranded nucleic acid
molecule in contact with individual nucleotides to subject the
first single-stranded nucleic acid molecule to a nucleic acid
incorporation reaction which 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
comprises detectable labels, and (ii) while or subsequent to
conducting the nucleic acid incorporation reaction, using the given
sensor to detect signals from the detectable labels indicative of
incorporation of the individual nucleotides into the second
single-stranded nucleic acid molecule, thereby determining a
sequence and/or a length of the first single-stranded nucleic acid
molecule.
[0037] In some embodiments, during use, the plurality of
single-stranded nucleic acid molecules is 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 double layer is adjacent to a surface of
the given bead. In some embodiments, during use, the plurality of
single-stranded nucleic acid molecules is 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 the given sensor and the charge double layer is adjacent to the
surface. In some embodiments, the given sensor comprises at least
two electrodes. In some embodiments, the detectable labels are
electrostatic moieties.
[0038] In some embodiments, during use, the signals indicative of
incorporation of the individual nucleotides are steady state
signals. In some embodiments, the signals indicative of
incorporation of the individual nucleotides are detected once after
incorporation of the individual nucleotide. In some embodiments,
the signals indicative of incorporation of the individual
nucleotides are detected at least twice after incorporation of the
individual nucleotide. In some embodiments, during use, the signals
indicative of incorporation of the individual nucleotides are
transient signals. In some embodiments, during use, the signals
indicative of incorporation of the individual nucleotides are
electrical signals generated by an impedance or impedance change in
the charge double layer.
[0039] In another aspect, the present disclosure provides systems
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 a plurality of
single-stranded nucleic acid molecules is disposed 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) bring the first single-stranded nucleic acid
molecule in contact 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
detectable labels and exchanging the individual nucleotides of the
first plurality of nucleotides with individual nucleotides of a
second plurality of nucleotides that do not comprise detectable
labels and (ii) while or subsequent to conducting the nucleic acid
incorporation reaction, using the given sensor to detect signals
indicative of a change in charge or conductivity from a double
layer comprising the detectable labels, thereby determining a
sequence and/or a length of the first single-stranded nucleic acid
molecule.
[0040] In some embodiments, during use, the given sensor is
electrically coupled to a charge double layer comprising the first
single-stranded nucleic acid molecule. In some embodiments, during
use, the plurality of single-stranded nucleic acid molecules is
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 double layer is
adjacent to a surface of the given bead. In some embodiments,
during use, the plurality of single-stranded nucleic acid molecules
is 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 the given sensor and the charge
double layer is adjacent to the surface. In some embodiments, the
given sensor comprises at least two electrodes. In some
embodiments, the detectable labels are electrostatic moieties.
[0041] In some embodiments, during use, the signals indicative of
incorporation of the individual nucleotides are steady state
signals. In some embodiments, the signals indicative of
incorporation of the individual nucleotides are detected once after
incorporation of an individual nucleotide. In some embodiments, the
signals indicative of incorporation of the individual nucleotides
are detected at least twice after incorporation of an individual
nucleotide. In some embodiments, during use the signals indicative
of incorporation of the individual nucleotides are transient
signals. In some embodiments, during use the signals indicative of
incorporation of the individual nucleotides are electrical signals
generated by an impedance or impedance change in the charge double
layer.
[0042] Additional aspects and advantages of the present disclosure
will become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present 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.
INCORPORATION BY REFERENCE
[0043] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The novel features 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 "figure" and
"FIG." herein), of which:
[0045] FIG. 1 shows a model illustration of unstructured template
nucleic acid molecules coupled to a bead;
[0046] FIG. 2 shows a model illustration of structured template
nucleic acid molecules coupled to a bead;
[0047] FIG. 3 shows an example process flow for relaxed template
sequencing;
[0048] FIGS. 4A-4D show examples of double-stranded sequencing
methods and sequencing results using labeled and non-labeled
nucleotides; FIG. 4A shows an example comparison between
single-stranded and double-stranded sequencing results; FIG. 4B
shows example sequencing results of double-stranded sequencing
using polyanion electrostatic moieties; FIG. 4C shows example
sequencing results of double-stranded sequencing using polycation
electrostatic moieties; FIG. 4D shows example sequencing results of
double-stranded sequencing using both polyanion and polycation
electrostatic moieties;
[0049] FIG. 5 shows an example method for double-stranded
sequencing;
[0050] FIG. 6 shows an example method for double-stranded
sequencing using random hexamers;
[0051] FIGS. 7A and 7B show example methods for double-stranded
sequencing with reversible terminators; FIG. 7A shows an example
method for double-stranded sequencing using reversible terminators
and flap endonucleases; FIG. 7B shows an example method for
double-stranded sequencing using reversible terminators and nucleic
acid subunits;
[0052] FIG. 8 shows an example sequencing method using a different
type of electrostatic moiety for each type of nucleotide;
[0053] FIG. 9 shows an example sequencing method using a single
type of electrostatic moiety for each type of nucleotide;
[0054] FIG. 10 shows an example method for sequencing using
electrostatic moieties and reversible terminators;
[0055] FIGS. 11A and 11B show example methods for double-stranded
sequencing using detectable labels on the second single-stranded
nucleic acid molecule; FIG. 11A shows an example sequencing method
using detectable labels that are cleaved by a flap endonuclease;
FIG. 11B shows an example sequencing method using detectable labels
and reversible terminators;
[0056] FIGS. 12A and 12B shows example methods for double-stranded
sequencing using detectable labels and flap endonucleases; FIG. 12A
shows an example method for double-stranded sequencing using
detectable labels and a mesophilic flap endonuclease; FIG. 12B
shows an example method for double-stranded sequencing using
detectable labels and a thermostable flap endonuclease;
[0057] FIGS. 13A and 13B shows example methods for double-stranded
sequencing method using detectable labels, a flap endonuclease, and
reversible terminators; FIG. 13A shows an example method for
double-stranded sequencing using detectable labels, a mesophilic
flap endonuclease, and reversible terminators; FIG. 13B shows an
example method for double-stranded sequencing using detectable
labels, a thermostable flap endonuclease, and reversible
terminators;
[0058] FIG. 14A shows an example method for double-stranded
sequencing using detectable labels and nucleic acid subunits;
[0059] FIG. 14B shows an example method for double-stranded
sequencing using detectable labels, nucleic acid subunits, and
reversible terminators;
[0060] FIG. 15 shows an example method for pyrophosphorolysis
mediated terminator exchange sequencing;
[0061] FIG. 16 shows a computer system that is programmed or
otherwise configured to implement methods provided herein;
[0062] FIG. 17 shows an example of a modified nucleotide comprising
a detectable label or effector coupled to a nucleobase via a
linker;
[0063] FIGS. 18A-C show examples of detectable labels; FIG. 18A
shows an example of a polycation electrostatic moiety with a lysine
residue; FIG. 18B shows an example of a polyanion electrostatic
moiety with a carboxylic acid group; FIG. 18C shows an example of a
switch label comprising histidine imidazole residues that can
switch between a neutral state and a positive state in response to
pH of the buffer;
[0064] FIG. 19 shows activity of polymerizing enzymes in different
salt concentrations and in the presence or absence of polyethylene
glycol (PEG);
[0065] FIG. 20 illustrates a method for correcting phase error
during a sequencing reaction;
[0066] FIG. 21 is an example of a method for correcting phase
error; and
[0067] FIG. 22 is another example of a method for correcting phase
error.
DETAILED DESCRIPTION
[0068] While various embodiments of the 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 may 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.
[0069] The term "adjacent to," as used herein, generally refers to
next to, in proximity to, or in sensing or electronic vicinity (or
proximity) of. For example, a first object adjacent to a second can
be in contact with the second object, or may not be in contact with
the second object but may be in proximity to the second object. In
some examples, a first object to a second object is within about 0
micrometers ("microns"), 0.001 microns, 0.01 microns, 0.1 microns,
0.2 microns, 0.3 microns, 0.4 microns, 0.5 microns, 1 microns, 2
microns, 3 microns, 4 microns, 5 microns, 10 microns, or 100
microns of the second object.
[0070] The term "nucleic acid," as used herein, generally refers to
a molecule comprising one or more nucleic acid subunits. A nucleic
acid may include one or more subunits selected from adenosine (A),
cytosine (C), guanine (G), thymine (TO, and uracil (U), or variants
thereof. A nucleotide can include A, C, G, T, or U, or variants
thereof. A nucleotide can include any subunit that can be
incorporated into a growing nucleic acid strand. Such subunit can
be A, C, G, T, or U, or any other subunit that is specific to one
of 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, a nucleic acid may
be single-stranded or double stranded, in some cases, a nucleic
acid molecule is circular.
[0071] The terms "nucleic acid molecule," "nucleic acid sequence,"
"nucleic acid fragment," "oligonucleotide," "oligo," and
"polynucleotide," as used herein, generally refer to a polymeric
form of nucleotides that may have various lengths, either
deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs
thereof. An oligonucleotide is typically composed of a specific
sequence of four nucleotide bases: adenine (A); cytosine (C);
guanine (G); and thymine (T) (uracil (U) for thymine (T) when the
polynucleotide is RNA). Thus, the term "oligonucleotide sequence"
is the alphabetical representation of a polynucleotide molecule;
alternatively, the term may be applied to the polynucleotide
molecule itself. This alphabetical representation can be input into
databases in a computer having a central processing unit and used
for bioinformatics applications such as functional genomics and
homology searching. Oligonucleotides may include one or more
non-standard nucleotide(s), nucleotide analog(s) and/or modified
nucleotides. In some cases, an oligo may refer to a short
single-stranded nucleic acid sequence with at most 300 base pairs
(bp), at most 200 bp, at most 100 bp, at most 90 bp, at most 80 bp,
at most 70 bp, at most 60 bp, at most 50 bp, at most 40 bp, at most
30 bp, at most 20 bp, at most 10 bp or less. In some cases, an
oligo may have a --C6-NH.sub.2 functional group at its 3' or 5' end
suitable for conjugation.
[0072] Examples of modified nucleotides include, but are not
limited to diaminopurine, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methyl cytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-D46-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine
or the like. Nucleic acid molecules may also be modified at the
base moiety (e.g., at one or more atoms that typically are
available to form a hydrogen bond with a complementary nucleotide
and/or at one or more atoms that are not typically capable of
forming a hydrogen bond with a complementary nucleotide), sugar
moiety or phosphate backbone. Nucleic acid molecules may also
contain amine-modified groups, such as aminoallyl-dUTP (aa-dUTP)
and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent
attachment of amine reactive moieties, such as N-hydroxy
succinimide esters (NHS). Alternatives to standard DNA base pairs
or RNA base pairs in the oligonucleotides of the present disclosure
can provide higher density in bits per cubic mm, higher safety
(resistant to accidental or purposeful synthesis of natural
toxins), easier discrimination in photo-programmed polymerases, or
lower secondary structure. Such alternative base pairs compatible
with natural and mutant polymerases for de novo and/or
amplification synthesis are described in Betz K, Malyshev D A,
Lavergne T, Welte W, Diederichs K, Dwyer T J, Ordoukhanian P,
Romesberg F E, Marx A (2012).
[0073] The term "nucleotide," as used herein, generally refers to
an organic molecule that serves as the monomer, or subunit, of a
nucleic acid molecule, such as a deoxyribonucleic (DNA) molecule or
ribonucleic acid (RNA) molecule. In some embodiments, a nucleotide
may also be a peptide nucleic acid (PNA) nucleotide, a locked
nucleic acid (LNA) nucleotide, or a dideoxynucleotide.
[0074] The term "primer," as used herein, generally refers to a
strand of nucleic acid that serves as a starting point for nucleic
acid synthesis, such as polymerase chain reaction (PCR). In an
example, during replication of a DNA sample, an enzyme that
catalyzes replication starts replication at the 3'-end of a primer
attached to the DNA sample and copies the opposite strand.
[0075] The term "polymerizing enzyme," as used herein, generally
refers to any enzyme capable of catalyzing a polymerization
reaction. Examples of polymerases include, without limitation, a
nucleic acid polymerase. The polymerase can be naturally occurring
or synthesized. An example polymerase is a .PHI.29 polymerase or
derivative thereof. A polymerase can be a polymerization enzyme. In
some cases, a transcriptase or a ligase is used (i.e., enzymes
which catalyze the formation of a bond). Examples of polymerases
include a DNA polymerase, and RNA polymerase, a thermostable
polymerase, a wild-type polymerase, a modified polymerase, E. coli
DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA
polymerase .PHI.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 polymerases, 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,
modified products and derivatives thereof. In some embodiments, the
polymerase is a single subunit polymerase. The polymerase can have
high processivity, namely the capability of the polymerase to
consecutively incorporate nucleotides in a nucleic acid template
without releasing the nucleic acid template.
[0076] The term "detectable label," as used herein, generally
refers to any detectable moiety that is coupled to a molecule to be
detected. Non-limiting examples of detectable labels may include
electrostatic moieties, fluorescence moieties, chemiluminescence
moieties, radio moieties, colorimetric moieties, or any combination
thereof. Detectable labels may be reversibly or irreversibly
coupled to a molecule to be detected. Such moieties may be labels.
Examples of electrostatic moieties include charge labels.
Detectable labels may be coupled to a nucleobase at a C5 or C7
position. For example, a reversible electrostatic moiety may be
coupled to a nucleotide that is incorporated into a nucleic acid
molecule.
[0077] A detectable label may be coupled to a nucleobase via a
linker. A linker may be coupled to a nucleobase at a C5 or C7
position. The linker may be a non-nucleotide molecule. The linker
may be acid labile, photolabile or contain a disulfide linkage. 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 an enzyme. In some examples, the detectable
linker is at a distance of at least about 1 nanometer (nm), 2 nm, 3
nm, 4 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm,
300 nm, 400 nm, 500 nm, or greater from the nucleotide. FIG. 17
shows an example of a modified nucleotide with a detectable label
coupled to a nucleotide via a linker. In this example, the
detectable label may also be referred to as an effector molecule
since the detectable label may affect the charge distribution
around the nucleotide.
[0078] The term "electrostatic moiety," as used herein, generally
refers to a detectable label comprising a net positive or negative
charge, or a moiety attached to a chemical or biological unit that
renders the chemical or biological unit detectable. For example, an
electrostatic moiety may include a charged functional group, a part
of a functional group having a charge, a charge label, or a charged
molecule as a detectable label. The electrostatic moiety may be
monovalent (e.g., have a +1 or -1 charge) or polyvalent (e.g., have
a +2, +3, +4, +5, +6, etc. or -1, -2, -3, -4, -5, -6, etc. charge).
The electrostatic moiety may have a net positive charge or a
negative charge. The electrostatic moiety may have one or more
anionic or cationic charge groups. In an example, the electrostatic
moiety has both anionic and cationic charge groups 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 an example, the
electrostatic moiety switches or changes charge as a function of
solution conditions (e.g., pH, temperature, etc.).
[0079] The term "clonal," as used herein, generally refers to at
least some, substantially all, or all, of the populations of a
sensor area being of the same nucleic acid sequence. There may be
two population associated with a single sample nucleic acid
fragment, as may be used for "mate pairs," "paired ends", or other
similar methodologies; the populations may be present in roughly
similar numbers in the sensor area, and may be randomly distributed
over the sensor area.
[0080] The term "phase error," as used herein, generally refers to
an error or difference between a given polynucleotide sequence
(e.g., second or third single-stranded nucleic acid molecule) and a
template nucleic acid molecule from which the given polynucleotide
sequence is derived. The given polynucleotide sequence may be a
part of a clonal population and the given nucleotide sequence may
have a longer or shorter sequence than the consensus state (e.g.,
reference sequence) of the clonal population. A phase error may be
a leading or a lagging phase error. A leading phase error may
include additional nucleotide bases that are not present in the
consensus (e.g., reference) sequence. A lagging phase error may
include fewer nucleotide bases relative to the consensus (e.g.,
reference) sequence. Phase error may be a product of
misincorporation or lack of incorporation of nucleotide bases by a
polymerizing enzyme. Phase error may limit the read length of a
sequencing system.
[0081] The term "flap," as used herein, generally refers to a
portion of a single-stranded nucleic acid molecule that is not
hybridized or associated with another single-stranded nucleic acid
molecule while a portion of the single-stranded nucleic acid
molecule is hybridized or associated with the other single-stranded
nucleic acid molecule. A flap may be at least about 1, 2, 3, 4, 5,
6, 8, 10, 12, 15, 20, 30, 40, 50 or more nucleotide bases in
length.
[0082] Whenever the term "at least," "greater than," or "greater
than or equal to" precedes the first numerical value in a series of
two or more numerical values, the term "at least," "greater than"
or "greater than or equal to" applies to each of the numerical
values in that series of numerical values. 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.
[0083] Whenever the term "no more than," "less than," or "less than
or equal to" precedes the first numerical value in a series of two
or more numerical values, the term "no more than," "less than," or
"less than or equal to" applies to each of the numerical values in
that series of numerical values. 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.
Methods for Nucleic Acid Sequencing
[0084] In an aspect, the present disclosure provides a method for
nucleic acid sequencing. The method may comprise providing a
plurality of double-stranded nucleic acid molecules adjacent to a
sensor array. A given, or individual, double-stranded nucleic acid
molecule may be disposed adjacent to a given, or individual, sensor
of the sensor array. The double-stranded nucleic acid molecule may
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 may have sequence
complementarity with one another. The sensor may be electrically
coupled to a charge double layer (e.g., within a Debye length) of
the double-stranded nucleic acid molecule. A portion of the second
single-stranded nucleic acid molecule may be released 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. The
segment may be brought in contact with an individual nucleotide.
The individual nucleotide may be subject to a nucleic acid
incorporation reaction that generates a third single-stranded
nucleic acid molecule. The third single-stranded nucleic acid
molecule may have sequence complementarity with the first
single-stranded nucleic acid molecule. During the nucleic acid
incorporation reaction, the sensor may be used to detect signals
indicative of incorporation of the individual nucleotides into the
third single-stranded nucleic acid molecule, thereby determining a
sequence or a length of the non-hybridized segment.
[0085] In another aspect, the present disclosure may provide
methods for detecting a nucleic acid molecule. The method may
comprise providing a plurality of single-stranded nucleic acid
molecules adjacent to a sensor array, bringing the first
single-stranded nucleic acid molecule in contact with individual
nucleotides to subject the first single-stranded nucleic acid
molecule to a nucleic acid incorporation reaction which generates a
second single-stranded nucleic acid molecule from the individual
nucleotides, and while or subsequent to conducting the nucleic acid
incorporation reaction, using the given sensor to detect signals
from the detectable labels indicative of incorporation of the
individual nucleotides into the second single-stranded nucleic acid
molecule, thereby determining a sequence and/or a 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 may be disposed adjacent to
a given sensor of the sensor array. The given sensor may be
electrically coupled to a charge double layer (e.g., within a Debye
length) of the first single-stranded nucleic acid molecule. The
second single-stranded nucleic acid molecule may have sequence
complementarity with the first single-stranded nucleic acid
molecule. At least a subset of the individual nucleotides may
comprise detectable labels.
[0086] In another aspect, the present disclosure may provide
methods for nucleic acid sequencing. The methods may comprise
providing a plurality of single-stranded nucleic acid molecules
adjacent to a 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 as a
growing strand complementary to the first single-stranded nucleic
acid molecule, and while or subsequent to conducting the nucleic
acid incorporation reaction, using the given sensor to detect
signals from the detectable labels indicative of incorporation of
the individual nucleotides of the first plurality of nucleotides
into the second single-stranded nucleic acid molecule, thereby
determining a sequence or a 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
may be disposed adjacent to a given sensor of the sensor array. The
nucleic acid incorporation reaction may comprise alternately and
sequentially (i) incorporating individual nucleotides of a first
plurality of nucleotides comprising detectable labels, and (ii)
incorporating individual nucleotides of a second plurality of
nucleotides that do not comprise detectable labels.
[0087] The systems and methods described herein may be used to
detect biological molecules and reactions. For example, the systems
and methods described may be used to detect binding events,
reactions and reaction products, and/or the presence or absence of
biological molecule. In an example the systems and methods may be
used to determine a sequence of a nucleic acid molecule. In another
example, the systems and methods may be used to determine a length
(e.g., the number of nucleotides) of a nucleic acid molecule. In an
example, the systems and method may be used to determine both a
sequence and a length of a target nucleic acid molecule. The
systems and methods may be used to detect nucleic acid
polymorphisms such as, but not limited to, misincorporated
nucleotides, changes in fragment size, repeated nucleotide
sequences, and/or deleted nucleotide sequences. Determining a
length of a nucleic acid molecule may have applications for
healthcare, such as diagnostics (e.g., cancer detection). For
example, the systems and methods may be used to detect
microsatellite instability by detecting increases in fragment
length.
[0088] Sequencing or determining a length of nucleic acid molecules
may utilize nucleic acid templates free in solution or coupled to a
support. The support may include a bead, planar surface, well, or
any other structure capable of coupling to a nucleic acid molecule.
The support may be positioned near a sensor of a sensor array.
Alternatively, or in addition to, the support may be a part of a
sensor of a sensor array (e.g., an electrode, passivation layer,
dielectric layer, etc.). The nucleic acid template coupled to the
support may be unstructured (e.g., extend linearly from the support
surface) or may be structured (e.g., form loops, hairpins, and/or
other secondary structure). FIG. 1 shows an example of an
unstructured nucleic acid template coupled to a bead. The bead may
be coupled to a single nucleic acid template or coupled to multiple
nucleic acid templates. The unstructured templates may not interact
with one another around the surface of the bead. Alternatively, or
in addition to, the unstructured nucleic acid templates may
interact with each other around the surface of the bead. Nucleic
acid templates that do not interact may generate monotonic signals
(e.g., each nucleotide incorporated generates a constant signal)
during sequencing. FIG. 2 shows an example of a structured nucleic
acid template 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 to it. In an
example, the bead is coupled to multiple nucleic acid templates and
the nucleic acid templates may interact with each other. Nucleic
acid templates that interact with each other may generate
non-monotonic signals (e.g., each nucleotide incorporated generates
a different, non-linear signal) during sequencing.
[0089] Structured nucleic acid templates may be unstructured or
relaxed prior to sequencing to generate monotonic signals. The
template structure may be relaxed prior to nucleotide incorporation
(e.g., a primer extension reaction) or prior to reading or
detecting an incorporation event. FIG. 3 shows an example method
for relaxed template sequencing. The structured template may
include random coils, secondary structure, and/or hairpins. In an
example, the template includes a self-priming loop. The
self-priming loop may be a hairpin structure that permits the
single-stranded nucleic acid structure to be extended without a
separate primer sequence. In the structured state, the self-priming
loop may be arranged to facilitate a primer extension reaction
through Loop-mediated amplification (LAMP). The self-priming loop
may facilitate the incorporation of a nucleotide into the 3-prime
end of the nucleic acid template. Alternatively, or in addition to,
the self-priming loop may incorporate a nucleotide into the 5-prime
end of the nucleic acid template. After incorporation of a
nucleotide, the structure of the nucleic acid template may be
relaxed. The template structure may be relaxed by altering the
solution conditions, including, but not limited to, applying heat,
altering the pH, altering the ionic strength, and/or introducing
one or more organic solvents (e.g., formamide or urea) to the
solution. The relaxed nucleic acid template may then be read to
detect the nucleotide incorporation. The detected signal may be a
linear, or monotonic, signal.
[0090] Signal linearity may be increased using double-stranded
sequencing. Double-stranded sequencing may include a
double-stranded nucleic acid template free in solution or coupled
to a support. The double-stranded nucleic acid template may have a
secondary structure, such as a double helix structure. The double
helix structure may reduce or prevent interactions between
double-stranded nucleic acid templates coupled to the same support.
Reducing or preventing interactions between the double-stranded
nucleic acid templates may increase the linearity of the signal
detected during sequencing. Additionally, combining double-stranded
sequencing with nucleotides comprising detectable labels may both
increase linearity and increase the signal-to-noise ratio. FIGS.
4A-4D show examples of double-stranded sequencing methods and
examples of sequencing results using labeled and non-labeled
nucleotides. FIG. 4A shows an example comparison between
single-stranded 401 and double-stranded 402 sequencing results. The
single-stranded sequencing 401 example shows signal that is both
positive and negative with respect to the y-axis of the plot and
varies non-monotonically. The double-stranded sequencing 402
example shows signal that is positive with respect to the y-axis of
the plot and varies monotonically with the number of nucleotides
incorporated. FIG. 4B shows example sequencing results for
double-stranded sequencing using polyanion electrostatic moieties.
Polyanion electrostatic moieties may comprise one or more of a
phosphate, phosphonate, sulfate, sulfonate, boronate, or
carboxylate group. The detected signal in this example is both
positive with respect to the y-axis and monotonic. Additionally,
the detected signal may be outside the detectable signal noise
(e.g., has a high signal-to-noise ratio). FIG. 4C shows example
sequencing results of double-stranded sequencing using polycation
electrostatic moieties. Polycation electrostatic moieties may
comprise one or more of a pyridinium, imidazolium, guanidinium,
iminium, primary amine, secondary amine, tertiary amine, or
quaternary ammonium. As with polyanion electrostatic moieties,
polycation electrostatic moieties may generate signals that are
outside the detectable signal noise. However, polycation
electrostatic moieties may generate signals that are negative or
opposite to the signals generated with a polyanion electrostatic
moiety. FIG. 4D shows example sequencing results of double-stranded
sequencing using both polyanion and polycation electrostatic
moieties. The polyanion and polycation electrostatic moieties may
generate detectable signals that are outside the detectable signal
noise and that are both positive and negative (e.g., opposite
signal direction with respect to one another).
[0091] Polycation electrostatic moieties may be useful for
improving single-to-noise ratio, such as during sequencing.
Detectable labels, such as polycation or polyanion electrostatic
moieties, may be useful in generating a monotonic signal, i.e. a
linear signal when compared with signals from unmodified
nucleotides. The linearizing signal may be due to structural
transitions of a nucleic acid molecule caused by a detectable
label. The structural transitions may lead to the changes in ion
distribution around the nucleic acid molecule, resulting in a
signal that is of the same magnitude as the signal generated by
single nucleotide incorporation.
[0092] Polycation electrostatic moieties may comprise amines or
amino acid residues, such as lysine, histidine, arginine, or any
combination thereof. Polycation electrostatic moieties may displace
or repel other polycations, such as magnesium ions (Mg'), from the
vicinity of a nucleic acid molecule. The displacement of other
polycations may result in a lower conduction current, which may be
detected as a negative signal by a sensor. Polyanion electrostatic
moieties, such as carboxylic acid groups, may attract or
concentrate polycations, such as Mg', around a nucleic acid
molecule. The detectable label may comprise a charge group. The
detectable label may be monovalent (e.g., have a single positive or
negative charge, such as, e.g., +1 or -1) or polyvalent (e.g., have
multiple positive or negative charges, such as, e.g., +2 or -2).
The detectable label, such as a polycation or polyanion detectable
label, may have from about one to about fifty or more positive or
negative charges. In some cases, the detectable label may 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 include from 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 an example, a
detectable label may be a polycation electrostatic moiety
comprising three lysine residues, six lysine residues, or more than
six lysine residues. In another example, a detectable label may be
a polyanion electrostatic moiety comprising three carboxylic acid
groups, six carboxylic acid groups or more than six carboxylic acid
groups. The higher concentration of polycation electrostatic
moieties may result in a higher conduction current, which may be
detected as a positive signal by a sensor. The number of
polycations or polyanions in a detectable label may correlate with
the strength of a signal as detected by a sensor. For example, a
detectable label with six lysine residues (e.g., K6 label) may
produce a stronger negative signal compared to a detectable label
with three lysine groups (i.e., K3 label). Similarly, six
carboxylic acid groups may produce a stronger positive signal as
compared to three carboxylic acid groups in a detectable label. A
larger charge state of a detectable label may lead to greater
non-specific binding to surfaces, such as glassware. For example, a
K6 label may have a higher charge state than a K3 label and,
therefore, the K6 label may have greater non-specific binding
compared to the K3 label. An example of polycation electrostatic
moiety with a lysine residue is shown in FIG. 18A. An example of
polyanion electrostatic moiety with a carboxylic acid group is
shown in FIG. 18B.
[0093] Detectable labels may be switchable between a charged state
and a neutral state or between one charge state and another charge
state (e.g., positive to negative charge or negative to positive
charge). The detectable label may switch a charge state in response
to solution conditions, such as buffer conditions, e.g., such as pH
or ionic strength of the buffer. Switchable detectable labels may
be in a charged state during nucleic acid incorporation reaction
(e.g., during signal detection) and may be in a neutral state rest
of the time. In an example, nucleotide incorporation is detected
during an incorporation event and the detectable labels may be
charged during incorporation. In another example, nucleotide
incorporation may be detected subsequent to nucleotide
incorporation and the detectable label may not be charged during
incorporation, but may be switched such that the detectable labels
are charged during detection. In another example, the detectable
labels have one charge during incorporation (e.g., positive,
negative, or neutral) and are switched to have another charge
during detection (e.g., negative or positive). Switch labels may be
useful in reducing non-specific binding compared to the detectable
label that remain in a charged state throughout the process, a K6
label, for example. An example of a histidine switch label is shown
in FIG. 18C. As shown in FIG. 18C, a switch label may comprise
histidine imidazole residues that can switch between a neutral
state and a positive state in response to pH of the buffer. Example
switch labels may include detectable labels with greater than or
equal to 1, 2, 3, 4, 5, 6, 8, 10, 12, or more histidine groups. In
an example, a detectable label has three histidine groups (e.g.,
H3), six histidine groups (e.g., H6), or more than six histidine
groups. The switch label may be in a neutral state when the pH is
equal to or greater than 7. The switch label may be in a positive
state when the pH is equal to or less than 5. When the switch label
is in a neutral state, the label may not non-specifically bind to
surfaces and may have greater mobility when compared to the label
in a positive state. Switch labels may be kept in a positive state
during signal detection by a sensor in order immobilize the label.
Switch labels may be maintained in a neutral state when the
nucleotide is directed towards and/or away (e.g., when the
nucleotide is mobile within the system) from a target nucleic acid
molecule.
[0094] Non-specific binding of detectable labels may be reduced by
altering reaction conditions. For example, a K6 label may be used
along with a high concentration of low affinity peptides, such as a
K3 label. In such situation, the K6 label may exhibit reduced
non-specific binding due to competition for binding surfaces from
the low affinity peptides. In some cases, non-specific binding may
be reduced by using high ionic strength buffers. For example,
buffer with 200 mM potassium chloride (KCl) may reduce non-specific
binding by a K6 label, in turn, mobilizing the K6 label to maintain
the K6 label in solution.
[0095] Polymerizing enzymes may be kinetically active in altered
reaction conditions used with the switch labels and/or with the
nucleotides comprising the detectable label. In some cases,
polymerizing enzymes may be selected based on the kinetic activity
and/or compatibility with the detectable label. For example, Type B
polymerases, such as 9.degree. N, RB69, KOD polymerases with larger
binding pockets may be used with large detectable labels. In some
cases, polymerizing enzymes that can tolerate high ionic strength
buffers, such as Type B polymerases, Therminator IX, Bst 3.0,
.PHI.29, Taq polymerase, may be used with high salt buffers and
with polycation electrostatic moieties, such as a K6 label.
Tolerance of polymerizing enzymes may be improved by adding volume
excluders, such as, for example, polyethylene glycol (PEG),
dextran, or similar compounds. As shown in FIG. 19, polymerizing
enzymes may exhibit improved salt tolerance during nucleic acid
incorporation reaction in the presence of PEG. A template may be
coupled to a bead. A primer may be complementary to the 3' end of
the template strand. A primer may be fluorescently labeled with
6-FAM fluorophore and extended by incorporation of individual
nucleotides. The primer extension reaction may be detected by a
sensor.
[0096] The primer extension reaction may be facilitated by a
polymerizing enzyme, such as, for example, thermostable
polymerizing enzymes. Examples of polymerase enzymes that may be
used for extension reactions include, but not limited to, Thermus
thermophilus HB8, mutant Thermus oshimai, Thermus scotoductus;
Thermus thermophilus 1B21, Thermus thermophilus GK24, Thermus
aquaticus polymerase (AmpliTaq.RTM. FS or Taq (G46D, F667Y), Taq
(G46D, F667Y, E6811), and Taq (G46D, F667Y, T664N, R660G),
Pyrococcus furiosus polymerase, Thermococcus gorgonarius
polymerase, Pyrococcus species GB-D polymerase, Thermococcus sp.
(strain 9.degree. N-7) polymerase, Bacillus stearothermophilus
polymerase (Bst), Bacillus caldotenax DNA polymerase (Bca) Tsp
polymerase, ThermalAce.TM. polymerase (Invitrogen), Thermus flavus
polymerase, Thermus litoralis polymerase, Thermus Z05 polymerase,
delta Z05 polymerase (e.g. delta Z05 Gold DNA polymerase),
Sulfolobus DNA Polymerase IV, or mutants, variants, or derivatives
thereof. Additional examples of polymerase enzymes that may be used
for primer extension reactions are non-thermostable polymerases,
including, but not limited to, DNA polymerase I, mutant DNA
polymerase I, including, but not limited to, Klenow fragment and
Klenow fragment (3' to 5' exonuclease minus), T4 DNA polymerase,
mutant T4 DNA polymerase, T7 DNA polymerase, mutant T7 DNA
polymerase, phi29 DNA polymerase, and mutant phi29 DNA
polymerase.
[0097] In some examples, the primer extension reaction may be
performed in various salt concentrations, such as three salt
concentrations (about 0 mM, 100 mM, and 200 mM), and in the
presence or absence of PEG. For example, one set of experiments may
be conducted with PEG and another set may be conducted without PEG.
FIG. 19 shows example results of a primer extension reaction
conducted in various salt and PEG concentrations with different
types of polymerizing enzymes. When the buffer lacks KCl (e.g., has
0 mM KCl), Bst 2.0 polymerase may incorporate nucleotides
regardless of the presence (e.g., +PEG) or absence (e.g., -PEG) of
PEG, as indicated by the presence of peaks in both +PEG and -PEG.
When the buffer lacks KCl (e.g., has 0 mM KCl), TIX polymerase may
not incorporate nucleotides regardless of the presence or absence
of PEG, as indicated by the absence of peaks in both +PEG and -PEG.
When the buffer comprises 100 mM KCl, both the polymerizing enzymes
may incorporate nucleotides regardless of the presence or absence
of PEG, as indicated by the presence of peaks in both +PEG and
-PEG. When the buffer comprises 200 mM KCl, both the polymerizing
enzymes may incorporate nucleotides in the presence of PEG, as
indicated by peaks in +PEG, but may not incorporate nucleotides in
the absence of PEG.
[0098] In some cases, signal-to-noise ration may be improved by
including molecules that can improve conduction current produced by
polycations, such as Mg', Ca', Zn'. Such molecules may associate
with polycations that may lead to increased conduction current.
Non-limiting examples of molecules the may improve conduction
current include, but are not limited to, phosphodiester backbone of
a nucleic acid molecules (e.g., dT3, dT6, dT12, etc.),
carboxyglutamic acid (Gla (e.g., the .gamma.-carboxyglutamic acids
Gla3, Gla6, Gla12, etc.), specific peptides (e.g., peptides with
the sequences DIETDIET, FDGDFDGD, and/or STLPLPP), or small
molecules (e.g., pyridines, NTA, IDA, or phosphanes).
[0099] A target nucleic acid molecule may be sequenced and/or a
length of the target nucleic acid molecule may be determined. The
target nucleic acid molecule may be a fragmented nucleic acid
molecule or may be a non-fragmented nucleic acid molecule. The
target nucleic acid molecule may be amplified prior to detection.
The target nucleic acid molecule may be amplified in solution
and/or on a support. The target nucleic acid molecule amplified on
a support may be immobilized to the support prior to amplification.
The target nucleic acid molecule may be amplified by bridge
amplification, wild fire amplification, recombinase polymerase
amplification, isothermal amplification, or using any other
amplification technique. Sequencing or determining a length of the
target nucleic acid molecule may comprise providing a plurality of
double-stranded nucleic acid molecules adjacent to a sensor array.
A given, or individual, double-stranded nucleic acid molecule may
be disposed adjacent to a given, or individual, sensor of the
sensor array. The double-stranded nucleic acid molecule may
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 may have sequence
complementarity with one another. The sensor may be electrically
coupled to a charge double layer (e.g., within a Debye length) of
the double-stranded nucleic acid molecule. A portion of the second
single-stranded nucleic acid molecule may be released 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. The
segment may be brought in contact with an individual nucleotide.
The individual nucleotide may be subject to a nucleic acid
incorporation reaction that generates a third single-stranded
nucleic acid molecule. The third single-stranded nucleic acid
molecule may have sequence complementarity with the first
single-stranded nucleic acid molecule. During the nucleic acid
incorporation reaction, the sensor may be used to detect signals
indicative of incorporation of the individual nucleotides into the
third single-stranded nucleic acid molecule, thereby determining a
sequence of the non-hybridized segment.
[0100] The double-stranded nucleic acid molecule may be coupled to
a support. The support may be a bead or one or more surfaces of the
sensor array. A plurality of double-stranded nucleic acid molecules
may be coupled to a plurality of beads or a plurality of locations
on the surface of the sensor array. Each bead of the plurality of
beads may be disposed adjacent to a given sensor. The charge double
layer (e.g., Debye length) may be adjacent to the surface of the
bead. Alternatively, or in addition to, the 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 may be coupled to a surface of a given sensor. The
charge double layer (e.g., Debye length) may be adjacent to the
surface of the given sensor. The double-stranded nucleic acid
molecule coupled to the support may be clonally amplified prior to
sequencing so that support surface is coupled to a clonal
population of double-stranded nucleic acid molecules.
[0101] A given sensor may comprise at least one, at least two, at
least three, or at least four electrodes, or more electrodes. In an
example, a given sensor comprises at least two electrodes. In
another example, a given sensor comprises two electrodes. The
electrodes may be exposed to the solution in which the primer
extension reaction takes place. Alternatively, or in addition to,
the electrodes may be buried within the sensor array and,
therefore, may not be exposed to the solution in which the primer
extension reaction takes place. The electrodes of a given sensor
may detect signals indicative of incorporation of individual
nucleotides into the double-stranded nucleic acid molecule. Signals
indicative of incorporation events may include changes in
impedance, conductance, or charge in the electronic double layer.
In an example, signals indicative of incorporation of individual
nucleotides are electrical signals generated by an impedance or
impedance change in the charge double layer. The signals indicative
of incorporation of individual nucleotides may be steady state
signals, transient signals, or a combination of steady state and
transient signals. Signals may be detected transiently or during
steady state conditions. In a transient signal detection modality,
the detection occurs during or closely after nucleotide
incorporation. In steady state detection, reading of the sensor may
occur after the completion of the incorporation event. A steady
state change in signal may be constant until a change is introduced
to the environment around the sensor.
[0102] FIG. 5 shows an example method for double-stranded
sequencing. The double-stranded nucleic acid template may have a
uniform structure that produces a linear, substantially linear, or
semi-linear response to a change in charge due to nucleotide
incorporation. The double-stranded nucleic acid may comprise a
priming site adjacent to the 3-prime end of the first
single-stranded nucleic acid (e.g., the nucleic acid template to be
sequenced). A primer 503 may have complementarity with the 3-prime
end of the first single-stranded nucleic acid molecule and may
hybridize with the 3-prime end of the first single-stranded nucleic
acid molecule. Alternatively, or in addition to, the second double
stranded nucleic acid may be nicked to provide a primer and a
strand to be displaced (e.g., displacement 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
may be nicked by any enzyme capable of cleaving a uracil (e.g.
uracil DNA glycosylase). A polymerizing enzyme 502 may bind to the
double-stranded nucleic acid and facilitate a primer extension
reaction. In an example, the polymerizing enzyme 502 is a
polymerase, such as Bst DNA polymerase. The primer extension
reaction may displace an end of the second single-stranded nucleic
acid and create a single-stranded flap 505 and a segment of the
first single-stranded nucleic acid molecule that is not hybridized
to the second single-stranded nucleic acid molecule. A segment may
be a portion of the first single-stranded nucleic acid molecule
that is not hybridized to the second or third single-stranded
nucleic acid molecule. The segment may not comprise the entire
first-single stranded nucleic acid molecule. The segment may be a
single nucleotide in length or may be multiple nucleotides in
length. A 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. The flap 505 may
induce the polymerizing enzyme 502 to stutter and lead to phasing
during sequencing. The flap 505 may be recognized and cleaved by a
flap endonuclease (FEN) 501. The FEN 501 may be thermostable or
mesophilic. The thermostable FEN may remain associated with the
nucleic acid after cleavage of the flap 505 and during subsequent
nucleic acid incorporation reactions. The mesophilic FEN may be
inactivated during the primer extension reaction and may be
replenished to the system after each incorporation and detection
cycle. The flap may be cleaved after detecting signals indicative
of nucleotide 504 incorporation and prior to incorporation of
subsequent nucleotides. Incorporation of the nucleotide 504 may
generate a gain in negative charge of the double-stranded nucleic
acid molecule. Cleaving the flap 505 may generate a loss in
negative charge of the double-stranded nucleic acid. Therefore,
incorporation of a nucleotide followed by cleavage of the flap may
generate a net neutral change in charge, resulting in little or no
detectable signal.
[0103] The second single-stranded nucleic acid of the
double-stranded nucleic acid may comprise subunits. FIG. 6 shows an
example method for double-stranded 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 subunits
601 may 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 an example, the nucleic acid subunits 601 comprise
at least 5 nucleotides. In an example, the nucleic acid subunits
601 comprise at least 6 nucleotides. The nucleic acid subunits 601
may all be the same length or may vary in length. The library of
nucleic acid subunits may comprise DNA subunits, peptide nucleic
acid (PNA) subunits, RNA subunits, or lock nucleic acid (LNA)
subunits. Association between the nucleic acid subunits 601 and the
first single-stranded nucleic acid (e.g., nucleic acid template
molecule) may generate a double-stranded nucleic acid molecule and
linearize the nucleic acid template. Nucleotide 504 incorporation
(e.g., via a primer extension reaction) may displace the subunits
and provide a segment of non-hybridized single-stranded nucleic
acid template. In an example, the nucleic acid subunits are
non-charged and, therefore, displacement of a nucleic acid subunit
601 does not alter the charge state of the double-stranded nucleic
acid molecule. In an example, the nucleic acid subunits are charged
and displacement of the subunit 601 alters 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 a FEN.
[0104] The individual nucleotides may comprise reversible
terminators. The reversible terminators may prevent the addition of
subsequent nucleotides into the third single-stranded nucleic acid
molecule. Alternatively, or in addition to, the reversible
terminator may prevent an additional nucleotide from stably
hybridizing with the first single-stranded nucleic acid molecule.
The reversible terminator may reduce the formation of homopolymers
and/or incorporation of more than one nucleotide during an
incorporation cycle. The reversible terminator may be coupled to
the oxygen atom of the 3-prime hydroxyl group of the nucleotide
pentose (e.g., 3'-O-blocked reversible terminator). Alternatively,
or in addition to, the reversible terminator may be coupled to the
nucleobase of the nucleotide (e.g., 3'-unblocked reversible
terminator). The reversible terminator may include a detectable
label. The reversible terminator may comprise an allyl,
hydroxylamine, acetate, benzoate, phosphate, azidomethyl, or amide
group. The reversible terminator may be removed by treatment with a
reducing agent, acid or base, organic solvents, ionic surfactants,
photons (photolysis), or any combination thereof. Removal of the
reversible terminator of a 3'-O-blocked reversible terminator may
return the hydroxyl group to pentose of the nucleotide and allow
for the incorporation of subsequent nucleotides into the third
single-stranded nucleic acid molecule.
[0105] FIGS. 7A and 7B show example methods for double-stranded
sequencing using reversible terminators 701. FIG. 7A shows an
example method for double-stranded sequencing using reversible
terminators and a FEN 501. The second single-stranded nucleic acid
of the double-stranded nucleic acid may comprise a uracil
nucleotide that is nicked by a uracil-DNA glycosylase.
Alternatively, or in addition to, the second single-stranded
nucleic acid molecule may comprise a displacement strand and a
primer. A polymerizing enzyme 502 may bind to the primer 503. The
polymerizing enzyme may be an enzyme that enables incorporation
with high efficiency and fidelity. The polymerizing enzyme may be,
without limitation, a Bst polymerase, reverse transcriptase, type A
polymerase, type B polymerase, or type C polymerase. The
polymerizing enzyme may incorporate an individual nucleotide
comprising a reversible terminator 701. Incorporation of the
nucleotide 701 may generate a flap. The incorporated nucleotide 701
may be detected and, subsequent to detection, the flap may be
cleaved by a FEN 501. The FEN 501 may be mesophilic. The mesophilic
FEN 501 may be brought into contact with the flap after detection
of the incorporated nucleotide and may be removed prior to the next
incorporation cycle. The FEN 501 may be replenished with each
nucleotide incorporation cycle. The reversible terminator may be
reversed during or after cleavage of the flap. The reversible
terminator may be reversed by introducing a reducing agent to the
solution. In an example, the reducing agent is dithiothreitol
(DTT). After the reversible terminator is reversed, the cycle of
nucleotide incorporation, detection, cleavage of the flap, and
reversing the terminator is repeated until a portion of or the
entire first single-stranded nucleic acid is sequenced.
[0106] FIG. 7B show an example method for double-stranded
sequencing using reversible terminators and nucleic acid subunits.
The second single-stranded nucleic acid molecule may comprise
random nucleic acid subunits. The second single-stranded nucleic
acid molecule may additionally comprise a primer 503. A
polymerizing enzyme 502 may bind to the primer and incorporate an
individual nucleotide 701 into the third single-stranded nucleic
acid molecule. The individual nucleotide may displace a portion of
or an entire nucleic acid subunit. The incorporated nucleotide may
be detected after incorporation into the third single-stranded
nucleic acid molecule. The individual nucleotide may include a
reversible terminator. The reversible terminator may be reversed
after detection of the incorporated nucleotide. The reversible
terminator may be reversed by introducing a reducing agent to the
solution. In an example, the reducing agent is DTT. After the
reversible terminator is reversed, the cycle of nucleotide
incorporation, detection, and reversing the terminator may be
repeated until a portion of or the entire first single-stranded
nucleic acid is sequenced.
[0107] The double-stranded nucleic acid molecule may comprise
detectable labels. The detectable labels may be electrostatic
moieties, fluorescent labels, colorimetric labels, chemiluminescent
labels, radio labels, or any combination thereof. The detectable
labels may be coupled to the second single-stranded nucleic acid
molecule, the nucleotides to be incorporated into the third
single-stranded nucleic acid, or any combination thereof. The
detectable label may be coupled to the phosphate of a nucleotide,
the nucleobase of a nucleotide, or to a reversible terminator
couple to a nucleotide. In an example, the detectable label is
coupled to the nucleobase of the nucleotide. The detectable label
may be reversibly coupled or irreversibly coupled to a nucleotide.
The detectable label may generate signals indicative of nucleotide
incorporation into the third single-stranded nucleic acid molecule
or cleavage of a nucleotide from the second single-stranded nucleic
acid molecule. The signals from the detectable label may be
detected by the sensor array.
[0108] In an example, each different type of nucleotide may be
coupled to a different detectable label. Each type of detectable
label may indicate the nucleotide base to which it is bound. For
example, each of guanine, cytosine, adenine, thymine, and uracil
may have different detectable labels that are resolvable from each
other. FIG. 8 shows example nucleotides each having different
electrostatic moieties and a reversible terminator 801. The
electrostatic moieties may be polyanion or polycation electrostatic
moieties. One or more individual nucleotides may have no
electrostatic moiety. One or more individual nucleotides may have a
polycation electrostatic moiety. The polycation electrostatic
moieties may have varying degrees of charge. One or more individual
nucleotides may have a polyanion electrostatic moiety. The
polyanion electrostatic moieties may have varying degrees of
charge. The presence of excess charge on the double-stranded
nucleic acid molecule may reduce the efficiency of the polymerizing
enzyme. The polymerizing enzyme may be an enzyme that enables
incorporation of nucleotides with detectable labels with high
efficiency and fidelity. The polymerizing enzyme may be, without
limitation, a 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 nucleotides electrostatic moieties may be
coupled to the second single-stranded nucleic acid molecule or to
the individual nucleotides that are incorporated into the third
single-stranded nucleic acid. In an example, the electrostatic
moieties are coupled the individual nucleotides that are
incorporated into the third single-stranded nucleic acid molecule.
The individual nucleotides may be directed to contact the
double-stranded nucleic acid individually and sequentially (e.g.,
contacted with A, followed by T, followed by C, followed by G, and
so forth) and the sensor may detect nucleotide incorporation
between each addition. Alternatively, or in addition to, the
nucleotides may be directed to contact the double-stranded nucleic
acid molecule simultaneously (e.g., contacted with a solution
comprising all of G, A, T, and C at once) and the sensor may detect
a change in charge after nucleotide incorporation. Different
electrostatic moieties coupled to different types of individual
nucleotides may allow for each type of individual nucleotides
incorporated into the third single-stranded nucleic acid molecules
to be detected and distinguished from each other. The individual
nucleotides may be resolved using a single read per incorporation
cycle. After 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
organic solvents (e.g., formamide or urea), cleavage by ionic
surfactant, or combination thereof.
[0109] In an example, each different type of nucleotide may have
the same detectable label. FIG. 9 shows example nucleotides each
having the same electrostatic moieties and different, reversible
coupling mechanisms. The coupling mechanisms may be decoupled, or
cleaved, by a reduction reaction, acid or base cleavage, cleavage
in organic solvents, cleavage by ionic surfactant, or combination
thereof. Additionally, the electrostatic moieties may be coupled to
the individual nucleotides by a variety of coupling mechanisms
including, but not limited to, covalent bonds,
association-disassociation interactions, ligand and binding pair
interactions (e.g., Streptavidin-Biotin interaction), hybridization
interaction, or any combination thereof. The individual nucleotides
may be directed to contact the double-stranded nucleic acid
individually and sequentially (e.g., contacted with A, followed by
T, followed by C, followed by T, and so forth) and the sensor may
detect nucleotide incorporation between each addition.
Alternatively, or in addition to, the nucleotides may be directed
to contact the double-stranded nucleic acid simultaneously (e.g.,
contacted with a solution of all A, T, C, and G at once) and the
sensor may detect a change in charge after nucleotide
incorporation. The change in charge may be used to determine a
length of the nucleic acid target molecule. In an example (see FIG.
9), the nucleotides G, A, and T all have the same polyanion
electrostatic moiety that is cleaved by condition one. Nucleotide C
may not have a electrostatic moiety. The electrostatic moiety for A
may further comprise a second coupling mechanism that is cleaved
under condition two. The electrostatic moiety for T may initially
not be present during nucleotide incorporation, but may be coupled
to the nucleotide using a third coupling mechanism (e.g.,
Streptavidin-Biotin). The double-stranded nucleic acid molecule may
be contacted with all four nucleotides at once. One or more
nucleotides may be incorporated into a plurality of third
single-stranded nucleic acid molecules adjacent to the sensor
array. After the incorporation reaction, nucleotide incorporation
may be read or detected. In this example, nucleotides G and A may
have a polyanion electrostatic moiety during the initial read
(e.g., detection cycle) and both G and A may generate a detectable
signal. Nucleotides T and C may initially not comprise a
electrostatic moiety nor generate a detectable signal. The
electrostatic moiety for A may be removed by contacting the
nucleotide with the second cleavage condition and T may obtain a
electrostatic moiety by the introduction of a electrostatic moiety
comprising the third coupling mechanism (e.g., a streptavidin
group). A second read (e.g., detection cycle) may be performed and
both G and T may generate a detectable signal and A and C may not
generate a detectable signal. The incorporated nucleotides may then
be resolved and distinguished from each other by combining signals
from the first and second read and matching the signal to the
corresponding nucleotides. After the first and second read, the
electrostatic moieties may be cleaved using cleavage condition
one.
[0110] FIG. 10 shows an example method for sequencing using
electrostatic moieties and reversible terminators. The nucleic acid
template to be sequences may be subjected to a nucleic acid
incorporation reaction (e.g., primer extension reaction). The
incorporated nucleotide may have a cleavable electrostatic moiety
and a reversible terminator. The reversible terminator may prevent
the addition of subsequent nucleotides from incorporating into the
extended primer. After incorporation of the nucleotide the
incorporated nucleotide may be read or detected. Following reading,
the electrostatic moiety may be cleaved and the reversible
terminator may be removed. The electrostatic moiety may be cleaved
and the terminator may be removed using the same chemistry. Example
chemistries include treatment with a reducing agent such as
dithiothreitol (DTT) or tris92-carboxyethyl0phosphine (TCEP).
Alternatively, or in addition to, the electrostatic moiety may be
cleaved and the terminator may be removed using different cleavage
and removal chemistries.
[0111] The detectable labels may be coupled to the nucleotides
incorporated during the primer extension reaction or may be coupled
to the nucleotides of the second single-stranded nucleic acid
molecule. In an example, the second single stranded nucleic acid
(e.g., the displacement strand) may comprise one or more detectable
labels. FIGS. 11A and 11B show example methods for double-stranded
sequencing using detectable labels coupled to the second
single-stranded nucleic acid molecule. FIG. 11A shows an example
sequencing method using detectable labels that are cleaved by a
flap endonuclease 501. The second single-stranded nucleic acid
molecule of the double-stranded nucleic acid molecule may comprise
detectable labels. The detectable labels may be electrostatic
moieties. Each nucleotide of the second single-stranded nucleic
acid molecule may be coupled to a electrostatic moiety. Each
different type of nucleotide in the second single-stranded nucleic
acid molecule may be coupled to a different electrostatic moiety or
coupled to the same electrostatic moiety. The polymerizing enzyme
502 may bind to the primer 503 adjacent to an end of the
displacement strand. The polymerizing enzyme 503 may incorporate a
nucleotide 504 into the third single-stranded nucleic acid
molecule. The incorporated nucleotide may or may not have a
electrostatic moiety. Incorporation of the nucleotide may create a
flap. The flap may be cleaved by a FEN 501. The FEN 501 may be a
thermostable or mesophilic FEN. Cleavage of the flap by a FEN 501
may remove a electrostatic moiety from the displacement strand,
thereby altering the charge state of the double-stranded nucleic
acid molecule. The change in charge state may be detected by the
sensor array to generate a sequence of the first single-stranded
nucleic acid molecule. Cycles of nucleotide incorporation, cleavage
of the flap, and detection of the change in charge may be repeated
until the sequence of the first single-stranded nucleic acid
molecule is determined.
[0112] FIG. 11B shows an example double-stranded sequencing method
using both detectable labels coupled to the second single-stranded
nucleic acid molecule and reversible terminators. The detectable
labels may be electrostatic moieties. Each nucleotide of the second
single-stranded nucleic acid molecule may be coupled to a
electrostatic moiety. Each different type of nucleotide in the
second single-stranded nucleic acid molecule may be coupled to a
different electrostatic moiety or coupled to the same electrostatic
moiety. The polymerizing enzyme 502 may bind to the primer 503
adjacent to an end of the displacement strand. The polymerizing
enzyme 502 may incorporate a nucleotide 701 into the third
single-stranded nucleic acid molecule. The incorporated nucleotide
may or may not have a electrostatic moiety and a reversible
terminator. The flap may be cleaved by a FEN. The FEN may be
thermostable or mesophilic. Cleavage of the flap by a FEN may
remove a electrostatic moiety from the displacement strand, thereby
altering the charge state of the double-stranded nucleic acid
molecule. The change in charge state may be detected to generate a
sequence of the first single-stranded nucleic acid molecule. After
detecting nucleotide incorporation, the newly incorporated
nucleotide may undergo a cleavage reaction to remove the reversible
terminator. Removal of the reversible terminator may permit the
incorporation of subsequent nucleotides into the third
single-stranded nucleic acid molecule. Cycles of nucleotide
incorporation, cleavage of the generated 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 may 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 cycles of nucleotide incorporation and
detection. Nucleotide incorporation and detection may be conduct
for a set number of cycles or may be conducted until the primer
extension reaction is complete.
[0113] The detectable label may be coupled to individual
nucleotides that are incorporated into the third single-stranded
nucleic acid molecule. FIG. 12A shows an example method for
double-stranded sequencing using individual nucleotide coupled
electrostatic moieties 1201 and a mesophilic FEN 501. A
polymerizing enzyme may bind to the primer of the second
single-stranded nucleic acid molecule to facilitate the
incorporation of an individual nucleotide 1201. The individual
nucleotide 1201 may comprise a electrostatic moiety bound to the
nucleobase of the nucleotide. Incorporation of an individual
nucleotide may generate a flap. The flap may be cleaved by a FEN.
The FEN may be a mesophilic FEN and may be regenerated after each
incorporation cycle. The flap may be cleaved after detection of the
nucleotide incorporation event. The electrostatic moiety may be
reversibly couple to the nucleotide. The electrostatic moiety may
be cleaved at the same time or subsequent to cleavage of the flap.
Cycles of nucleotide incorporation, detection of nucleotide
incorporation, cleavage of the generated flap, and cleavage of the
electrostatic moiety may be repeated until the sequence of at least
a portion of the first single-stranded nucleic acid molecule is
determined.
[0114] FIG. 12B shows an example method for double-stranded
sequencing using electrostatic moieties and a thermostable FEN. A
polymerizing enzyme 502 may bind to the primer 503 of the second
single-stranded nucleic acid molecule to facilitate the
incorporation of an individual nucleotide 1201. The individual
nucleotide 1201 may comprise a electrostatic moiety bound to the
nucleobase of the nucleotide. Incorporation of an individual
nucleotide may generate a flap. The flap may be cleaved by a FEN
501. The FEN 501 may be a thermostable FEN and may remain
associated with the double-stranded nucleic acid molecule after
cleavage of the flap. The thermostable FEN may not be regenerated
after each incorporation cycle. The flap may be cleaved prior to
detection of the nucleotide incorporation event. The electrostatic
moiety may be reversibly coupled to the nucleotide and may be
cleaved after detection of the nucleotide incorporation event.
Cycles of nucleotide incorporation, cleavage of the generated flap,
detection of nucleotide incorporation, and removal of the
electrostatic moiety may be repeated until the sequence of at least
a portion of the first single-stranded nucleic acid molecule is
determined.
[0115] The individual nucleotides may comprise both a detectable
label and a reversible terminator. FIG. 13A shows an example method
for double-stranded sequencing using electrostatic moieties,
reversible terminators, and a mesophilic FEN. A polymerizing enzyme
502 may bind to the primer 503 of the second single-stranded
nucleic acid molecule to facilitate the incorporation of an
individual nucleotide 1301. The individual nucleotide 1301 may
comprise a electrostatic moiety bound to the nucleobase of the
nucleotide and a reversible terminator bound to the 3-prime side of
the pentose. The reversible terminator may reduce homopolymer
formation and/or the incorporation of multiple nucleotides per
cycle. Incorporation of an individual nucleotide may generate a
flap. The flap may be cleaved by a FEN 501. The FEN 501 may be a
mesophilic FEN and may be regenerated after each incorporation
cycle. The flap may be cleaved after detection of the nucleotide
incorporation event. The electrostatic moiety may be reversibly
couple to the nucleotide. The electrostatic moiety may be cleaved
at the same time or subsequent to cleavage of the flap. The
reversible terminator may be reversed prior to, simultaneously
with, or subsequent to cleavage of electrostatic moiety. In an
example, the electrostatic moiety is cleaved and the reversible
terminator reversed by a reducing agent, such as DTT or TCEP.
Cycles of nucleotide incorporation, detection of nucleotide
incorporation, cleavage of the generated flap, cleavage of the
electrostatic moiety, and reversing 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 may
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 cycles of nucleotide
incorporation and detection. Nucleotide incorporation and detection
may be conduct for a set number of cycles or may be conducted until
the primer extension reaction is complete.
[0116] FIG. 13B shows an example method for double-stranded
sequencing using electrostatic moieties, a thermostable FEN, and
reversible terminators. A polymerizing enzyme 502 may bind to the
primer 503 of the second single-stranded nucleic acid molecule to
facilitate the incorporation of an individual nucleotide 1301. The
individual nucleotide 1301 may comprise a electrostatic moiety
bound to the nucleobase of the nucleotide and a reversible
terminator on the 3-prime side. The reversible terminator may
reduce homopolymer formation and/or the incorporation of multiple
nucleotides per cycle. Incorporation of an individual nucleotide
may generate a flap. The flap may be cleaved by a FEN 501. The FEN
501 may be a thermostable FEN may remain associated with the
double-stranded nucleic acid molecule after cleavage of the flap.
The thermostable FEN may not be regenerated after each
incorporation cycle. The flap may be cleaved prior to detection of
the nucleotide incorporation event. The electrostatic moiety may be
reversibly coupled and may be cleaved after detection of the
nucleotide incorporation event. The reversible terminator may be
reversed simultaneously with cleavage of electrostatic moiety or
subsequent to cleavage of the electrostatic moiety. In an example,
the electrostatic moiety is cleaved and the reversible terminator
reversed by a reducing agent, such as DTT or TCEP. Cycles 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 may 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 cycles of nucleotide incorporation and
detection. Nucleotide incorporation and detection may be conduct
for a set number of cycles or may be conducted until the primer
extension reaction is complete.
[0117] FIG. 14A shows an example method for double-stranded
sequencing using detectable labels and nucleic acid subunits. The
detectable labels may be electrostatic moieties. The detectable
labels may be reversibly coupled to the individual nucleotides. The
second single-stranded nucleic acid may comprise random nucleic
acid subunits and a primer 503. A polymerizing enzyme 502 may bind
to the primer 503. The polymerizing enzyme 502 may incorporate an
individual nucleotide with a electrostatic moiety into the third
single-stranded nucleic acid molecule. Incorporation of the
individual nucleotide 1201 may displace a portion of or an entire
random nucleic acid subunit. The sensor array may detect signals
indicative of incorporation events after the incorporation of the
individual nucleotide into the third single-stranded nucleic acid
molecule. After detection of the incorporation event, the
reversible electrostatic moiety may be cleaved. In an example, the
reversible electrostatic moiety is cleaved with a reducing agent
such as DTT or TCEP. Cycles of nucleotide incorporation, nucleic
acid subunit displacement, individual nucleotide detection, and
cleavage of the electrostatic moiety may be repeated until the
sequence of at least a portion of the first single-stranded nucleic
acid molecule is determined. The method may 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 cycles of nucleotide incorporation and
detection. Nucleotide incorporation and detection may be conduct
for a set number of cycles or may be conducted until the primer
extension reaction is complete.
[0118] FIG. 14B shows an example method for double-stranded
sequencing using detectable labels, nucleic acid subunits, and
reversible terminators. The detectable labels may be electrostatic
moieties. The detectable labels may be reversibly coupled to the
individual nucleotides 1301. The individual nucleotides may
comprise reversible terminators on the 3-prime side. The second
single-stranded nucleic acid may comprise random nucleic acid
subunits and a primer 503. A polymerizing enzyme 502 may bind to
the primer 503. The polymerizing enzyme 502 may incorporate an
individual nucleotide 1301 with a electrostatic moiety and
reversible terminator into the third single-stranded nucleic acid
molecule. Incorporation of the individual nucleotide may displace a
portion of or an entire random nucleic acid subunit. The sensor
array may detect signals indicative of incorporation events after
the incorporation of the individual nucleotide into the third
single-stranded nucleic acid molecule. After detection of the
incorporation event, the reversible electrostatic moiety may be
cleaved. The reversible terminator may be removed simultaneously
with or sequentially to the cleavage of the electrostatic moiety.
In an example, the reversible electrostatic moiety and reversible
terminator are cleaved simultaneously with a reducing agent such as
DTT or TCEP. Cycles of nucleotide incorporation, nucleic acid
subunit displacement, individual nucleotide detection, 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 may 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 cycles of
nucleotide incorporation and detection. Nucleotide incorporation
and detection may be conduct for a set number of cycles or may be
conducted until the primer extension reaction is complete.
[0119] A target nucleic acid molecule may be sequenced and/or a
length of the target nucleic acid molecule may be determined. The
target nucleic acid molecule may be a fragmented nucleic acid
molecule or may be a non-fragmented nucleic acid molecule. The
target nucleic acid molecule may be amplified prior to detection.
The target nucleic acid molecule may be amplified in solution
and/or on a support. The target nucleic acid molecule amplified on
a support may be immobilized to the support prior to amplification.
The target nucleic acid molecule may be amplified by bridge
amplification, wild fire amplification, recombinase polymerase
amplification, isothermal amplification, or using any other
amplification technique. Sequencing or determining a length of the
target nucleic acid molecule may comprise 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 may be disposed adjacent to
a given sensor of the sensor array. The given sensor may be
electrically coupled to a charge double layer (e.g., within a Debye
length) containing the first single-stranded nucleic acid molecule.
The first single-stranded nucleic acid molecule may be brought into
contact with individual nucleotides to subject the first
single-stranded nucleic acid molecule to a nucleic acid
incorporation reaction. The nucleic acid incorporation reaction
(e.g., primer extension reaction) may generate a second
single-stranded nucleic acid molecule from the individual
nucleotides. The second single-stranded nucleic acid molecule may
have sequence complementarity with the first single-stranded
nucleic acid molecule. At least a subset of the individual
nucleotides may comprise detectable labels. A given sensor of the
sensor array may be used to detect signals from the detectable
labels indicative of incorporation of the individual nucleotides
during or subsequent to conducting the nucleic acid incorporation
reaction. The detected signals may be used to determine the
sequence of the first single-stranded nucleic acid molecule.
[0120] The plurality of single stranded-nucleic acid molecules may
be coupled to a plurality of supports. The plurality of supports
may be a plurality of beads or a plurality of surfaces on the
sensor array. In an example, the 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 double layer may be adjacent to a surface of
the given bead. The single-stranded nucleic acid molecule may be
amplified on the surface of the bead. The amplification products
may be coupled to the surface of the bead. The amplification
products may form a clonal colony of single-stranded nucleic acid
molecules on the surface of the bead. The clonal colony of
single-stranded nucleic acid molecules may be sequenced.
[0121] In an example, the plurality of single-stranded nucleic acid
molecules may be coupled to a plurality of surfaces on the sensor
array and a given single-stranded nucleic acid molecule is coupled
to a surface of a given sensor. The charge double layer may be
adjacent to the surface of the given sensor. The single-stranded
nucleic acid molecule may be amplified on the surface of the
sensor. The amplification products may be coupled to the surface of
the sensor. The amplification products may form a clonal colony of
single-stranded nucleic acid molecules of the surface of the
sensor. The clonal colony of single-stranded nucleic acid molecules
may be sequenced.
[0122] A given sensor of the sensor array may comprise at least
one, at least two, at least three, at least four, or more
electrodes. In an example, a given sensor comprises at least two
electrodes. In another example, a given sensor comprises two
electrodes. The electrodes may be exposed to the solution in which
the primer extension reaction takes place. Alternatively, or in
addition to, the electrodes may be buried within the sensor array
and, therefore, may not be exposed to the solution in which the
primer extension reaction takes place. The sensor may detect
signals indicative of nucleotide incorporation events. The sensor
may detect the detectable label coupled to the individual
nucleotides. The sensor may detect the detectable label during
transient or steady state conditions. Nucleotide incorporation may
be detected once, twice, three times, four times, or more than four
times per incorporation cycle during steady state conditions. In an
example, nucleotide incorporation may be detected at least twice
per incorporation cycle during steady state conditions. The sensor
array may detect electrical signals during transient or steady
state conditions. The electrical signals may include, but are not
limited to, changes in charge state of a molecule, changes in the
conductivity of a surrounding solution, impedance signals, or
changes in impedance signals. The sensor may detect a change in
charge and/or conductivity or a change in impedance. The sensor may
detect the change in charge and/or conductivity or impedance within
a charge double layer (e.g., Debye length) of the sensor, support,
or nucleic acid molecule (e.g., the sample). The detectable labels
coupled to the individual nucleotides may alter the electrical
environment surrounding the single-stranded nucleic acid molecules
and a given sensor may detect the electrical change.
[0123] The second single-stranded nucleic acid molecule may
comprise a priming site adjacent to the first-single stranded
nucleic acid molecule. The priming site may be a primer with
sequence complementarity with the first single-stranded nucleic
acid molecule. The second single-stranded nucleic acid molecule may
be generated by a primer extension reaction originating from the
primer. In an example, the primer is a self-priming loop. The
self-priming loop may be in a structure or looped configuration
during the primer extension reaction. Subsequent to the
incorporation of an individual nucleotide, the structure of the
self-priming loop may be relaxed to form a linear nucleic acid
molecule. Incorporation of the individual nucleotide may be
detected during the relaxed, unstructured state. The self-priming
loop may be relaxed by increasing the reaction temperature,
changing the solution pH, changing the solution ionic strength,
introducing formamide to the solution, or by any other method that
denatures the nucleic acid structure.
[0124] The different types of individual nucleotides may be brought
into contact with the single-stranded nucleic acid molecule
sequentially (e.g., a single nucleotide at a time). Signals
indicative of nucleotide incorporation may be detected after each
type of individual nucleotide is brought into contact with the
single-stranded nucleic acid molecule. In an example, the
single-stranded nucleic acid molecule may be contacted with A
nucleotides followed by the detection of signals indicative of
nucleotide incorporation. The single-stranded nucleotide may then
be contacted with T nucleotides followed by signal detection. The
single-stranded nucleotide may then be contacted with G nucleotides
followed by signal detection. The single-stranded nucleotide may be
contacted with C nucleotides followed by signal detection. This
cycle may repeat until the entire or a portion of the sequence of
the singe-stranded nucleic acid molecule is determined. The method
may 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 cycles of
nucleotide incorporation and detection.
[0125] The single-stranded nucleic acid molecule may be contacted
with different types of nucleotides simultaneously. For example,
the single-stranded nucleic acid molecule may be contacted with all
A, T, C, and G in at one time. Alternatively, or in addition two,
the single-stranded nucleic acid molecule may be contacted with any
combination of A, T, C, and G at one time. For example, the
single-stranded nucleic acid molecule may 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
an example, the 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 the sequence of the nucleic
acid molecule. This cycle may be repeated until all or a portion of
the sequence of the single-stranded nucleic acid molecule is
determined.
[0126] The individual nucleotides may comprise detectable labels.
The detectable labels may be reversibly or irreversibly coupled to
the individual nucleotides. The detectable labels may be coupled to
the nucleobase of an individual nucleotide. The individual
nucleotides may comprise different types of nucleotide. In an
example, the detectable labels may be electrostatic moieties. 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. The detectable label may
be selectively coupled to or cleaved from an individual nucleotide.
For example, an individual nucleotide may comprise a detectable
label when contacted with the single-stranded nucleic acid
molecule. After incorporation the signal of the individual
nucleotide may be detected and generate a positive signal (e.g., a
signal is detected). The detectable label may be removed under a
given cleavage condition. After removal of the detectable label the
incorporated nucleotide may generate a null signal (e.g., no signal
is detected). In an example using a double read sequencing approach
(see FIG. 9), an individual nucleotide may have a positive/null
signal, positive/positive signal, a null/null signal, or a
null/positive signal. In an example, a single-stranded nucleic acid
molecule may be contacted with four different types of nucleotides
simultaneously and a polymerizing enzyme may facilitate nucleotide
incorporation. Each of the nucleotides may have the same or a
different detectable label. In an 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,
signals indicative of nucleotide incorporation may be measure. The
incorporated nucleotides may generate a variety of positive and
null signals. The single-stranded nucleic acid molecules may be
treated with a cleavage and/or coupling condition. After treatment
with the cleavage and/or coupling condition, signals indicative of
nucleotide incorporation may again be measured. The incorporated
nucleotides may generate a variety of positive and null signals.
The pattern of positive and null signals may be used to determine
which type of nucleotide was incorporated into the second
single-stranded nucleic acid molecule. After the second detection
cycle, the electrostatic moieties may be removed using a second
cleavage condition.
[0127] In an example, each different individual nucleotide may be
coupled to a different detectable label. The detectable labels may
be electrostatic moieties. The electrostatic moieties may include
polyanion, polycation, or neutral electrostatic moieties. The
single-stranded nucleic acid molecule may be contacted with the
different nucleotides sequentially or simultaneously. In an
example, the single-stranded nucleic acid molecules may be
contacted with the different individual nucleotides simultaneously
and each individual nucleotide of the different individual
nucleotides may be coupled to a different electrostatic moiety. A
polymerizing enzyme may facilitate incorporation of the individual
nucleotides into the second single-stranded nucleic acid molecule.
After incorporation of the individual nucleotides, the sensor array
may detect the signals indicative of individual nucleotide
incorporation. The different individual nucleotides may generate
signals representing the different charge groups and signals
representing different magnitudes of charge respective to the
electrostatic moiety to which they are coupled. The detectable
label may be removed after signal detection.
[0128] The individual nucleotides may comprise reversible
terminators, detectable labels, and both reversible terminators and
detectable labels. The reversible terminator may be coupled to the
3-prime side of an individual nucleotide. The reversible terminator
may prevent additional nucleotides from stably hybridizing to the
first single-stranded nucleic acid molecule. The reversible
terminator may be removed after detection of signals indicative of
nucleotide incorporation. Removal of the reversible terminator may
permit incorporation of subsequent nucleotides. In an example, a
single-stranded nucleic acid molecule may be contacted with
different individual nucleotides comprising different detectable
moieties and reversible terminators simultaneously. A polymerizing
enzyme may facilitate incorporation of a single individual
nucleotide into a second single-stranded nucleic acid molecule. The
sensor may detect signals indicative of nucleotide incorporation.
Following signal detection, the detectable label and reversible
terminator may be removed either simultaneously or sequentially.
This cycle may be repeated until the sequence of all or part of the
first single-stranded nucleic acid molecule is determined.
[0129] The cleavage of the detectable label may leave a scar on the
individual nucleotide after cleavage. The scar may comprise
portions of the detectable label that are not fully removed during
cleavage of the label. In an example, the scar may reduce the
efficiency of the polymerizing enzyme. In an example, the scar may
inhibit the polymerizing enzyme. Pyrophosphorolysis mediated
terminator exchange (PMTE) sequencing may reduce the amount of scar
build up during sequencing.
[0130] A target nucleic acid molecule may be sequenced and/or a
length of the target nucleic acid molecule may be determined. The
target nucleic acid molecule may be a fragmented nucleic acid
molecule or may be a non-fragmented nucleic acid molecule. The
target nucleic acid molecule may be amplified prior to detection.
The target nucleic acid molecule may be amplified in solution
and/or on a support. The target nucleic acid molecule amplified on
a support may be immobilized to the support prior to amplification.
The target nucleic acid molecule may be amplified by bridge
amplification, wild fire amplification, recombinase polymerase
amplification, isothermal amplification, or using any other
amplification technique. Sequencing or determining a length of the
target nucleic acid molecule may comprise 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 may be disposed adjacent to
a given sensor of the sensor array. The first single-stranded
nucleic acid molecule may be subjected to a nucleic acid
incorporation reaction to generate a second single-stranded nucleic
acid molecule. The nucleic acid incorporation reaction may comprise
alternately and sequentially incorporating individual nucleotide of
a first plurality of nucleotide comprising delectable labels and
followed by incorporation of a second plurality of individual
nucleotides without detectable labels. The nucleic acid
incorporation reaction may comprise alternately and sequentially
incorporating individual nucleotide of a first plurality of
nucleotide comprising delectable labels and exchanging (e.g.,
removal of the first nucleotide) the first plurality of individual
nucleotides with individual nucleotides of a second plurality of
individual nucleotides without detectable labels. The first
plurality of nucleotides may be covalently incorporated into the
growing nucleic acid strand or may be transiently bound (e.g., not
covalently bound) to the growing nucleic acid strand. The second
plurality of individual nucleotides may not comprise detectable
labels. The given sensor may detect signals from the detectable
labels while or subsequent to conducting the nucleic acid
incorporation reaction. The detected signals may be generated from
the detectable labels and may be indicative of incorporation of the
first plurality of individual nucleotides into the second
single-stranded nucleic acid molecule, thereby determining a
sequence of the first single-stranded nucleic acid molecule.
[0131] The plurality of single stranded-nucleic acid molecules may
be coupled to a plurality of supports. The plurality of supports
may be a plurality of beads. In an example, the 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 double layer comprising the first
single-stranded nucleic acid molecule. The charge double layer may
be adjacent to a surface of the given bead. The single-stranded
nucleic acid molecule may be amplified on the surface of the bead.
The amplification products may be coupled to the surface of the
bead. The amplification products may form a clonal colony of
single-stranded nucleic acid molecules on the surface of the bead.
The clonal colony of single-stranded nucleic acid molecules may be
sequenced.
[0132] In an example, the plurality of single-stranded nucleic acid
molecules may be coupled to a plurality of surfaces on the 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 double layer comprising the first
single-stranded nucleic acid molecule. The charge double layer may
be adjacent to the surface of the given sensor. The single-stranded
nucleic acid molecule may be amplified on the surface of the
sensor. The amplification products may be coupled to the surface of
the sensor. The amplification products may form a clonal colony of
single-stranded nucleic acid molecules of the surface of the
sensor. The clonal colony of single-stranded nucleic acid molecules
may be sequenced and/or a length of the single-stranded nucleic
acids may be determined.
[0133] A given sensor of the sensor array may comprise at least
one, at least two, at least three, at least four, or more
electrodes. In an example, a given sensor comprises at least two
electrodes. In another example, a given sensor comprises two
electrodes. The electrodes may be exposed to the solution in which
the primer extension reaction takes place. Alternatively, or in
addition to, the electrodes may be buried within the sensor array
and, therefore, may not be exposed to the solution in which the
primer extension reaction takes place. The sensor may detect
signals indicative of nucleotide incorporation events. The sensor
may detect the detectable label coupled to the individual
nucleotides. The sensor may detect the detectable label during
transient or steady state conditions. Nucleotide incorporation may
be detected once, twice, three times, four times, or more than four
times per incorporation cycle during steady state conditions. In an
example, nucleotide incorporation may be detected at least twice
per incorporation cycle during steady state conditions. The sensor
array may detect electrical signals during transient or steady
state conditions. The electrical signals may include, but are not
limited to, changes in charge state of a molecule, changes in the
conductivity of a surrounding solution, impedance signals, or
changes in impedance signals. The sensor may detect a change in
charge and/or conductivity or a change in impedance. The sensor may
detect the change in charge and/or conductivity or impedance within
a charge double layer (e.g., Debye length) of the sensor, support,
or nucleic acid molecule (e.g., the sample). The detectable labels
coupled to the individual nucleotides may alter the electrical
environment surrounding the single-stranded nucleic acid molecules
and a given sensor may detect the electrical change.
[0134] The second single-stranded nucleic acid molecule may
comprise a priming site adjacent to the first-single stranded
nucleic acid molecule. The priming site may be a primer with
sequence complementarity with the first single-stranded nucleic
acid molecule. The second single-stranded nucleic acid molecule may
be generated by a primer extension reaction originating from the
primer. In an example, the primer is a self-priming loop. The
self-priming loop may be in a structure or looped configuration
during the primer extension reaction. Subsequent to the
incorporation of an individual nucleotide, the structure of the
self-priming loop may be relaxed to form a linear nucleic acid
molecule. Incorporation of the individual nucleotide may be
detected during the relaxed, unstructured state. The self-priming
loop may be relaxed by increasing the reaction temperature,
changing the solution pH, changing the solution ionic strength,
introducing formamide to the solution, or by any other method that
denatures the nucleic acid structure.
[0135] The first plurality of nucleotides may comprise a terminator
that prevents an additional nucleotide from stably hybridizing to
the first single-stranded nucleic acid molecule. The terminator may
be a reversible terminator or an irreversible terminator. In an
example, the terminator is an irreversible terminator. The
terminator may reduce the occurrence of homopolymers and/or the
incorporation of multiple individual nucleotides per incorporation
cycle. The first plurality of individual nucleotides may comprise
dideoxynucleotides (ddNTP) or 3-fluorodeoxynucleotides. The ddNTP
may be a chain-elongating inhibitor. The first plurality of
nucleotides may comprise detectable labels. The detectable labels
may not be removed after detection of nucleotide incorporation. The
detectable labels may be electrostatic moieties, fluorescent
labels, chemiluminescent labels, colorimetric labels, radio labels,
or any other detectable label. In an example, the detectable labels
are electrostatic moieties. The detectable labels may be coupled to
the nucleobases of the first plurality of nucleotides.
[0136] The first plurality of nucleotides may comprise different
types of nucleotides. In an example, the different types of
nucleotides may 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 may be contacted with all the different types of
nucleotides simultaneously. The sensor array may then detect the
different detectable electrostatic moieties coupled to the
different individual nucleotides. Alternatively, or in addition to,
each type of nucleotide may have the same detectable label and the
sensor may detect the addition of a nucleotide without resolving
the different nucleotides (e.g., determine a sequence length). In
this example, a single read may be used per incorporation cycle. In
an example, each type of individual nucleotide may be coupled to
the same detectable label. The first single-stranded nucleic acid
molecule may be contacted with each type of nucleotide sequentially
(e.g., contacted with one type of nucleotide, followed by contact
with another type of nucleotide). After incorporation of a
nucleotide of one type, the sensor array may detect signals
indicative of nucleotide incorporation. The first single-stranded
nucleic acid molecule may 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).
[0137] The first plurality of individual nucleotides may be
exchanged for a second plurality of individual nucleotides. The
exchange reaction may be accomplished by driving the polymerization
reaction in reverse with an excess of pyrophosphate, triphosphate,
or tetraphosphate. The second plurality of individual nucleotides
may not comprise detectable labels. Exchanging the first plurality
of individual nucleotides for the second plurality of individual
nucleotide may reduce scar formation. The second plurality of
individual nucleotides may comprise reversible terminators. The
reversible terminators may be reversed by contact with a reducing
agent, by changing solution pH, by changing solution ionic
strength, by contact with ionic surfactants, or by any other
terminator removal method.
[0138] FIG. 15 shows an example PMTE sequencing method. A first
single-stranded nucleic acid molecule may be coupled to a bead. The
first single-stranded nucleic acid molecule may have a priming
site. The priming site may be complementary to a portion of the
first single-stranded nucleic acid molecule. The first
single-stranded nucleic acid molecule may be contacted with a first
plurality of individual nucleotides 1501. The first plurality of
individual nucleotides 1501 may comprise single type of nucleotide.
The first plurality of individual nucleotides 1501 may comprise an
irreversible terminator and an irreversible detectable
electrostatic moiety. The irreversible detectable electrostatic
moiety may be the same for each different type of nucleotide. A
polymerizing enzyme 502 may facilitate incorporation of the first
individual nucleotide 1501 into a second single-stranded nucleic
acid molecule. A given sensor may detect the presence or absence of
nucleotide incorporation via the presence or absence of the
detectable label. The first plurality of individual nucleotides
1501 may then be exchanged for a second plurality of individual
nucleotides 701. The second plurality of individual nucleotides 701
may be the same type of nucleotides as the first plurality 1501.
The second plurality of individual nucleotides 701 may not have a
detectable label and may have a reversible terminator. After
incorporation of the second plurality of individual nucleotides
into the second single-stranded nucleic acid molecule the
reversible terminator may be removed or reversed. The terminator
may be reversed by a reducing agent. This cycle may be repeated
until the sequence of all or a part of the first single-stranded
nucleic acid molecule is determined. The method may 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 cycles of nucleotide
incorporation and detection.
[0139] In an example (see FIG. 15), a first single-stranded nucleic
acid molecule may be coupled to a bead. The first single-stranded
nucleic acid molecule may have a priming site coupled to a primer
502. The priming site may be complementary to a portion of the
first single-stranded nucleic acid molecule. The first
single-stranded nucleic acid molecule may be contacted with a first
plurality of individual nucleotide. The first plurality of
individual nucleotides 1501 may comprise multiple different types
of nucleotides. The first plurality of individual nucleotides 1501
may comprise an irreversible terminator and an irreversible
detectable electrostatic moiety. The irreversible detectable
electrostatic moiety may be different for each type of individual
nucleotide. A polymerizing enzyme 502 may facilitate incorporation
of the first individual nucleotide 1501 into a second
single-stranded nucleic acid molecule. A given sensor may detect
the type of nucleotide incorporated into the second single-stranded
nucleic acid molecule via the type of the detectable label present
at the sensor. The first plurality of individual nucleotides 1501
may then be exchanged for a second plurality of individual
nucleotides 701. The second plurality of individual nucleotides 701
may include same types of nucleotides as the first plurality. The
second plurality of individual nucleotides 701 may not have
detectable labels and may have a reversible terminator. After
incorporation of the second plurality of individual nucleotides
into the second single-stranded nucleic acid molecule the
reversible terminator may be removed or reversed. The terminator
may be reversed by a reducing agent. This cycle may be repeated
until the sequence of all or a part of the first single-stranded
nucleic acid molecule is determined.
[0140] The PMTE sequencing approach described above may 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 may be contacted with
a polymerizing enzyme. The polymerizing enzyme may incorporate an
individual nucleotide comprising an irreversible terminator and a
detectable label into a third single-stranded nucleic acid
molecule. Incorporation of the individual nucleotide may generate a
flap. The flap may be cleaved before or after detection of
individual nucleotide incorporation. The double-stranded nucleic
acid molecule may be contacted with the different types of
individual nucleotides simultaneously or sequentially. The
different types of individual nucleotides may comprise the same or
different detectable label. After incorporation of the individual
nucleotides the incorporation event may be detected by the sensor
array. After detection, the individual nucleotide comprising the
detectable label and irreversible terminator may be exchanged for
an individual nucleotide comprising a reversible terminator. The
reversible terminator may then be reversed to allow for
incorporation of subsequent individual nucleotides.
[0141] The method may further comprise monitoring and/or correcting
for phase error. A nucleic acid molecule with phase error may be
extended more or less than the consensus state (e.g. reference
sequence) of a clonal population for which the nucleic acid
molecule is a member of or a template nucleic acid molecule for
which the nucleic acid molecule is a copy or representative
sequence of. For fragments including a base incorporated
incorrectly (e.g., an extra base or incorrect base added to the
growing strand), this phase error may be considered to be leading.
For other nucleic acid molecules where a base is not incorporated
into the growing strand relative to a consensus sequence, the
polynucleotide can be considered to be lagging. As polymerases may
be imperfect, some phase error can occur within a colony that has a
long extension reaction as a part of a colony based sequencing
process. Phase error may limit the read lengths of commercial
clonal sequencing systems.
[0142] Phase errors may lead to leading sequencing incorporation
errors. Leading sequencing error may refer to sequences that are
longer than the dominant sequence due to incorrect or excess (e.g.,
homopolymer) additions of nucleotides. The incorrect or excess
additions may result from polymerase errors, particularly when high
concentrations of dNTPs are used in a noncompetitive reaction.
Alternatively or in addition to, the leading sequencing
incorporation error may result from inadequate washing or
nonspecific binding of dNTPs, which may be subsequently released
and incorporated. Leading sequencing incorporation errors may
result from the incorporation of nucleotides without effective 3'
terminators, thereby causing the incorporation event to proceed one
cycle ahead. For example, leading sequencing incorporation errors
may be caused by the presence of a trace amount of unprotected or
unblocked 3'-OH nucleotides during a nucleic acid incorporation
event. The unprotected 3'-OH nucleotides may be generated during
the manufacturing processes or possibly during storage and reagent
handling processes.
[0143] Phase errors may lead to lagging sequencing errors. Lagging
sequencing incorporation errors may refer to sequences that are
shorter than the dominant sequence through missed additions of the
correct nucleotide. Lagging sequencing errors may occur due to
non-optimal reaction conditions, steric hindrance, secondary
structure, or other sources of polymerase inhibition. Non-limiting
examples of processes that may cause lagging sequencing errors
include: incomplete removal of the reversible terminators,
detectable labels, a flap derived from a second single-stranded
nucleic acid molecules, modified nucleotides, and/or linkers.
Longer cycle times can allow more opportunities for the polymerase
to incorporate the wrong nucleotide. Similarly, less accessible
nucleic acid molecules (e.g., DNA) may result in inadequate
opportunities to incorporate the correct nucleotide. It is
anticipated that temperature, step times, polymerase selection,
nucleotide concentration, salt concentration and buffer selection
may be optimized to minimize incorporation errors.
[0144] For example, a nucleic acid (e.g., DNA) sample may have a
sequence of TGTTC in a first region after a region which is
complementary to a primer. A fluidic cycle may first introduce
deoxycytidine triphosphate (dCTP), secondly followed by
deoxythymidine triphosphate (dTTP), thirdly followed by
deoxyadenosine triphosphate (dATP), and fourthly followed by
deoxyguanosine triphosphate (dGTP), interspersed with wash steps.
In the first part of a fluidic cycle, dCTP molecules which flow in
as part of the first cycle may not be properly washed out and away
from the nucleic acid template. In a second part of a fluidic
cycle, dTTP molecules which flow in as part of the second cycle may
not be properly washed out a well structure. During the first and
second part of the first fluidic cycle, dNTPs may not be
incorporated. During a third part of a fluidic cycle, dATPs may be
introduced and may be incorporated, as dATP is complementary to T,
the first base of the sample. Any nonspecifically bound dCTP
molecules which cease to be nonspecifically bound may also be
incorporated during this third portion of a fluidic cycle. These
unbound dCTP molecules may be incorporated subsequent to
incorporation of a dATP molecule. Subsequent to incorporation of a
dCTP molecule, two more dATP molecules may be incorporated, which
may result in some of the molecules of a monoclonal bead having
leading sequencing phase errors. Thus some molecules of a
monoclonal bead may become out of phase.
[0145] Phase errors may be detected by comparing sequences of a
plurality of double- or single-stranded molecules from a clonal
population and/or by comparing with a reference sequence. For
example, sequences may be analyzed for miscalls, such as
substitution-type or indel-type miscalls. Miscalls may be detected
by measuring signal intensities during nucleic acid incorporation
reaction using single-stranded molecules as templates for
generating complementary single-stranded molecules. The double- or
single-stranded molecules may comprise a clonal population. In an
example, a portion of the clonal population may have substantially
low detectable signal intensity, such as less than threshold,
compared to the rest of the clonal population. This may indicate
that the nucleotides may be incorporated in fewer than all of the
available positions and may result in indel-type miscall.
Indel-type miscalls may be caused by the incomplete extension of
the single-stranded molecules and may lead to lagging sequencing
errors. In another example, a portion of the clonal population may
have a substituted base when compared to a reference sequence.
Substitution-type miscalls may be caused by leading sequencing
errors due to incorporation of an additional nucleotide in nucleic
acid incorporation reaction. The additional nucleotide may be
different than the nucleotide in the reference sequence.
[0146] Phase errors may be reduced by carrying out nucleic acid
incorporation reaction in competitive conditions. For example,
concentration of nucleotides may be reduced in order to mitigate
leading sequencing errors. In another example, cycle time and/or
number of cycles may be reduced for each read in order to avoid
wrong incorporation of nucleotides causing leading sequencing
errors. In some cases, phase errors may be reduced by using
polymerizing enzymes based on the nucleotides. For example, Type A
polymerase, such as Bst polymerase, may be used when incorporating
unmodified nucleotides in order to reduce phase errors. Type B
polymerase, such as Therminator (NEB), may be used when
incorporating modified nucleotides in order to reduce phase
errors.
[0147] In an aspect, phase errors may be reduced by incorporating
unmodified nucleotides, subsequently or simultaneously, with
modified nucleotides in a nucleic acid incorporation reaction.
[0148] A method for nucleic acid sequencing may comprise providing
a plurality of double- or single-stranded nucleic acid molecules
adjacent to a sensor array. A first double- or single-stranded
nucleic acid molecule of the plurality of single-stranded nucleic
acid molecules may be disposed adjacent to a given sensor of the
sensor array.
[0149] The first single-stranded nucleic acid molecule may 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 may comprise alternately
and sequentially (i) incorporating individual nucleotides of a
first plurality of nucleotides comprising detectable labels, and
(ii) incorporating individual nucleotides of a second plurality of
nucleotides that do not comprise detectable labels. The given
sensor may be used to detect signals from the detectable labels
which may be indicative of incorporation of the individual
nucleotides of the first plurality of nucleotides into the second
single-stranded nucleic acid molecule, thereby determining a
sequence of the first single-stranded nucleic acid molecule. The
first plurality of nucleotides may be exchanged with the second
plurality of nucleotides. The incorporation of the second plurality
of nucleotides may correct phase error. The incorporation of the
second plurality of nucleotides may correct phase error by
incorporating an individual nucleotide from the second plurality of
nucleotides at a location along the first single-stranded nucleic
acid molecule in which an individual nucleotide from the first
plurality of nucleotides has not been incorporated. The nucleic
acid incorporation reaction may be continued by using the
individual nucleotides from the first plurality of nucleotides. The
first single-stranded nucleic acid molecule may have sequence
homology to a template single-stranded nucleic acid molecule.
[0150] An example of a method for nucleic acid sequencing in order
to correct phase error is illustrated in FIG. 20. A plurality of
single-stranded nucleic acid molecules may be coupled to a bead.
The plurality of single-stranded nucleic acid molecules may
comprise a clonal population of a given single-stranded nucleic
acid molecule. A first single-stranded nucleic acid molecule may
have priming sites coupled to individual primers. The priming site
may be complementary to a portion of the first single-stranded
molecule. The first single-stranded molecule may be contacted with
a first plurality of individual nucleotides. The first plurality of
individual nucleotides may comprise multiple different types of
nucleotides. The first plurality of individual nucleotides may
comprise single type of nucleotide. The first plurality of
individual nucleotides may be modified nucleotides. The first
plurality of individual nucleotides may comprise an irreversible
terminator and an irreversible detectable electrostatic moiety. The
irreversible detectable electrostatic moiety may be different for
each type of individual nucleotide. A polymerizing enzyme may
facilitate incorporation of the first individual nucleotide into a
second single-stranded nucleic acid molecule. A given sensor may
detect the type of nucleotide incorporated into the second
single-stranded nucleic acid molecule via the type of the
detectable label present at the sensor. The first plurality of
individual nucleotides may then be exchanged for a second plurality
of individual nucleotides. The second plurality of individual
nucleotides may include same types of nucleotides as the first
plurality. The second plurality of individual nucleotides may not
have detectable labels and may have a reversible terminator. The
addition of the second plurality of nucleotides may correct phase
error by incorporating an individual nucleotide from the second
plurality of nucleotides at a location along the first
single-stranded nucleic acid molecule in which an individual
nucleotide from the first plurality of nucleotides has not been
incorporated.
[0151] As shown in FIG. 20, the phase error can be a leading
sequencing error as indicated by the addition of an extra
nucleotide (n+1) in a leading second single-stranded molecule in
Read1. An individual nucleotide from the second plurality of
nucleotides may be incorporated into a lagging second
single-stranded molecule, such that the lagging molecule may be in
sync with the leading molecule, as indicated by "n+1" in the next
cycle before Read2. After incorporation of the second plurality of
individual nucleotides into the second single-stranded nucleic acid
molecule the reversible terminator may be removed or reversed. The
terminator may be reversed by a reducing agent. Once both the
molecules may be synced-in with the same number of incorporated
nucleotides that is (n+1), the nucleic acid incorporation reaction
may be continued using the individual nucleotides from the first
plurality of nucleotides. In the next cycle, Read2, the detectable
label in the first plurality of nucleotides may be cleaved for
detection by a sensor. The detectable label may be cleaved by using
a phosphate reagent, such as tris(hydroxypropyl)phosphine (THPP).
The cleavage of the detectable label may leave a scar on the
individual nucleotide after cleavage. The scar may comprise
portions of the detectable label that are not fully removed during
cleavage of the label. The first single-stranded nucleic acid
molecule may be alternately provided with the first plurality of
individual nucleotides and the second plurality of individual
nucleotides to generate the second single-stranded nucleic acid
molecule until the sequence of all or a part of the first
single-stranded nucleic acid molecule is determined.
[0152] FIGS. 21 and 22 show example results of using the method for
reducing phase error during nucleic acid sequencing. A first
single-strand molecule may be contacted with a first plurality of
nucleotides with a detectable label, such as three lysine amino
acid residues, in order to generate a second single-strand
molecule. The first plurality of nucleotides may be exchanged with
a second plurality of nucleotides. The incorporation of the first
plurality of nucleotides may cause a leading phase error (n+1) in
the second single-strand molecule in Read1. The phase error may be
corrected by incorporating the second plurality of nucleotides in
the lagging molecules such that the lagging molecules may be
in-sync with leading molecules. As shown in FIG. 21, X-axis shows
flow number corresponding to the number of nucleotides incorporated
and Y-axis shows signal derived from cleaving of the detectable
label. In Read1, the detectable label may be coupled to the first
plurality of nucleotides which may result in a negative signal on
the Y-axis. The negative signal may be due to the displacement of
cations, such as Mg.sup.2+, by the lysine residues of the
detectable label. In Read2, the detectable label may be cleaved
from the first plurality of nucleotides resulting in a positive
signal on the Y-axis derived from the scarred nucleotides. The
positive signal may be derived from the concentration of cations,
such as Mg.sup.2+, upon removal of the detectable label comprising
lysine residues. In both Read1 and Read2, the second plurality of
individual nucleotides may not have detectable labels, which may
result in a signal close to zero on the Y-axis. The changes in
signals during the sequencing process are shown in FIG. 22. The
presence of a detectable label, three lysine residues, in the first
plurality of nucleotides may result in a net negative signal on the
Y-axis, as indicated by delta K3 in Read1. Addition of the
unmodified nucleotides may result in a neutral signal, close to
zero on the Y-axis as indicated by delta chase in Read2. Cleavage
of the detectable label by THPP may result in a surge of a net
positive signal as indicated by delta THPP in Read3. Upon cleavage
of the detectable label, the neutral signal due to the unmodified
nucleotides may shift to a positive signal as indicated by arrows
during delta chase. The positive signal may be due to negative
phosphate groups in the nucleotides, in turn, may concentrate
Mg2.sup.+ cations which may produce a net positive signal.
Systems for Nucleic Acid Sequencing
[0153] The present disclosure provides a system for nucleic acid
sequencing that may include various components. The system may be
used in various applications, such as sequencing a nucleic acid
sample from a living subject. For example, a sensor array with
sites occupied by beads or with sites directly occupied by a
plurality of nucleic acid templates comprising clonal populations
may be contacted with a fluid comprising a primer(s) that hybridize
to clonal nucleic acids. The sensor array may then be washed and
contacted with a fluid comprising one or more types of nucleotides,
polymerizing enzymes, and/or any co-factors in a suitable buffer.
The array may then be washed and the incorporated nucleotides may
be detected. The incorporate, wash, detect cycle may be repeated
until sample nucleic acids bound to the bead or bound to a surface
of the sensor have been sequenced.
[0154] The sensor array may be incorporated into an integrated
sequencing platform. An integrated sequencing platform may 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 separate and/or in modular format. In some embodiments, the
integrated sequencing platform can include one, two, three, four,
or all five of these systems. In some cases, the modules can be
integrated within a single unit (e.g., a microfluidic device), a
single array (e.g., a sensor array that may be re-usable) 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 entirely
incorporated herein by reference.
[0155] In another aspect, the present disclosure provides a system
for nucleic acid sequencing. The system may comprise a sensor array
comprising a plurality of individual sensors. During use a given
double-stranded nucleic acid molecule of a plurality of
double-stranded nucleic acid molecules may be disposed adjacent to
a given sensor of the sensor array. The given double-stranded
nucleic acid molecule may comprise a first single-stranded nucleic
acid molecule and a second-single stranded nucleic acid molecule.
The given sensor may be electrically coupled to a charge double
layer (e.g., within a Debye length of) the given double-stranded
nucleic acid molecule. The system may further comprise one or more
computer processors that are operatively coupled to the sensor
array. The one or more computer processors may be programmed to
bring a non-hybridized segment of the first single-stranded nucleic
acid molecule in contact with individual nucleotides to subject the
non-hybridized segment to a nucleic acid incorporation reaction
that generates a third single-stranded nucleic acid molecule for
the individual nucleotides. The third single-stranded nucleic acid
molecule may have sequence complementarity with the first
single-stranded nucleic acid molecule. During or subsequent to the
nucleic acid incorporation reaction, the given sensor may detect
signals indicative of incorporation of the individual nucleotides
not the third single-stranded nucleic acid molecule, thereby
determining a sequence of the non-hybridized segment.
[0156] The double-stranded nucleic acid molecule may be coupled to
a support. The support may be a bead or a surface of the sensor
array. A plurality of double-stranded nucleic acid molecules may be
coupled to a plurality of beads or a plurality of locations on the
surface of the sensor array. Each bead of the plurality of beads
may be disposed 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 double-stranded nucleic
acid molecule to the bead. The charge double layer (e.g., Debye
length) may be adjacent to the surface of the bead. Alternatively,
or in addition to, the 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 may be coupled
to a surface of a given sensor. The charge double layer (e.g.,
Debye length) may be adjacent to the surface of the given sensor.
The double-stranded nucleic acid molecule coupled to the bead or
surface of the sensor array may be clonally amplified prior to
sequencing so that each bead is coupled to a clonal population of
double-stranded nucleic acid molecules or so that each surface of a
given sensor is coupled to a clonal population of double-stranded
nucleic acid molecules.
[0157] A given sensor may comprise at least one, at least two, at
least three, or at least four electrodes. In an example, a given
sensor comprises at least two electrodes. The electrodes of a given
sensor may detect signals indicative of incorporation of individual
nucleotides into the double-stranded nucleic acid molecule. Signals
indicative of incorporation events may include changes in
impedance, conductance, or charge in the electronic double layer.
In an example, signals indicative of incorporation of individual
nucleotides are electrical signals garneted by an impedance or
impedance change in the charge double layer. The signals indicative
of incorporation of individual nucleotides may be steady state
signals, transient signals, or a combination of steady state and
transient signals. Signals may be detected transiently or during
steady state conditions. In a transient signal detection modality,
the detection occurs during or closely after nucleotide
incorporation. In steady state detection, reading of the sensor may
occur after the "completion" of the incorporation event. A steady
state change in signal may remain until a change is introduced to
the environment around the sensor.
[0158] The one or more computer processors may be programed to
direct fluid flow across the sensor array. The double-stranded
nucleic acid molecules may be stably coupled to one or more
surfaces during fluid flow conditions. The double-stranded nucleic
acid molecules may be stably coupled to a plurality of beads. The
beads may be stably disposed adjacent to the sensor array. The
beads may be held adjacent to the sensor array by a magnetic or
electric field. The fluid flow may not disrupt or move the beads.
The fluid directed across the sensor array may include nucleic acid
molecules, primers, polymerizing enzymes, individual nucleotides,
co-factors used for a nucleotide incorporation reaction (e.g.,
primer extension reaction), and/or buffers. The fluid may be a
washing fluid comprising buffers. In an example, a fluid may be
directed to the sensor array and incubated with the 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.
[0159] In another aspect, the present disclosure provides a system
for nucleic acid sequencing. The system may comprise a sensor array
comprising a plurality of sensors. During use a first
single-stranded nucleic acid molecule of a plurality of
single-stranded nucleic acid molecules may be disposed adjacent to
a given sensor of the sensor array. The given sensor may be
electrically coupled to a charge double layer (e.g., within a Debye
length of) the first single-stranded nucleic acid molecule. The
system may comprise one or more computer processors couple to the
sensor array. The one or more computer processors may be programed
to bring the first single-stranded nucleic acid molecule into
contact with individual nucleotides to subject the first
single-stranded nucleic acid molecule to a nucleic acid
incorporation reaction which generates a second single-stranded
nucleic acid molecule from the individual nucleotides. The second
single-stranded nucleic acid molecule may have sequence
complementarity with the first single-stranded nucleic acid
molecule. At least a subset of the individual nucleotides may
comprise detectable labels. A given sensor may detect signals from
the detectable labels during or subsequent to the nucleic acid
incorporation reaction. The signals may be indicative of
incorporation of the individual nucleotides into the second
single-stranded nucleic acid molecule. The signals may be used to
determine a sequence of the first single-stranded nucleic acid
molecule.
[0160] The single-stranded nucleic acid molecule may be coupled to
a support. The support may be a bead or a surface of the 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 the sensor array. Each bead of the plurality of beads
may be disposed 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 molecule to the bead. The charge double layer (e.g., Debye
length) may be adjacent to the surface of the bead. Alternatively,
or in addition to, the 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 may be coupled
to a surface of a given sensor. The charge double layer (e.g.,
Debye length) may be adjacent to the surface of the given sensor.
The single-stranded nucleic acid molecule coupled to the bead or
surface of the sensor array may be clonally amplified prior to
sequencing so that each bead is coupled to a clonal population of
single-stranded nucleic acid molecules or so that each surface of a
given sensor is coupled to a clonal population of single-stranded
nucleic acid molecules.
[0161] A given sensor may comprise at least one, at least two, at
least three, or at least four electrodes. In an example, a given
sensor comprises at least two electrodes. In another example, a
given sensor comprises two electrodes. The electrodes may be
exposed to the solution in which the primer extension reaction
takes place. Alternatively, or in addition to, the electrodes may
be buried within the sensor array and, therefore, may not be
exposed to the solution in which the primer extension reaction
takes place. The electrodes of a given sensor may detect signals
indicative of incorporation of individual nucleotides into the
single-stranded nucleic acid molecule. Signals indicative of
incorporation events may include changes in impedance, conductance,
or charge in the electronic double layer. In an example, signals
indicative of incorporation of individual nucleotides are
electrical signals garneted by an impedance or impedance change in
the charge double layer. The signals indicative of incorporation of
individual nucleotides may be steady state signals, transient
signals, or a combination of steady state and transient signals.
Signals may be detected transiently or during steady state
conditions. In a transient signal detection modality, the detection
occurs during or closely after nucleotide incorporation. In steady
state detection, reading of the sensor may occur after the
completion of the incorporation event. A steady state change in
signal may remain until a change is introduced to the environment
around the sensor. The sensor may detect incorporation events
(e.g., count incorporation events) or may individually resolve
incorporated nucleotides (e.g., determine which nucleotide is
incorporated).
[0162] The one or more computer processors may be programed to
direct fluid flow across the sensor array. The single-stranded
nucleic acid molecules may be stably coupled to one or more
surfaces during fluid flow conditions. The single-stranded nucleic
acid molecules may be stably coupled to a plurality of beads. The
beads may be stably disposed adjacent to the sensor array. The
beads may be held adjacent to the sensor array by a magnetic or
electric field. The fluid flow may not disrupt or move the beads.
The fluid directed across the sensor array may include nucleic acid
molecules, primers, polymerizing enzymes, individual nucleotides,
co-factors used for a nucleotide incorporation reaction (e.g.,
primer extension reaction), and/or buffers. The fluid may be a
washing fluid comprising buffers. In an example, a fluid may be
directed to the sensor array and incubated with the 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.
[0163] In another aspect, the present disclosure provides a system
for nucleic acid sequencing. The system may comprise a sensor array
comprising a plurality of sensors. During use a first
single-stranded nucleic acid molecule of a plurality of
single-stranded nucleic acid molecules may be disposed adjacent to
a given sensor of the sensor array. The system may comprise one or
more computer processors operatively coupled to the sensor array.
The one or more computer processors may be programmed to subject
the first single-stranded nucleic acid molecule to a nucleic acid
incorporation reaction that comprises alternately and sequentially
incorporating individual nucleotides of a first plurality of
nucleotides comprising detectable labels and exchanging the
individual nucleotides of the first plurality of nucleotides with
individual nucleotides of a second plurality of nucleotides that do
not comprise detectable labels. A given sensor may detect signals
from the detectable labels during or subsequent to the nucleic acid
incorporation reaction. The signals may be indicative of
incorporation of the individual nucleotides into the second
single-stranded nucleic acid molecule. The signals may be used to
determine a sequence of the first single-stranded nucleic acid
molecule.
[0164] The plurality of single stranded-nucleic acid molecules may
be coupled to a plurality of supports. The plurality of supports
may be a plurality of beads or a plurality of surfaces on the
sensor array. In an example, the 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
double layer comprising the first single-stranded nucleic acid
molecule. The charge double layer may be adjacent to a surface of
the given bead or on the surface of a given sensor. The
single-stranded nucleic acid molecule may be amplified on the
surface of the support. The amplification products may be coupled
to the surface of the support. The amplification products may form
a clonal colony of single-stranded nucleic acid molecules on the
surface of the support. The clonal colony of single-stranded
nucleic acid molecules may be sequenced.
[0165] A given sensor of the sensor array may comprise at least
one, at least two, at least three, at least four, or more
electrodes. In an example, a given sensor comprises at least two
electrodes. In another example, a given sensor comprises two
electrodes. The electrodes may be exposed to the solution in which
the primer extension reaction takes place. Alternatively, or in
addition to, the electrodes may be buried within the sensor array
and, therefore, may not be exposed to the solution in which the
primer extension reaction takes place. The sensor may detect
signals indicative of nucleotide incorporation events. The sensor
may detect the detectable label coupled to the individual
nucleotides. The sensor may detect the detectable label during
transient or steady state conditions. Nucleotide incorporation may
be detected once, twice, three times, four times, or more than four
times per incorporation cycle during steady state conditions. In an
example, nucleotide incorporation may be detected at least twice
per incorporation cycle during steady state conditions. The sensor
array may detect electrical signals during transient or steady
state conditions. The electrical signals may include, but are not
limited to, changes in charge state of a molecule, changes in the
conductivity of a surrounding solution, impedance signals, or
changes in impedance signals. The sensor may detect a change in
charge and/or conductivity or a change in impedance. The sensor may
detect the change in charge and/or conductivity or impedance within
a charge double layer (e.g., Debye length) of the sensor, support,
or nucleic acid molecule (e.g., the sample). The detectable labels
coupled to the individual nucleotides may alter the electrical
environment surrounding the single-stranded nucleic acid molecules
and a given sensor may detect the electrical change. The sensor may
detect incorporation events (e.g., count incorporation events) or
may individually resolve incorporated nucleotides (e.g., determine
which nucleotide is incorporated).
[0166] The one or more computer processors may be programed to
direct fluid flow across the sensor array. The single-stranded
nucleic acid molecules may be stably coupled to one or more
surfaces during fluid flow conditions. The single-stranded nucleic
acid molecules may be stably coupled to a plurality of beads. The
beads may be stably disposed adjacent to the sensor array. The
beads may be held adjacent to the sensor array by a magnetic or
electric field. The fluid flow may not disrupt or move the beads.
The fluid directed across the sensor array may include nucleic acid
molecules, primers, polymerizing enzymes, individual nucleotides,
co-factors used for a nucleotide incorporation reaction (e.g.,
primer extension reaction), and/or buffers. The fluid may be a
washing fluid comprising buffers. In an example, a fluid may be
directed to the sensor array and incubated with the 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 Systems
[0167] The present disclosure provides computer systems that are
programmed to implement methods of the disclosure. FIG. 16 shows a
computer system 1601 that is programmed or otherwise configured to
sequence nucleic acid molecules. The 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 the sensor array, controlling flow of individual
nucleotides to the sensor array, and controlling incorporation
reaction conditions. The computer system 1601 can be an electronic
device of a user or a computer system that is remotely located with
respect to the electronic device. The electronic device can be a
mobile electronic device.
[0168] The computer system 1601 includes a central processing unit
(CPU, also "processor" and "computer processor" herein) 1605, which
can be a single core or multi core processor, or a plurality of
processors for parallel processing. The computer system 1601 also
includes memory or memory location 1610 (e.g., random-access
memory, read-only memory, flash memory), electronic storage unit
1615 (e.g., hard disk), communication interface 1620 (e.g., network
adapter) for communicating with one or more other systems, and
peripheral devices 1625, such as cache, other memory, data storage
and/or electronic display adapters. The memory 1610, storage unit
1615, interface 1620 and peripheral devices 1625 are in
communication with the CPU 1605 through a communication bus (solid
lines), such as a motherboard. The storage unit 1615 can be a data
storage unit (or data repository) for storing data. The computer
system 1601 can be operatively coupled to a computer network
("network") 1630 with the aid of the communication interface 1620.
The network 1630 can be the Internet, an internet and/or extranet,
or an intranet and/or extranet that is in communication with the
Internet. The network 1630 in some cases is a telecommunication
and/or data network. The network 1630 can include one or more
computer servers, which can enable distributed computing, such as
cloud computing. The network 1630, in some cases with the aid of
the computer system 1601, can implement a peer-to-peer network,
which may enable devices coupled to the computer system 1601 to
behave as a client or a server.
[0169] The CPU 1605 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
1610. The instructions can be directed to the CPU 1605, which can
subsequently program or otherwise configure the CPU 1605 to
implement methods of the present disclosure. Examples of operations
performed by the CPU 1605 can include fetch, decode, execute, and
writeback.
[0170] The CPU 1605 can be part of a circuit, such as an integrated
circuit. One or more other components of the system 1601 can be
included in the circuit. In some cases, the circuit is an
application specific integrated circuit (ASIC).
[0171] The storage unit 1615 can store files, such as drivers,
libraries and saved programs. The storage unit 1615 can store user
data, e.g., user preferences and user programs. The computer system
1601 in some cases can include one or more additional data storage
units that are external to the computer system 1601, such as
located on a remote server that is in communication with the
computer system 1601 through an intranet or the Internet.
[0172] The computer system 1601 can communicate with one or more
remote computer systems through the network 1630. For instance, the
computer system 1601 can communicate with a remote computer system
of a user (e.g., laptop or cellular phone of a user). Examples of
remote computer systems include personal computers (e.g., portable
PC), slate or tablet PC's (e.g., Apple.RTM. iPad, Samsung.RTM.
Galaxy Tab), telephones, Smart phones (e.g., Apple.RTM. iPhone,
Android-enabled device, Blackberry.RTM.), or personal digital
assistants. The user can access the computer system 1601 via the
network 1630.
[0173] Methods as described herein can be implemented by way of
machine (e.g., computer processor) executable code stored on an
electronic storage location of the computer system 1601, such as,
for example, on the memory 1610 or electronic storage unit 1615.
The machine executable or machine readable code can be provided in
the form of software. During use, the code can be executed by the
processor 1605. In some cases, the code can be retrieved from the
storage unit 1615 and stored on the memory 1610 for ready access by
the processor 1605. In some situations, the electronic storage unit
1615 can be precluded, and machine-executable instructions are
stored on memory 1610.
[0174] The code can be pre-compiled and configured for use with a
machine having a processer adapted to execute the code, or can be
compiled during runtime. The code can be supplied in a programming
language that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
[0175] Aspects of the systems and methods provided herein, such as
the computer system 1601, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such as memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless links, optical links or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
[0176] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, a tangible storage medium, a carrier wave medium or
physical transmission medium. Non-volatile storage media include,
for example, optical or magnetic disks, such as any of the storage
devices in any computer(s) or the like, such as may be used to
implement the databases, etc. shown in the drawings. Volatile
storage media include dynamic memory, such as 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 may take the form of electric or electromagnetic signals, or
acoustic or light waves such as those generated during radio
frequency (RF) and infrared (IR) data communications. Common forms
of computer-readable media therefore 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 may 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.
[0177] The computer system 1601 can include or be in communication
with an electronic display 1635 that comprises a user interface
(UI) 1640 for providing, for example, current operating conditions
of the system or sequencing results. Examples of UI's include,
without limitation, a graphical user interface (GUI) and web-based
user interface.
[0178] Methods and systems of the present disclosure can be
implemented by way of one or more algorithms. An algorithm can be
implemented by way of software upon execution by the central
processing unit 1605. The algorithm can, for example, convert
signals indicative of nucleotide incorporation into a nucleic acid
sequence.
[0179] 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. It is not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
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. Furthermore, it shall be understood that all aspects of
the 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 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.
* * * * *