Forced polynucleotide scanning for mapping features in nanopore device

Abstract

To satisfy a (1) need for consistent linearization of translocating molecules, (2) need to reduce effect of molecular fluctuations that introduce random error, and (3) need to develop strategies to perform accurate genomic distance calibration.SOLUTION: The present disclosure provides an automated method of mapping one or more features of a target polynucleotide. Also provided are automated methods of sequencing a polynucleotide sequence. Also provided are methods for extended recapture of a polynucleotide in a nanopore device. Also provided are devices and systems for implementing the methods of the present disclosure.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] The present disclosure provides an automated method for mapping one or more features of a target polynucleotide. An automated method for sequencing a polynucleotide sequence is also provided in the present disclosure. A method for extended recapture of a polynucleotide in a nanopore device is also provided in the present disclosure. Devices and systems for carrying out the methods of the present disclosure are also provided in the present disclosure.

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 003,129, filed March 31, 2020, U.S. Provisional Application No. 62 / 883,449, filed August 6, 2019, and U.S. Provisional Application No. 62 / 962,838, filed January 17, 2020, which are incorporated by reference in their entireties herein. [Background technology]

[0003] Precise mapping of binding positions of molecular motifs along long individual dsDNA strands in highly heterogeneous samples is central to a wide range of genomics applications outside of sequencing. One candidate approach to mapping molecular properties is based on measuring the modulation of ionic currents that arise when double-stranded DNA (dsDNA) is driven electrically through solid-state pores (ss-pores). Nanopores are attractive because they have a purely electrical readout, leading to a small footprint and substantially reduced costs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 012881 [Patent Document 2] International Publication No. 2018 / 236673 [Patent Document 3] U.S. Patent Application Publication No. 2017 / 0145481 [Patent Document 4] U.S. Patent No. 9,863,912 [Patent Document 5] US Special Application No. 2018 / 0023115 [Patent Document 6] US Special Offer No. 2013-0233709

Non-licensed documents

[0005]

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[0006] (1) The need for consistent linearization of transferred molecules; (2) the need to reduce the effects of molecular variations that introduce random errors; and (3) the need to develop strategies for accurate genomic distance calibration. [Means for solving the problem]

[0007] An embodiment of the present disclosure discloses a method for partially or completely recapturing a polynucleotide in a previously captured nanopore device, the method comprising the steps of: a) providing a device having at least one nanopore, the device comprising: (i) a first fluidic volume that is a chamber and a geometrically constrained enclosure; (ii) a first fluid volume having an inlet and an outlet that are fluid-filled and accessible to an electrode, the first pore being connected to the first fluid volume at a location between the inlet and the outlet, (iii) at least one electrode disposed within the first fluid volume and at least one electrode disposed within the chamber, (iv) a sensor configured to provide a voltage between an electrode in the first fluid volume and an electrode in the chamber, and a current measurement to detect capture and translocation of a polynucleotide into and through the first pore; b) introducing the polynucleotide into the chamber of the device; and c) applying a first voltage to the first fluid volume. d) capturing and translocating the polynucleotide in a first direction from the chamber through the first pore into the first fluid volume; d) detecting a current with a first sensor as the polynucleotide translocates in the first direction through the first pore; e) applying a second voltage equal to zero mV for a predetermined time while the polynucleotide is contained within the first fluid volume; f) applying a third voltage to capture and partially or completely translocate the polynucleotide from the first fluid volume through the first pore into the chamber; and g) detecting a current in the first sensor as the polynucleotide translocates partially or completely through the first pore.

[0008] In some embodiments, the nanopore device further comprises a second pore.

[0009] In some embodiments, the first fluid volume is a fluid channel.

[0010] In some embodiments, the polynucleotide exhibits a minimum time duration during the detection step (d) in which the polynucleotide was captured and transferred through the first pore prior to performing step (e), indicating that the polynucleotide is of greater than a minimum length.

[0011] In some embodiments, the polynucleotide is identified as a target if the minimum duration is greater than a threshold, and is identified as a non-target if the minimum duration is less than a threshold.

[0012] In some embodiments, the detecting step of step (d), in which the polynucleotide is captured at and transferred from the first pore at a first voltage, maintains the first voltage for a time range of from 30 ms to 500 ms or more.

[0013] In some embodiments, the second voltage equal to zero in step (e) is maintained for a time range from 10 ms to 5 seconds or more.

[0014] In some embodiments, the second voltage equal to zero in step (e) is maintained for a period of time sufficient to allow the molecules to relax to an entropically equilibrium configuration.

[0015] In some embodiments, the first end of the polynucleotide is positioned away from at least the first pore by a distance ranging from 5 microns to 5 millimeters or more.

[0016] In some embodiments, the second end of the polynucleotide is positioned away from the first pore by a distance ranging from 2 microns to 5 millimeters or more.

[0017] In some embodiments, the method includes repeating steps c) through g).

[0018] In some embodiments, the chamber is disposed above at least the first pore.

[0019] In some embodiments, the chambers are connected to a common ground relative to a first voltage.

[0020] In some embodiments, the nanopore device further comprises a second pore.

[0021] In some embodiments, the second pore is fluidly connected to a second fluid volume, said second fluid volume being a geometrically constrained enclosure with a second inlet and a second outlet, and the second pore is fluidly connected to the second fluid volume between said inlet and outlet.

[0022] In some embodiments, the second fluid volume is a second fluid channel.

[0023] In some embodiments, a second pore is connected to said chamber and said second volume.

[0024] In some embodiments, the chamber is disposed above the first and second pores.

[0025] In some embodiments, the first voltage is applied between the first fluid volume and the chamber.

[0026] In some embodiments, the nanopore device comprises at least one electrode disposed within a second fluid volume, the at least one electrode configured to provide a voltage at a second pore that is controllable independently of a voltage at the first pore.

[0027] In some embodiments, the nanopore device comprises dual-amplifier electronics configured to control the voltage and measure the current at the first pore and the second pore.

[0028] In some embodiments, the method further comprises detecting a second sensor current.

[0029] In some embodiments, the method further comprises adjusting the first voltage at the first pore after detecting the second sensor current, setting the first voltage at the second pore such that at least a portion of a polynucleotide translocates through the first pore and the second pore.

[0030] In some embodiments, the first voltage at the second pore is greater than the first voltage at the first pore.

[0031] In some embodiments, the first voltage at the second pore is greater than the third voltage at the first pore.

[0032] In some embodiments, the third voltage is greater than the first voltage.

[0033] In some embodiments, the second voltage is 0 mV.

[0034] In some embodiments, the first voltage is in the range of 50 to 900 mV.

[0035] In some embodiments, the first voltage is in the range of 50 to 900 mV.

[0036] In some embodiments, the third voltage is in the range of 50 to 900 mV.

[0037] In some embodiments, the first voltage at the second pore is in the range of 50 to 900 mV.

[0038] In some embodiments, the first voltage at the first pore, the third voltage at the first pore, and the first voltage at the second pore are independently in the range of 50 to 900 mV in magnitude.

[0039] In some embodiments, the first fluid volume, which is a geometrically constrained housing, is opposite the first pore, and in some embodiments, the second geometrically constrained fluid volume is opposite the second pore.

[0040] In some embodiments, the polynucleotide is substantially linearized. In some embodiments, the polynucleotide is substantially linearized by the effect of adjusting the first voltage, the second voltage, the third voltage, or a combination thereof.

[0041] In some embodiments, the polynucleotide is translocated in a second direction in step f), the second direction being from the first fluid volume through the first pore.

[0042] In some embodiments, the method includes adjusting a third voltage at the first pore, a first voltage at the second pore, or both, to change the orientation of the polynucleotide such that at least a portion of the polynucleotide translocates from the second pore through the first pore in a first direction.

[0043] In some embodiments, adjusting the third voltage at the first pore, adjusting the first voltage at the second pore, or both, such that at least a portion of the polynucleotide moves in a first direction and / or a second direction is repeated for a period ranging from 30 ms to 5 minutes or more until the polynucleotide exits the device.

[0044] In some embodiments, a first voltage is applied between the chamber and the second fluid volume of the device.

[0045] In some embodiments, the first voltage is applied for a time range of 30 ms to 500 ms or more.

[0046] In some embodiments, the method further comprises detecting a first set of characteristics of the polynucleotide when the polynucleotide is within both pores in the first orientation.

[0047] In some embodiments, the method comprises detecting a second set of characteristics of the polynucleotide when the polynucleotide is in both pores simultaneously in the second orientation.

[0048] In some embodiments, the method comprises adjusting the first voltage such that the polynucleotide translocates through the first pore for a period ranging from 30 ms to 500 ms or more.

[0049] In some embodiments, the polynucleotide passes through the first pore, the chamber, and the second pore.

[0050] In some embodiments, the method further comprises detecting a third sensor current at the first pore and a fourth sensor current at the second pore when the polynucleotide is in both pores in the first or second orientation.

[0051] In some embodiments, the method further comprises detecting a fifth sensor current at the first pore and a sixth sensor current at the second pore when the polynucleotide is in both pores in the first or second orientation.

[0052] In some embodiments, the first voltage creates a voltage gradient across the first pore and along the length of the first fluid volume.

[0053] In some embodiments, the third voltage creates a voltage gradient across the second pore and along the length of the second fluid volume.

[0054] In some embodiments, the resistance of the first fluid channel is inversely proportional to the width of the first fluid channel.

[0055] In some embodiments, the resistance of the second fluid channel is inversely proportional to the width of the second fluid channel.

[0056] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel.

[0057] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel.

[0058] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of the first fluid channel and / or the second fluid channel.

[0059] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of a cross-section of the first fluid channel and / or the second fluid channel.

[0060] In some embodiments, the polynucleotide is substantially linearized.

[0061] In some embodiments, the polynucleotide is substantially linearized by the effect of adjusting the first voltage, the second voltage, the third voltage, or a combination of the first voltage, the second voltage, and the third voltage.

[0062] In some embodiments, the method further comprises controlling, by the controller, when the polynucleotide requires rescanning of one or more features of the polynucleotide a second or third time.

[0063] In some embodiments, the first fluid channel and / or the second fluid channel comprise a geometrically constrained volume.

[0064] In some embodiments, the controller determines whether one or more features of the polynucleotide are present and performs additional recapture of the one or more features in the first orientation and / or the second orientation.

[0065] In some embodiments, the method further comprises moving away from one or more features of the previously recaptured polynucleotide.

[0066] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 0 mV to 1000 mV.

[0067] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 0 mV to 100 mV.

[0068] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 100 mV to 200 mV.

[0069] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 200 mV to 300 mV.

[0070] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 300 mV to 400 mV.

[0071] In some embodiments, the first voltage, the second voltage, and the third voltage are in the range of 400 mV to 500 mV.

[0072] In some embodiments, the first voltage to the first pore is lower than the second voltage.

[0073] In some embodiments, the first voltage to the first pore is higher than the second voltage.

[0074] In some embodiments, the first voltage to the first pore is the same as the second voltage.

[0075] In some embodiments, the first voltage to the first pore and the second voltage to the first pore are the same in the first direction.

[0076] In some embodiments, the first voltage across the first pore is lower than the second voltage across the first pore in the second direction.

[0077] In some embodiments, the first voltage to the first pore is lower than the third voltage to the second pore in the third orientation.

[0078] In some embodiments, the first voltage to the first pore is greater than the third voltage to the second pore in the fourth direction.

[0079] In some embodiments, the method further comprises controlling a direction of the polynucleotide through the first and / or second pore by a controller, a processor, and a non-transitory computer readable medium comprising instructions to the processor to change the direction of the polynucleotide.

[0080] In some embodiments, the processor comprises a field-programmable gate array (FPGA) or an application-specific integrated circuit (ASIC).

[0081] In some embodiments, the controller comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0082] In some embodiments, the controller is a microcontroller.

[0083] In some embodiments, the recapture provides for detection of polynucleotides comprising polynucleotide sequences having lengths ranging from 300 base pairs to about 3,000,000 base pairs.

[0084] In some embodiments, the first fluid channel and / or the second fluid channel have a length from the inlet to the outlet in a range from about 0.05 mm to about 8 mm.

[0085] In some embodiments, the first fluid channel and / or the second fluid channel have a width in the range of 20-500 μm.

[0086] In some embodiments, the first fluid channel and / or the second fluid channel have a depth in the range of 0.5 μm to about 2 μm.

[0087] In some embodiments, the sensor is further configured to provide a voltage between the electrode in the second fluid volume and the electrode in the chamber, and to provide a current measurement to detect capture and transfer of polynucleotides into and through the first pore and the second pore.

[0088] In some embodiments, the length of the polynucleotide is at least 32 times, at least 3 times, at least 4 times, or at least 5 times the distance between the first pore and the second pore, between the chamber and the first fluid volume, and / or between the chamber and the second fluid volume.

[0089] In some embodiments, the first voltage is maintained for a period in the range of 0 to 1000 milliseconds, 0 to 20 milliseconds, 20 to 50 milliseconds, 50 to 100 milliseconds, 100 to 500 milliseconds, or 500 to 1000 milliseconds.

[0090] In some embodiments, the first voltage is maintained for a period in the range of 20 ms or more, 50 ms or more, 50 ms or more, 120 ms or more, 150 ms or more, 300 ms or more, 500 ms or more, 1000 ms or more, 20 seconds or more, 60 seconds or more, 120 seconds or more, 150 seconds or more, 300 seconds or more, 500 seconds or more, or 1000 seconds or more.

[0091] In some embodiments, the first voltage is maintained for a period of time after the target polynucleotide is captured and translocated through the first pore.

[0092] Aspects of the disclosure include a method of mapping one or more characteristics of a target polynucleotide, the method comprising: a) providing a device for controlling the movement of a target polynucleotide through first and second pores simultaneously, the device comprising: (i) a first pore disposed in fluid communication between a chamber and a first fluid volume, the first fluid volume being a geometrically constrained enclosure; (ii) a second pore disposed in fluid communication between a chamber and a second fluid volume, the second fluid volume being a geometrically constrained enclosure; and (iii) a second pore disposed in fluid communication between the chamber and a second fluid volume, the second fluid volume being a geometrically constrained enclosure, each of which is filled with fluid and has access to an electrode. (iv) at least one electrode disposed in the first fluid volume, at least one electrode disposed in the second fluid volume, and at least one electrode disposed in the chamber; (v) a first fluid volume and a second fluid volume including an inlet and an outlet for connecting the first fluid volume and the second fluid volume to a geometrically constrained housing of the first fluid volume and a second fluid volume, the first fluid volume and the second fluid volume including an inlet and an outlet for connecting the first fluid volume and the second fluid volume to a geometrically constrained housing of the second fluid volume and the second fluid volume; (vi) at least one electrode disposed in the first fluid volume, at least one electrode disposed in the second fluid volume, and at least one electrode disposed in the chamber; (vi) at least one electrode disposed in the first fluid volume and the chamber; a) introducing a target polynucleotide into a chamber of the device, the target polynucleotide being introduced into the chamber; b) providing a voltage between the electrode within the chamber, the second electrode within the second fluid volume and the electrode within the chamber, and c) providing a current measurement to detect capture and partial or complete translocation of the polynucleotide into and through the first pore, and d) providing a current measurement to detect capture and partial or complete translocation of the polynucleotide into and through the second pore; and e) introducing a target polynucleotide into the chamber of the device. c) applying a first voltage at the first pore and a first voltage at the second pore to capture and transfer the target polynucleotide through the first pore to the first fluid volume; d) applying a second voltage at the first pore and a second voltage at the second pore to recapture and partially or completely transfer the target polynucleotide from the first fluid volume through the first pore; and e) applying a third voltage at the first pore and a third voltage at the second pore such that at least a portion of the target polynucleotide remains in the first pore and is captured by the second pore.f) applying a fourth voltage at the first pore and a fourth voltage at the second pore to control the direction of migration of the target polynucleotide; and g) detecting a first set of features of the polynucleotide at a first sensor current as each feature passes through the first pore and again at a second sensor current as each feature passes through the second pore.

[0093] In some embodiments, the method further comprises h) applying a fifth voltage at the first pore and a fifth voltage at the second pore to reverse the direction of movement of the target polynucleotide; and i) detecting a second set of features of the polynucleotide at a second sensor current as each feature passes through the second pore, and again at the first sensor current as each feature passes through the first pore.

[0094] In some embodiments, the first fluid volume, which is a geometrically constrained housing, is opposite the first pore, and in some embodiments, the second geometrically constrained fluid volume is opposite the second pore.

[0095] In some embodiments, the chamber is disposed over the first and second pores of the device.

[0096] In some embodiments, the third voltage at the second pore in step (e) is greater than the third voltage at the first pore.

[0097] In some embodiments, the third voltage at the first pore in step (e) is 0 mV.

[0098] In some embodiments, the target polynucleotide in step (d) is partially translocated from the first fluid volume through the first pore.

[0099] In some embodiments, the target polynucleotide in step (d) is completely translocated from the first fluid volume through the first fluidic pore.

[0100] In some embodiments, the method includes repeating steps (c) and (d) prior to step (e).

[0101] In some embodiments, the magnitude of the fourth voltage at the first pore is greater than the magnitude of the third voltage at the first pore to control the orientation of the target polynucleotide.

[0102] In some embodiments, the magnitude of the fourth voltage at the first pore is less than the magnitude of the fourth voltage at the second pore so that the target nucleotide continues to move toward the second pore, and the polarity of the fourth voltage at the first pore is such that the voltage pulls the target polynucleotide from the chamber toward the first fluid volume, and the polarity of the fourth voltage at the second pore is such that the voltage pulls the target polynucleotide from the chamber toward the second fluid volume.

[0103] In some embodiments, the magnitude of the fourth voltage at the first pore in step (f) is less than the magnitude of the fourth voltage at the second pore.

[0104] In some embodiments, the magnitude of the fifth voltage at the first pore in step (h) is greater than the magnitude of the fifth voltage at the second pore.

[0105] In some embodiments, the polarity of the first voltage at the first pore in step (c) is such that the voltage pulls the target polynucleotide out of the chamber towards the first fluid volume.

[0106] In some embodiments, the fluid volume has a positive polarity relative to the chamber during transfer of the target polynucleotide in step (c).

[0107] In some embodiments, the polarity of the second voltage at the first pore in step (d) is such that the voltage pulls the target polynucleotide from the first fluid volume towards the chamber.

[0108] In some embodiments, the fluid volume has a positive polarity relative to the first fluid volume during partial or complete translocation of the target polynucleotide in step (d).

[0109] In some embodiments, in step (c), the target polynucleotide is partially or completely translocated from the chamber through the first pore into the first fluid channel in a time ranging from 30 ms to 500 ms or more.

[0110] In some embodiments, in step (d), the target polynucleotide is partially or completely translocated in a first direction from the first fluidic channel through the first pore to the chamber in a time ranging from 30 ms to 500 ms or more.

[0111] In some embodiments, the method includes scanning one or more features of the polynucleotide prior to each detection step.

[0112] In some embodiments, the method further comprises detecting one or more features of the polynucleotide, each feature passing through the second pore in step g) and not yet detected when passing through the first pore.

[0113] In some embodiments, between steps c) and d), the method comprises scanning one or more features of the target polynucleotide and detecting a first set of features as the target polynucleotide passes through the first pore.

[0114] In some embodiments, steps c) through i) constitute a first cycle of feature detection in both directions.

[0115] In some embodiments, the method further comprises repeating steps c) through i) to detect a third and fourth set of features at the second aperture of detection of the features in both directions.

[0116] In some embodiments, a first voltage at the first pore is applied between the first fluid volume and the chamber, and a first voltage at the second pore is applied between the chamber and a second fluid volume of the device.

[0117] In some embodiments, the first fluid volume is a first fluid channel and the second fluid volume is a second fluid channel.

[0118] In some embodiments, the polynucleotide is partially or completely translocated from the first fluidic channel to the chamber through the first pore in a time ranging from 30 ms to 500 ms or more.

[0119] In some embodiments, a portion of the target polynucleotide is transferred from the first pore to the second pore in a time ranging from 30 ms to 500 ms or more.

[0120] In some embodiments, at least a portion of the target polynucleotide moves from the chamber through the second pore to the second fluid channel in a time ranging from 30 ms to 500 ms or more.

[0121] In some embodiments, the target polynucleotide translocates through the first pore, through the second pore, or through the first pore and the second pore.

[0122] In some embodiments, detecting the set of polynucleotide features is accomplished by an ionic current event from the first pore, the second pore, or both.

[0123] In some embodiments, the detecting step comprises detecting a first ionic current event when the one or more features pass through the first pore in a first direction in step (g) and a second ionic current event when the one or more features pass through the second pore in a first manner in step (g).

[0124] In some embodiments, the detecting step includes detecting a third ionic current event when the one or more features pass through the first pore in the second direction in step (h) and a fourth ionic current event when the one or more features pass through the second pore in the second direction in step (h).

[0125] In some embodiments, the first end of the polynucleotide is disposed in the first fluidic channel a distance away from the first pore ranging from 5 microns to 5 millimeters or more.

[0126] In some embodiments, the second end of the target polynucleotide is disposed in the first fluid channel at a distance ranging from 5 microns to 5 millimeters or more away from the first pore.

[0127] In some embodiments, the target polynucleotide moves through the second pore into the second fluid channel.

[0128] In some embodiments, at least a portion of the target polynucleotide moves from the second fluidic channel through the second pore and into the chamber in a period ranging from 5 ms to 500 ms.

[0129] In some embodiments, the first end of the target polynucleotide is disposed in the second fluid channel, away from the second pore, a distance ranging from 5 microns to 5 millimeters or more.

[0130] In some embodiments, the second end of the target polynucleotide is disposed in the second fluidic channel, away from the second pore, a distance ranging from 2 microns to 5 millimeters or more.

[0131] In some embodiments, the first voltage at the first pore is maintained for a period in the range of 0-1000 milliseconds, 0-20 milliseconds, 20-50 seconds, 50-100 seconds, 100-500 seconds, or 500-1000 seconds.

[0132] In some embodiments, the first voltage at the first pore is maintained for a period in the range of 20 ms or more, 50 ms or more, 120 ms or more, 150 ms or more, 300 ms or more, 500 ms or more, 1000 ms or more, 20 ms or more, 60 seconds or more, 120 seconds or more, 150 seconds or more, 300 seconds or more, 500 seconds or more, or 1000 seconds or more.

[0133] In some embodiments, the first voltage is maintained for a predetermined period of time after capturing and translocating the target polynucleotide through the first pore.

[0134] In some embodiments, the method further comprises adjusting the first voltage to an intermediate voltage of 0 mV for a period in the range of 0-1000 ms, 0-20 ms, 20-50 s, 50-100 s, 100-500 s, or 500-1000 s between steps c) and d).

[0135] In some embodiments, the method further comprises adjusting the first voltage to an intermediate voltage of 0 mV for a period ranging from 10 ms to 5 seconds or more between steps c) and d).

[0136] In some embodiments, the method further comprises adjusting the first voltage to an intermediate voltage of 0 mV for a period in the range of 10 ms to 5 seconds or more, 5 seconds to 50 seconds or more, 50 seconds to 60 seconds or more, 60 seconds to 120 seconds or more, 120 seconds to 180 seconds or more, 180 seconds to 240 seconds or more, or 240 seconds to 300 seconds or more, between steps c) and d).

[0137] In some embodiments, adjusting the first voltage to an intermediate voltage of 0 mV is maintained for a period of time sufficient to allow the polynucleotide to entropically relax to an equilibrium configuration.

[0138] In some embodiments, the polynucleotide is within the geometrically constrained enclosure of the first fluid volume or the second fluid volume for a period ranging from 10 ms to 5 seconds or more, from 5 seconds to 50 seconds or more, from 50 seconds to 60 seconds or more, from 60 seconds to 120 seconds or more, from 120 seconds to 180 seconds or more, from 180 seconds to 240 seconds or more, or from 240 seconds to 300 seconds or more.

[0139] In some embodiments, the polynucleotides are within the geometrically constrained enclosure of the second fluid volume for a period ranging from 10 ms to 5 seconds or more, from 5 seconds to 50 seconds or more, from 50 seconds to 60 seconds or more, from 60 seconds to 120 seconds or more, from 120 seconds to 180 seconds or more, from 180 seconds to 240 seconds or more, or from 240 seconds to 300 seconds or more.

[0140] In some embodiments, the first voltage, the second voltage, the third voltage, and / or the fourth voltage at the first pore are each independently in a range of magnitude from 0 mV to 900 mV, and the first voltage, the second voltage, the third voltage, and / or the fourth voltage at the second pore are each independently in a range of magnitude from 0 mV to 900 mV.

[0141] In some embodiments, the third voltage at the first pore in step (e) is 0 mV.

[0142] In some embodiments, the third voltage at the second pore in step (e) ranges from 25 mV to 600 mV.

[0143] In some embodiments, the method further comprises calculating, using a processor, a velocity of one or more features of the polynucleotide from the time difference between detection of the one or more features at the first pore and the second pore and the known distance between the first pore and the second pore.

[0144] In some embodiments, the method further comprises calculating, with a processor, a distance between one or more features by using the calculated velocity of the polynucleotide features from the time between one or more features detected in the sensor current from the first pore, the sensor current from the second pore, or both.

[0145] In some embodiments the polynucleotide exhibits a minimum duration between said detecting step (g) and detecting step (i) indicating that the polynucleotide exceeds a minimum length, at which time the polynucleotide is captured and partially or completely translocated through said first pore and / or through the second pore.

[0146] In some embodiments, a polynucleotide feature is identified as a target when the minimum duration is greater than a threshold, and a feature is identified as a non-target when the duration is less than the threshold.

[0147] In some embodiments, the method further comprises calculating, by the processor, the velocity of one or more features of the target polynucleotide for each scan.

[0148] In some embodiments, the method further comprises calculating, by the processor, velocity statistics for one or more features using the velocity distribution for each scan.

[0149] In some embodiments, the method further comprises calculating, by the processor, a time history of the velocity of the target polynucleotide using the velocities of all features of a given scan and a given scanning direction.

[0150] In some embodiments, the target polynucleotide is substantially linearized.

[0151] In some embodiments, the first fluid channel and / or the second fluid channel have a length in the range of about 0.01 mm to about 5 mm.

[0152] In some embodiments, the first fluid channel and / or the second fluid channel have a depth in the range of about 0.05 μm to about 2 μm.

[0153] In some embodiments, the first fluid channel and / or the second fluid channel have a width in the range of 50-500 μm.

[0154] In some embodiments, the target polynucleotide is substantially linearized by the effect of an adjustment to the first voltage, or the second voltage, or both.

[0155] In some embodiments, the first voltage creates a voltage gradient across at least the first pore and along the length of the first fluid channel.

[0156] In some embodiments, the second voltage creates a voltage gradient across at least the second pore and along the length of the second fluid channel.

[0157] In some embodiments, the resistance of the first fluid channel is inversely proportional to the width of the first fluid channel.

[0158] In some embodiments, the resistance of the second fluid channel is inversely proportional to the width of the second fluid channel.

[0159] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel.

[0160] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel.

[0161] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of the first fluid channel and / or the second fluid channel.

[0162] In some embodiments, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of a cross-section of the first fluid channel and / or the second fluid channel.

[0163] In some embodiments, the target polynucleotide is substantially linearized by the effect of adjusting the first voltage, the second voltage, the third voltage, and / or the fourth voltage in the first pore, the second pore, or both.

[0164] In some embodiments, the first fluid channel and / or the second fluid channel comprise a geometrically constrained volume.

[0165] In some embodiments, the controller determines whether one or more features of the target polynucleotide are captured and performs additional recapture of the one or more features in the first orientation and / or the second orientation.

[0166] In some embodiments, the method includes the step of decoupling one or more features of the already recaptured polynucleotide.

[0167] In some embodiments, the method further comprises controlling with a controller: a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, or h) a combination thereof.

[0168] In some embodiments, the method further comprises controlling the number of features scanned.

[0169] In some embodiments, the controller determines which of the one or more features to perform additional scans of.

[0170] In some embodiments, the method further comprises moving away from one or more features already scanned.

[0171] In some embodiments, the method further comprises scanning an area of ​​the polynucleotide that has not already been scanned.

[0172] In some embodiments, the method further comprises constructing, by the processor, a consensus map for each polynucleotide.

[0173] In some embodiments, the constructing step includes a machine learning algorithm trained to detect one or more features based on training data and probabilistic models.

[0174] In some embodiments, the method further comprises constructing, by the processor, a local map for each polynucleotide in real time.

[0175] In some embodiments, the method further comprises repeating steps c) through i) until the target polynucleotide exits the pore device.

[0176] In some embodiments, the method prevents the target polynucleotide from exiting the chamber of the device for a period ranging from 5 ms to 5 minutes.

[0177] In some embodiments, the method prevents the target polynucleotide from exiting the first fluid channel or the second fluid channel of the device for a period ranging from 5 ms to 5 minutes.

[0178] In some embodiments, the one or more features include a) one or more payload molecules bound to the polynucleotide, b) one or more payload molecules hybridized to the polynucleotide, c) one or more payload molecules incorporated into the genome of the polynucleotide, d) a molecular motif in the polynucleotide sequence of the target polynucleotide, or e) a combination thereof.

[0179] In some embodiments, the method further comprises determining the distance between one or more features of the target polynucleotide.

[0180] In some embodiments, the method further comprises determining the distance between each feature of the first set of features of the target polynucleotide.

[0181] In some embodiments, the method further comprises determining the distance between each feature of the second set of features of the target polynucleotide.

[0182] In some embodiments, the first cycle includes one or more scans performed by the processor to detect a first set of features.

[0183] In some embodiments, the first cycle comprises 2 or more scans, 3 or more scans, 4 or more scans, 5 or more scans, 6 or more scans, 7 or more scans, 8 or more scans, 9 or more scans, or 10 or more scans.

[0184] In some embodiments, the second cycle includes one or more scans performed by the processor to detect a third set of features.

[0185] In some embodiments, the second cycle comprises 2 or more scans, 3 or more scans, 4 or more scans, 5 or more scans, 6 or more scans, 7 or more scans, 8 or more scans, 9 or more scans, or 10 or more scans.

[0186] In some embodiments, the method further comprises repeating steps c) through i) for a third cycle, a fourth cycle, and a fifth cycle, or when the polynucleotides exit the device.

[0187] In some embodiments the first set of features is 1 feature or more, 2 features or more, 3 features or more, 4 features or more, 5 features or more, 6 features or more, 7 features or more, 8 features or more, 9 features or more, or 10 features or more.

[0188] In some embodiments, the second set of characteristics is greater than the first set of characteristics.

[0189] In some embodiments, the sets of features across a set of scans are combined to generate, for each target polynucleotide, a map of positions and distances between features.

[0190] In some embodiments, the one or more features comprise a DNA binding protein, a polypeptide, an anti-DNA antibody, streptavidin, a transcription factor, a histone, a peptide nucleic acid (PNA), a DNA-hairpin, a DNA molecule, an aptamer, a 5-methylcytosine (5mC) region, a 5-hydroxymethylcytosine (5hmC) region, a nucleotide base, two or more nucleotide bases, or a combination thereof.

[0191] In some embodiments, the polynucleotide sequence has a length ranging from 5 base pairs to about 3,000,000 base pairs.

[0192] In some embodiments, the target polynucleotide is selected from the group consisting of double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and a DNA-RNA hybrid.

[0193] In some embodiments, the method further comprises controlling a direction of the target polynucleotide through the first and second pores by a controller, a processor, and a non-transitory computer readable medium comprising instructions to the processor to change a direction of the target polynucleotide when the first set of features is detected.

[0194] In some embodiments, adjusting the first voltage and the second voltage occurs in real time, and the adjusting is performed by an active feedback controller using hardware and software.

[0195] In some embodiments, the method further includes controlling the first or second voltages with a feedback controller based on feedback of the first or second, or both, ionic current measurements.

[0196] In some embodiments, the processor comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0197] In some embodiments, the controller comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0198] In some embodiments, the controller is a microcontroller.

[0199] In some embodiments, the FPGA or ASIC executes control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0200] In some embodiments, the first set of features and the second set of features are the same.

[0201] In some embodiments, the first set of features and the second set of features are different from each other.

[0202] In some embodiments, the controller is configured to perform a control voltage frequency sweep of the polynucleotide in a first direction, a second direction, or both.

[0203] In some embodiments, the controller is configured to perform a control voltage amplitude sweep of the polynucleotide in a first direction, a second direction, or both.

[0204] In some embodiments, the controller is configured to regulate the rate of the polynucleotide.

[0205] In some embodiments, the rate ranges from 0.1 base pairs / millisecond to 10 base pairs / millisecond.

[0206] In some embodiments, the controller is configured to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple rates.

[0207] In some embodiments, performing multiple scans of the polynucleotide at multiple speeds improves the accuracy of detection of one or more features.

[0208] In some embodiments, the method includes performing multiple scans of the polynucleotide at multiple speeds.

[0209] In some embodiments, the controller is configured to control the range of velocities of the polynucleotide in a first direction, a second direction, or both.

[0210] In some embodiments, the controller is configured to control the voltage ranges of the first and second pores as the polynucleotide translocates through the first and second pores in the first direction, the second direction, or both.

[0211] In some embodiments, the controller is configured to determine an optimal velocity range for the polynucleotide in the first direction, the second direction, or both, wherein the optimal velocity range for the polynucleotide reduces the effects of Brownian motion of the polynucleotide.

[0212] In some embodiments, controlling the velocity range of the polynucleotide comprises determining an optimal velocity of the polynucleotide for sequencing. [Brief description of the drawings]

[0213] [Figure 1] FIG. 1 depicts DNA-based tagging of λ-DNA molecules. The depicted tag is in the same position as mono-streptavidin. The DNA is nicked using the same nicking enzyme. Instead of incorporating biotin-labeled dUTP at the nicking position, N3-dUTP is incorporated. Excess dUTP is removed by filtration. A 90-nucleotide oligo with a 5'DCBO group is reacted with the DNA overnight (e.g., using a copperless click reaction). The 5'DBCO nucleotide contains the nucleotide sequence AAA AAA AAA AGG GAA AGG GAA AGG GAA AGA AAA AAA AAA AAA AAA AGG GAA AGG GAA AAA AAA AAG AGA GAG AGA GAG AGA GAA GAG (SEQ ID NO:1). [Diagram 2] FIG. 2 shows the modulation of nanopore impedance by changing the size and shape of DNA attachment. [Figure 3A] FIG. 3A shows an example of the development of ionic current over time as a detection parameter for a DNA molecule in a single hole and confirmation of tagging the DNA molecule with one or more probes. [Figure 3B] FIG. 3B shows an example of the evolution of ionic current over time as a detection parameter for a DNA molecule in a single hole and confirmation of tagging the DNA molecule with one or more probes. [Figure 3C] FIG. 3C shows an example of the evolution of ionic current over time as a detection parameter for a DNA molecule in a single hole and confirmation of tagging the DNA molecule with one or more probes. [Figure 3D]FIG. 3D shows an example of the evolution of ionic current over time as a detection parameter for a DNA molecule in a single hole and confirmation of tagging the DNA molecule with one or more probes. [Figure 4] FIG. 4 is a diagram showing an example of reading the area of ​​a tagged molecule and then panning / gliding the molecule to different areas for detection. The algorithm involved in mapping one or more features of a molecule in this non-limiting example includes: (a) Initialize scan count=0; (b) The dual system detects Ntag (Ntag=2 in this example) tag first and starts flossing; (c) Increment scan count each time and start a new scan; (d) After a certain number of flossing scans Nscan (Nscan=4 in this example), the system scans Ntag+1 tag for the next scan; (e) The system holds flossing based on Ntag and the scan count restarts from 0; (f) Repeat steps (b) to (e) until the molecule exits. [Diagram 5] FIG. 5 is a flow chart of the processor steps, highlighting steps 8 and 15-19 related to rescanning and gliding; the states shown in bold text are key states that are highly relevant to rescanning and gliding. [Figure 6]6 is a diagram showing an example of a basic zoom showing bidirectional scanning and detection of multiple tags (e.g., multiple probes) of a biomolecule (DNA). The algorithm involved in mapping one or more features of a molecule in this non-limiting example includes: (a) initialize scan count=0, (b) the dual system first detects Ntag (Ntag=2 in this example), (c) increment scan count and start a new scan each time, (d) after a certain number of flossing scans Nscan (Nscan=4 in this example), the system increments the number of triggering tags to Ntag=Ntag+1, reset scan count to 0, (e) the system continues flossing based on Ntag, and (f) repeat steps (b) to (e) until the molecule exits. [Figure 7] FIG. 7 illustrates a non-limiting example of a terminated zoom with the following algorithm: (a) Initialize scan count=0; (b) The dual system first detects Ntag (Ntag=2 in this example) tag and starts flossing; (c) Increment scan count and start a new scan each time; (d) After a certain number of flossing scans Nscan (Nscan=4 in this example), the system increments the number of triggering tags to Ntag=Ntag+1; Reset scan count to 0; (e) The system continues flossing based on the new Ntag; (f) Repeat steps (b) to (e) until Ntag reaches Nmax (Nmax=4 in this example); (g) The system continues flossing based on Nmax; (h) Stop incrementing scan count and keep flossing based on Nmax until molecules leave. [Figure 8A]FIG. 8 shows a 3D schematic of a dual pore device. Two nanopores are placed on the same membrane. Two "V" shaped microchannels are fastened onto the glass and the entire surface is covered with a SiN membrane. The two channels lead the buffer to the center, where there is a micrometer bridge separating the two channels. A nanopore is drilled at the end of the channel. The same DNA molecule can span the two nanopores to achieve control of the two pores. C, D, E, are top views of the device at different magnifications. C shows the whole chip with a footprint size of 8 mm x 8 mm. D is a zoom of a field of view showing the elbow of the v-channel. E is a focus ion beam image showing the two nanopores. F is a side view showing the materials: glass substrate and SiN membrane. For clarity, the left side is shown as channel 1 and pore 1, and the right side is shown as channel 2 and pore 2. G is the placement of electrodes to simultaneously draw the same molecule into the channel. This design facilitates separate access to the two pores, as the electrodes are placed at the corners, within the access ports, and a common ground is placed in the center. [Figure 8B]Figure 8 shows a 3D schematic of a dual pore device. Two nanopores are placed on the same membrane. Two "V" shaped microchannels are fastened onto the glass and the entire surface is covered with a SiN membrane. The two channels lead to a buffer in the center, where there is a micrometer bridge separating the two channels. A nanopore is drilled at the end of the channel. The same DNA molecule can span the two nanopores to achieve control of the two pores. C, D, E, are top views of the device at different magnifications. C shows the whole chip with a footprint size of 8 mm x 8 mm. D is a zoom of a field of view showing the elbow of the v-channel. E is a focus ion beam image showing the two nanopores. F is a side view showing the materials: glass substrate and SiN membrane. For clarity, the left side is shown as channel 1 and pore 1, and the right side is shown as channel 2 and pore 2. G is the placement of electrodes to simultaneously draw the same molecule into the channel. This design facilitates separate access to the two pores, as the electrodes are placed at the corners, within the access ports, and a common ground is placed in the center. [Figure 9]Figure 9 shows DNA translocation due to the force of competing voltages within a dual pore device. (a) After DNA is co-captured, the DNA molecule threads from left to right (L to R) using voltages V1 < V2, where V1 and V2 are the voltages across pore 1 (left) and pore 2 (right), respectively. A single pass of DNA movement during this fixed polarity is termed a "scan". After automatic detection of a predefined number of tags, the direction of DNA movement is reversed by voltage V1 > V2, inducing movement of the molecule from right to left (R to L) and resulting in a second scan. This process is repeated in a cyclic fashion until the molecules exit the randomly co-captured state. (b) Raw data from multiple scanning events showing two detected mono-streptavidin (MS) tags for each scan movement is shown, up to DNA efflux after 50+ scans from pore 1→2 and pore 2→1. Here, the current trace I2 of multiple scans from pore 2 was recorded using logic where the predefined number of tags detected is 2, after which the controller induces a change in direction. Signals from 30 - 150 ms are omitted for visualization. [Figure 10A]Figure 10 shows representative dual current signals and scan count statistics generated during a flossing experiment with MS-tagged DNA. a Full signal trace for I1, V1, I2 and V2 is shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in the vertical scale at low and high ranges between low and high V1 values. B Zooming in on the first cycle, where the two-tag logic shows resolvable tags A and B in both signals. C Zooming in on the 41st and final cycle, showing the end of co-capture with no detected tag. d Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts over all co-capture events for the device used (bin width = 4). The red line for the probability data is the fitted model equation (1) with p = 0.89 for the probability of correctly detecting two tags for each scan. The chip used has a pore-to-pore distance of 0.61 μm, a pore 1 diameter of 27 nm, and a pore 2 diameter of 25 nm. [Figure 10B]Figure 10 shows representative dual current signals and scan count statistics generated during a flossing experiment with MS-tagged DNA. a Full signal trace for I1, V1, I2 and V2 is shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in the vertical scale at low and high ranges between low and high V1 values. B Zooming in on the first cycle, where the two-tag logic shows resolvable tags A and B in both signals. C Zooming in on the 41st and final cycle, showing the end of co-capture with no detected tag. d Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts over all co-capture events for the device used (bin width = 4). The red line for the probability data is the fitted model equation (1) with p = 0.89 for the probability of correctly detecting two tags for each scan. The chip used has a pore-to-pore distance of 0.61 μm, a pore 1 diameter of 27 nm, and a pore 2 diameter of 25 nm. [Figure 10C]Figure 10 shows representative dual current signals and scan count statistics generated during a flossing experiment with MS-tagged DNA. a Full signal trace for I1, V1, I2 and V2 is shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in the vertical scale at low and high ranges between low and high V1 values. B Zooming in on the first cycle, where the two-tag logic shows resolvable tags A and B in both signals. C Zooming in on the 41st and final cycle, showing the end of co-capture with no detected tag. d Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts over all co-capture events for the device used (bin width = 4). The red line for the probability data is the fitted model equation (1) with p = 0.89 for the probability of correctly detecting two tags for each scan. The chip used has a pore-to-pore distance of 0.61 μm, a pore 1 diameter of 27 nm, and a pore 2 diameter of 25 nm. [Figure 11A] FIG. 11 shows that flossing increases the linearization of DNA in the dual pore device. (a) Typical I1 trace of a single pore event containing both unfolded and folded examples. Only single pore events that occurred in co-capture were included in the next probability calculation (pre-1 step events in FIG. 16). (b) Typical I2 trace of a multiple scan event, where scan 1 shows folding and subsequent scans show unfolding. (c) Illustration of the mechanism by which the folded part (only early in I2) is removed by the 2nd scan when the molecule moves from R to L, as indicated by the letters. (d) Probability P (±95% error bars) is the fraction of unfolded events for the different transition types. In total 309 events were performed in all four types in sequence. [Figure 11B]FIG. 11 shows that flossing increases the linearization of DNA in the dual pore device. (a) Typical I1 trace of a single pore event containing both unfolded and folded examples. Only single pore events that occurred in co-capture were included in the next probability calculation (pre-1 step events in FIG. 16). (b) Typical I2 trace of a multiple scan event, where scan 1 shows folding and subsequent scans show unfolding. (c) Illustration of the mechanism by which the folded part (only early in I2) is removed by the 2nd scan when the molecule moves from R to L, as indicated by the letters. (d) Probability P (±95% error bars) is the fraction of unfolded events for the different transition types. In total, 309 events were performed in all four types in sequence. [Figure 12]FIG. 12 shows the estimation of the separation distance between tags from the dual current signals generated during a multiple scan experiment. (a) L to R and (b) R to L illustrations help visualize the relative tag positions indicated by the scan signals. (c) L to R and d R to L signals were from side-by-side scans of co-captured molecules scanned for 48 cycles. In L to R, pore 1 is the entry pore for the tag, while pore 2 is the exit pore. In R to L, pore 2 is the entry pore for the tag, while pore 1 is the exit pore. Thus, entry and exit relate to the direction of tag migration as the tag passes from pore to pore. The signal between pores in the common chamber and the estimated number of tags versus time are plotted. In the example (ai) A and B are visualized while in the common chamber, whereas (aii) B is pore-out but before C enters the common chamber, etc. The velocity plots show the calculated velocities at the inlet and outlet pores based on dividing the membrane thickness by the tag duration, and the pore-to-pore velocity of the tags was calculated by dividing the known distance between the pores by the pore-to-pore time. The prediction of the spacing distance (separation distance) between tags was calculated by multiplying the average pore-to-pore velocity within the scan by the time between pairs of detected tags, adding the membrane thickness as a correction (main text). The voltages were set at V1=250mV from L to R and 600mV from R to L, while V2 was held constant at 400mV. The FPGA monitored I2 for N=2 tags (exit signal L to R, inlet signal R to L), and 3 tags were visible at I1 in both directions. [Figure 13]Figure 13 shows Table 1 for five different multiple scan events with at least 30 cycles. The table lists the number of cycles equivalent to the number of scans in each direction, and the number of tag-pairs contributing to the evaluation of each separation distance. [Figure 14] Figure 14 shows that DNA methylation can be tagged with protein vs. antibody motifs and differentially detected. Concordance of multiple reads means that tag cell specificity is highly reliable. [Figure 15] Figure 15 shows restriction enzyme analysis of methylated λ-DNA for 0, 30 and 60 min incubation with HpaII (lanes 1-3) and Msp1 (lanes 4-6). (b) Dual pore rescanning data using 100 s scans for MeCP2 and antibody bound to the 5mC site of lambda DNA. (c) Comparison of tag signals for MS protein (three tags 301 bp, 323 bp apart) bound to biotin

[19] versus MeCP2 and antibody bound to 5mC. Proteins of size ~50 kDa give similar blockade, while antibodies of 150 kDa give deeper blockade, which can be avenues for multiplexing. [Figure 16] FIG. 16 shows the whole process of a tug-of-war event. (a) Each step is represented. (b) Current traces from both pore 1 and pore 2. I1 is in red while I2 is in blue. The y-axis of pore 1 is notched three times to fit different values ​​in one figure. The inset shows a zoom-in when the trigger occurred. (c) Average duration at different V1 including the duration of a single pore event. (d) Pair of current traces of one tug-of-war event with λDNA at V1=200 mV. [Figure 17A]Figure 17 shows the FPGA logic of the multiple scan experiment. (a) I2 and FPGA state signals from the end of the event shown in Figure 10. The red and black dotted lines indicate the trigger thresholds for tags and events, respectively. (b) Flowchart of the FPGA logic. The blue boxes indicate the states of the system and the magenta numbers represent the FPGA states in (a). The black boxes indicate decisions. The green boxes indicate actions. The arrows between the boxes show how the system flows between the different steps. [Figure 17B] Figure 17 shows the FPGA logic of the multiple scan experiment. (a) I2 and FPGA state signals from the end of the event shown in Figure 10. The red and black dotted lines indicate the trigger thresholds for tags and events, respectively. (b) Flowchart of the FPGA logic. The blue boxes indicate the states of the system and the magenta numbers represent the FPGA states in (a). The black boxes indicate decisions. The green boxes indicate actions. The arrows between the boxes show how the system flows between the different steps. [Figure 18A] Figure 18 shows four main cases where the FPGA cannot capture multiple scans. Plots are the State signals from the FPGA and I2 of the last cycle. The definitions of the states are in Figure 17b. (a) The tag is shown going up to the hold (18) state of the nth scan. The lines with arrow marks are the (n-1)th and nth scans. (b) A false positive spike at the (n-1)th scan. (c) A false negative spike at the nth scan. (d) A molecule exits pore 2 in the wait (17) state of the nth scan. [Figure 18B]Figure 18 shows four main cases where the FPGA cannot capture multiple scans. Plots are the State signals from the FPGA and I2 of the last cycle. The definitions of the states are in Figure 17b. (a) The tag is shown going up to the hold (18) state of the nth scan. The lines with arrow marks are the (n-1)th and nth scans. (b) A false positive spike at the (n-1)th scan. (c) A false negative spike at the nth scan. (d) A molecule exits pore 2 in the wait (17) state of the nth scan. [Figure 18C] Figure 18 shows four main cases where the FPGA cannot capture multiple scans. Plots are the State signals from the FPGA and I2 of the last cycle. The definitions of the states are in Figure 17b. (a) The tag is shown going up to the hold (18) state of the nth scan. The lines with arrow marks are the (n-1)th and nth scans. (b) A false positive spike at the (n-1)th scan. (c) A false negative spike at the nth scan. (d) A molecule exits pore 2 in the wait (17) state of the nth scan. [Figure 18D] Figure 18 shows four main cases where the FPGA cannot capture multiple scans. Plots are the State signals from the FPGA and I2 of the last cycle. The definitions of the states are in Figure 17b. (a) The tag is shown going up to the hold (18) state of the nth scan. The lines with arrow marks are the (n-1)th and nth scans. (b) A false positive spike at the (n-1)th scan. (c) A false negative spike at the nth scan. (d) A molecule exits pore 2 in the wait (17) state of the nth scan. [Figure 19A]Figure 19 shows a multiple scan experiment with three tag triggers. (a) Full signal traces of I1, V1, I2 and V2. V2 is set to V2=400 mV during the event, V1=200 mV for the L to R scan and V1=800 mV for the R to L scan. V1 is set. (b) Zoomed-in plot of the 2nd cycle. (c) Distribution of scan counts per event with theoretical fitting. [Figure 19B] Figure 19 shows a multiple scan experiment with three tag triggers. (a) Full signal traces of I1, V1, I2 and V2. V2 is set to V2=400 mV during the event, V1=200 mV for the L to R scan and V1=800 mV for the R to L scan. V1 is set. (b) Zoomed-in plot of the 2nd cycle. (c) Distribution of scan counts per event with theoretical fitting. [Figure 19C] Figure 19 shows a multiple scan experiment with three tag triggers. (a) Full signal traces of I1, V1, I2 and V2. V2 is set to V2=400 mV during the event, V1=200 mV for the L to R scan and V1=800 mV for the R to L scan. V1 is set. (b) Zoomed-in plot of the 2nd cycle. (c) Distribution of scan counts per event with theoretical fitting. [Figure 20]FIG. 20 illustrates the tag alignment procedure. (a) Example tag position vs. scan number for an event with two tags (blue circle, tag measured closest to the start of the scan in each scan). (b) Inter-tag spacing as a function of scan number for the event in (a). (c) Aligned tag positions for the event in (a). Distances were calibrated and aligned tag positions for the event in (a) by the final average tag position (where blue circle corresponds to measurements associated with tag A and red square corresponds to measurements associated with tag B). (e) Example tag position vs. scan number for an event with three tags (blue circle, tag position closest to the start of the event, red square at intermediate distance, magenta triangle tag observed further away from the start of the event). (f) Inter-tag distance for the event in (e). [Figure 21] FIG. 21 shows Table S3 showing statistics associated with the evaluation of tag separation from nine multiple scan events. [Figure 22] FIG. 22 illustrates a non-limiting example of a geometric configuration of a nanopore device of the present disclosure. [Diagram 23] FIG. 23 shows a graph plotting the relationship between channel resistance and channel width for devices with different geometric structures. [Figure 24] FIG. 24 shows a graph plotting the relationship between channel volume and channel width for devices with different geometric structures. [Diagram 25] FIG. 25 shows a graph plotting the relationship between polynucleotide delivery time and channel width. [Figure 26] 26 shows a non-limiting example of the wait periods during "pre-capture" shown in FIGS. 20A-20C, where the target polynucleotide in "pre-i" includes a wait period of about 30 ms, which pushes the target polynucleotide further into the first fluidic channel in a first direction, and in a second wait period "pre-ii", the voltage is adjusted to 0 mV "OFF" for about 20 ms before the target polynucleotide changes direction. The total period was about 50 ms. [Figure 27] FIG. 27 shows a non-limiting example of the distribution of target polynucleotides after channel-mediated and non-channel-mediated translocation. [Figure 28] FIG. 28 shows a non-limiting example where a target polynucleotide is forced through a first pore for about 30 ms and then the first voltage is adjusted to 0 mV "OFF" when the device contains a channel and when the device does not contain a channel. [Figure 29] FIG. 29 shows non-limiting examples of polynucleotide detection / capture times when the device includes a channel and when the device does not include a channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0214] The figures depict embodiments of the present invention for illustrative purposes only, and those skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be made without departing from the essence of the invention described herein.

[0215] definition The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases or other naturally occurring nucleotide bases, chemically or biochemically modified nucleotide bases, non-naturally occurring nucleotide bases, or derivatized nucleotide bases.

[0216] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), especially Chapter 11 and Table 11.1 (Non-Patent Document 1); and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) (Non-Patent Document 2). The conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0217] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0218] "Cleavage" refers to the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[0219] "Nuclease" and "endonuclease" are used interchangeably herein and refer to an enzyme that has catalytic activity for cleaving nucleic acids (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.). A "genome editing endonuclease" is an endonuclease that is used to edit the genome of a cell (e.g., by cleaving at a target location within the genomic DNA of the cell). Examples of genome editing endonucleases include, but are not limited to, class 2 CRISPR / Cas endonucleases, such as (a) type II CRISPR / Cas proteins, e.g., Cas9 protein, (b) type V CRISPR / Cas proteins, e.g., Cpf1 protein, C2c1 protein, C2c3 protein, etc., and (c) type V CRISPR / Cas proteins, e.g., C2c2 protein.

[0220] A "cleavage domain" or "activity domain" or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within a nuclease that has catalytic activity for cleaving nucleic acids. A cleavage domain may be contained within a single polynucleotide strand or the cleavage activity may result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.

[0221] In some instances, a component (e.g., a nucleic acid component, a protein component, etc.) includes a label moiety. As used herein, the terms "label," "detectable label," or "label moiety" refer to any moiety that provides signal detection and can vary widely depending on the particular nature of the assay. Label moieties of interest include both detectable labels (direct labels) (e.g., fluorescent labels) and indirectly detectable labels (indirect labels) (e.g., binding pair members). The fluorescent label can be any fluorescent label (e.g., a fluorescent dye (e.g., fluorescein, Texas Red, rhodamine, ALEXAFLUOR®, etc.), a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any fluorescent derivatives thereof), etc. Suitable detectable (direct or indirect) label moieties can include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, a suitable indirect label includes biotin (a member of a binding pair), which is bound by streptavidin (which itself can be directly or indirectly labeled). Labels also include radiolabels (direct labels) (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (indirect labels) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.), fluorescent proteins (direct labels) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any conventional derivatives thereof), metal labels (direct labels), colorimetric labels, binding pair members, and the like. By "binding pair partner" or "binding pair member" is meant one first and second moiety that has specific binding affinity for one another. Suitable binding pairs include, but are not limited to, antigen / antibody (e.g., digoxigenin / anti-digoxigenin, dinitrophenyl (DNP) / anti-DNF, dansyl-X-anti-dansyl, fluorescein / anti-fluorescein, lucifer yellow / anti-lucifer yellow, and rhodamine anti-rhodamine), biotin / avidin, (or biotin / streptavidin), and calmodulin binding protein (CBP) / calmodulin. Any binding pair member may be suitable for use as an indirectly detectable label moiety.

[0222] Any given component or combination of components may be unlabeled or detectably labeled with a labeling moiety. In some cases, when more than one component is labeled, they may be labeled with labeling moieties that are distinguishable from the others.

[0223] Common methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996) (Non-patent document 5), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999) (Non-patent document 6), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995) (Non-patent document 7); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997) (Non-Patent Document 8), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998) (Non-Patent Document 9), etc., the disclosures of which are incorporated herein by reference.

[0224] Before the present invention is further described, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention shall be limited only by the appended claims.

[0225] Where a range of numerical values ​​is provided, it is understood that intervening values ​​between the upper and lower limits of that range, and any other stated or intervening values ​​within that stated range, are included within the scope of the invention to one tenth of the unit of the lower limit, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included within the scope of the invention, subject to any specifically excluded limits in the stated ranges. Where a stated range includes one or both of the limits, ranges excluding either or both of the ranges included in the limits are also included in the invention.

[0226] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are described. All publications mentioned herein are incorporated by reference herein as if they disclose and describe the methods and / or materials to which they are cited.

[0227] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, "a ribonuclioprotein complex" includes a plurality of such complexes, and a reference to "a mutant dystrophin gene" includes mutant dystrophin genes and equivalents thereof known to those skilled in the art, and so forth. It should be noted that the claims may be drafted to exclude any optional element. In such cases, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements and for use in a "negative" limitation.

[0228] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination was individually and explicitly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0229] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which must be independently confirmed.

[0230] Detailed Description The present disclosure provides an automated method for mapping one or more features of a target polynucleotide. The present disclosure also provides an automated method for sequencing a polynucleotide sequence. The present disclosure also provides a method for differential detection of methylated and unmethylated regions in a polynucleotide sequence. The present disclosure also provides a method for extended recapture of a polynucleotide in a pore device. A method for sequencing a polynucleotide in a nanopore device is also provided. Devices and systems for carrying out the methods of the present disclosure are also provided in the present disclosure.

[0231] Systems and Devices The present disclosure includes devices and systems for carrying out the methods disclosed herein.The present disclosure provides devices and systems for mapping one or more features of the polynucleotide sequence of the target polynucleotide passing through the first and second pores.The present disclosure also provides devices and systems for capturing the target polynucleotide.The present disclosure also provides devices and systems for sequencing the polynucleotide sequence of the target polynucleotide passing through the first and second pores.

[0232] Aspects of the present disclosure include a dual pore, dual amplification device for sequencing a polynucleotide sequence of a target polynucleotide through a first and a second pore. In some cases, the device includes: (i) electrodes configured and connected to provide a first voltage at a first pore of the device and a second voltage at a second pore of the device; (ii) a first pore; (iii) a second pore, where the first and second pores are configured to allow the target polynucleotide to translocate simultaneously across both pores in a first direction or a second direction, and in a controlled manner; (iv) one or more sensors that enable identification of a first set of features from the target polynucleotide during translocation of the target polynucleotide through the first pore and the second pore in a first direction in a first cycle, and a second set of features from the target polynucleotide during translocation of the target polynucleotide through the second pore and the first pore in a second direction in a first cycle; (v) a processor; and (v i) a non-transitory computer readable medium comprising instructions to cause a processor to: a) determine from one or more sensors the simultaneous presence of a target polynucleotide in both pores; b) scan one or more features of the target polynucleotide; c) count a first set of features in a first cycle in a first direction and adjust one or both of the first and second voltages in response to the counts to produce a first force and an oppositely oriented second force acting on the target polynucleotide, wherein the first and second forces change the direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second direction; and d) repeat steps a) through c) and detect a third and fourth set of features in a second cycle.

[0233] In some embodiments, the device includes a first chamber. As used herein, the term "upper chamber" is used interchangeably with the term "fluidic channel", e.g., a first fluidic channel. In some embodiments, the device includes a middle chamber. As used herein, the term "middle chamber" is used interchangeably with the term "chamber". In some embodiments, the device includes a first pore connecting the upper chamber and the middle chamber. In some embodiments, the device includes a second pore connecting the middle chamber and the bottom chamber. As used herein, the term "lower chamber" is used interchangeably with the term "fluidic channel", e.g., a second fluidic channel. In some embodiments, the device includes a bottom chamber. In some embodiments, the device includes a second fluidic channel. In some embodiments, the first fluidic channel, the second fluidic channel, and / or the chamber contain one or more electrodes for connection to a power source such that separate voltages can be established across each of the pores between the chambers. In some embodiments, the device includes electrodes connected to a power source to provide a first voltage between a first fluidic channel and the chamber of the device, and to provide a second voltage between the chamber and a second fluidic channel of the device. In some embodiments, the chamber is disposed over the first and second pores. In some embodiments, the chamber is disposed over the first and second fluidic channels. In some embodiments, the chamber is disposed below the first and second pores. In some embodiments, the chamber is disposed between the first and second pores. In some embodiments, the chamber is disposed between the first and second fluidic channels.

[0234] In some embodiments, the first pore and the second pore are configured to allow the target polynucleotide to migrate simultaneously in a controlled manner across both pores in a first direction or a second direction. In some embodiments, the dual pore device includes one or more sensors. In some embodiments, the one or more sensors can identify a first set of features from the target polynucleotide. In some embodiments, the sensor can identify a first set of features in a first cycle (e.g., a first cycle having one or more scans) during migration of the target polynucleotide through the first pore and the second pore in a first direction (the first direction is from the first pore to the second pore). In some embodiments, the one or more sensors can identify a first set of features from the target polynucleotide. In some embodiments, the one or more sensors can identify a second set of features from the target polynucleotide in a first cycle during migration of the target polynucleotide through the second pore and the first pore in a second direction. In some embodiments, the first direction is from the first pore to the second pore. In some embodiments, the second direction is from the second pore to the first pore. In some embodiments, the dual pore device includes a processor. In some embodiments, the dual pore device includes a non-transitory computer readable medium including instructions that cause the processor to determine from one or more sensors that the target polynucleotide is simultaneously present in both pores. In some embodiments, the instructions cause the processor to scan one or more features of the target polynucleotide. In some embodiments, the instructions cause the processor to count a first set of features in a first cycle in a first direction and adjust one or both of the first and second voltages in response to the counts to produce a first force and an opposite second force acting on the target polynucleotide. In some embodiments, the first and second forces change the direction and speed of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in a second direction. In some embodiments, the process is repeated in a second cycle to detect a second set of features.In some embodiments, the process detects a third and fourth set of features in a second cycle, hi some embodiments, these steps are repeated until the polynucleotide exits the dual pore device.

[0235] Aspects of the present disclosure include a device for mapping one or more characteristics of a target polynucleotide through first and second pores, the device comprising: (i) a first pore disposed between a chamber and a first fluid volume, the first pore being fluidically connected to the chamber and to the first fluid volume, the first fluid volume being a geometrically constrained housing on an opposite side of the first pore; (ii) a second pore disposed between the chamber and a second fluid volume, the second pore being fluidically connected to the chamber and to the second fluid volume, the second fluid volume being a geometrically constrained housing on an opposite side of the first pore; (iii) housings for the first and second fluid volumes, each of which is filled with a fluid and includes an inlet and an outlet for accessing an electrode, the first pore being connected to the geometrically constrained housing of the first fluid volume at a location between the inlet and the outlet, and the second pore being connected to the geometrically constrained housing of the second fluid volume at a location between the inlet and the outlet; (iv) at least one electrode disposed within the first fluid volume; (v) one or more sensors configured to independently control a voltage at each pore and measure a current at each pore; (vi) a processor; and (vii) a non-transitory computer readable medium, the non-transitory computer readable medium being configured to: a) apply a first voltage at the first pore and a first voltage at the second pore to capture and transfer the target polynucleotide through the first pore to the first fluid volume; b) apply a second voltage at the first pore and a second voltage at the second pore to capture and transfer the target polynucleotide through the first pore to the first fluid volume; capturing and partially or completely translocating the target polynucleotide from the first pore through the first pore; c) applying a third voltage at the first pore and a third voltage at the second pore such that at least a portion of the target polynucleotide is captured by the second pore but remains in the first pore; d) applying a fourth voltage at the first pore and a fourth voltage at the second pore to control the direction of translocation of the target polynucleotide; e) detecting a first set of features of the polynucleotide in a first sensor current as each feature passes through the first pore.and again detecting, at a second sensor current, each feature as it passes through the second pore; f) applying a fifth voltage at the first pore and a fifth voltage at the second pore to reverse the direction of migration of the target polynucleotide; g) detecting, at a second sensor current, a second set of features of the target polynucleotide as it passes through the second pore, and again detecting, at the first sensor current, each feature as it passes through the first pore.

[0236] In some embodiments, after step a), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore, scanning to detect a first set of features as the target polynucleotide passes through the first pore.

[0237] In some embodiments, after step b), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore.

[0238] In some embodiments, after step d), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore, and to scan again each feature as it passes through the second pore.

[0239] In some embodiments, after step f), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the second pore, and to scan each feature again as it passes through the first pore.

[0240] In some embodiments, applying the voltage in step b) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore.

[0241] In some embodiments, adjusting the voltage in step c) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore.

[0242] In some embodiments, applying the voltage of step f) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through a first pore and when at least a portion of the target polynucleotide passes again through a second pore.

[0243] In some embodiments, the method further comprises instructions to cause the processor to repeat steps c) through g).

[0244] In some embodiments, the method further comprises instructions to cause the processor to detect one or more features of the target polynucleotide as each feature passes through the first pore, and to detect again those not yet detected as each feature passes through the second pore in step e).

[0245] In some embodiments, steps c) through g) comprise a first cycle of feature detection in both directions.

[0246] In some embodiments, the method further comprises repeating steps c) through g) to detect a third and fourth set of features in a second cycle of detecting features in both directions.

[0247] In some embodiments, the instructions cause the processor to further repeat c) until the target polynucleotide exits the device.

[0248] In some embodiments, the device includes at least one electrode disposed in the first fluid channel and at least one electrode disposed in the second fluid channel.

[0249] In some embodiments, the one or more sensors include dual amplifying electronics configured for voltage control and current measurement at the first pore and the second pore.

[0250] In some embodiments, the first voltage at the first pore and the second voltage at the second pore are independently controlled, and the first voltage and the second voltage range from 0 mV to 1000 mV.

[0251] In some embodiments, the first pore and the second pore are spaced from each other by about 10 nm to 2 μm.

[0252] In some embodiments, the device is a dual nanopore chip, which has a length of 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, or 8 mm or more, and a width of 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, or 8 mm or more.

[0253] In some embodiments, the pore diameter ranges from about 2 nm to about 50 nm.

[0254] In some embodiments, the pore diameter is about 20 nm.

[0255] In some embodiments, the first pore and the second pore are about 500 nm apart.

[0256] In some embodiments, the first pore has a depth of at least about 0.3 nm and separates the first channel and the chamber, and the second pore has a depth of at least about 0.3 nm and separates the chamber and the second channel.

[0257] In some embodiments, the chambers are connected to a common ground associated with a first voltage at the first pore and a second voltage at the second pore.

[0258] Any of the above claimed devices further includes a controller.

[0259] In some embodiments, the controller is configured to scan varying numbers of polynucleotide features.

[0260] In some embodiments, the controller is configured to vary the number of scans.

[0261] In some embodiments, the controller is configured to control the position of the polynucleotide that is scanned.

[0262] In some embodiments, the controller is configured to vary the region of the polynucleotide that is scanned.

[0263] In some embodiments, the controller is configured to control a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0264] In some embodiments, the processor comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0265] In some embodiments, the controller comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0266] In some embodiments, the controller comprises a microcontroller.

[0267] In some embodiments, the FPGA and ASIC execute control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0268] In some embodiments, the controller is configured to control the direction of movement of the polynucleotide.

[0269] In some embodiments, the method further includes instructions that cause the processor to construct a local map of regions of the polynucleotide that contain one or more features based on the scan.

[0270] In some embodiments, the method further includes instructions that cause the processor to construct a consensus map of regions of the polynucleotide that contain one or more features based on the scan.

[0271] In some embodiments, the constructing comprises a machine learning algorithm trained to detect one or more features based on training data and a probabilistic model.

[0272] In some embodiments, the method further includes instructions to cause the processor to calculate a velocity of the feature of the target polynucleotide from the time difference between detection of the feature at the first pore and detection of the feature at the second pore and the known distance between Pore 1 and Pore 2.

[0273] In some embodiments, the processor further includes instructions to calculate the distance between features from the velocity of the features of the target polynucleotide, i.e., from the time between the features detected in the current signal from the first pore, the second pore, or both.

[0274] In some embodiments, the method further comprises instructions to cause the processor to calculate, for every scan, the velocities of features of the target polynucleotide, and to calculate statistics of the feature velocities using the distribution of velocities.

[0275] In some embodiments, the method further includes instructions that cause the processor to combine all of the velocities of the features and calculate a time history of the velocity of the polynucleotide for a given scan and a given scanning direction.

[0276] In some embodiments, the method further comprises instructions to cause the processor to perform a frequency sweep of the polynucleotide in a first direction, a second direction, or both.

[0277] In some embodiments, the method further includes instructions that cause the processor to perform an amplitude sweep of the polynucleotide in a first direction, a second direction, or both.

[0278] In some embodiments, the method further comprises instructions that cause the processor to regulate the rate of the polynucleotide.

[0279] In some embodiments, the rate ranges from 1 base pair / millisecond to 10 base pairs / millisecond.

[0280] In some embodiments, the method further includes instructions to cause the processor to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple speeds.

[0281] In some embodiments, performing multiple scans of the polynucleotide at said multiple speeds improves accuracy of detection of one or more features.

[0282] In some embodiments, the method further comprises instructions that cause the processor to perform multiple scans of the polynucleotide at multiple speeds.

[0283] In some embodiments, the method further comprises instructions that cause the processor to control a range of velocity of the polynucleotide in a first direction, a second direction, or both.

[0284] In some embodiments, the processor further includes instructions to control a voltage range for the first and second pores as the polynucleotide translocates through the first and second pores in the first direction, the second direction, or both.

[0285] In some embodiments, the method further includes instructions for determining an optimal velocity range for the polynucleotide in a first direction, a second direction, or both, that reduces the effects of Brownian motion of the polynucleotide.

[0286] In some embodiments, controlling the speed range of the polynucleotide comprises determining an optimal speed range of the polynucleotide for sequencing.

[0287] Aspects of the present disclosure include systems for carrying out the methods described herein. Aspects of the present disclosure include a system, which includes: a) a device for mapping one or more characteristics of a target polynucleotide through a first and a second pore, the device comprising: (i) a first pore disposed between a chamber and a first fluid volume, the first pore being fluidically connected to the chamber and to the first fluid volume, the first fluid volume being a geometrically constrained housing on an opposite side of the first pore; (ii) a second pore disposed between the chamber and a second fluid volume, the second pore being fluidically connected to the chamber and to the first fluid volume, the first fluid volume being a geometrically constrained housing on an opposite side of the first pore; (iii) a housing for the first fluid volume and a housing for the second fluid volume, each housing for the first fluid volume and the second fluid volume including an inlet and an outlet for filling with a fluid and accessing an electrode, the first pore being connected to the geometrically constrained housing of the first fluid volume at a location between the inlet and the outlet, and the second pore being connected to the geometrically constrained housing of the second fluid volume at a location between the inlet and the outlet; (iv) (b) a device including at least one electrode disposed in the first fluid volume, at least one electrode disposed in the second fluid volume, and at least one electrode disposed in the chamber; (v) one or more sensors configured to independently control a voltage at each pore and to measure a current; (b) a processor; and (c) a non-transitory computer readable medium, the device including: (a) at least one electrode disposed in the first fluid volume, at least one electrode disposed in the second fluid volume, and at least one electrode disposed in the chamber; (b) a processor; and (c) a non-transitory computer readable medium, the device configured to: i) apply a first voltage at the first pore and a first voltage at the second pore to capture a target polynucleotide through the first pore and into the first fluid volume. ii) applying a second voltage at the first pore and a second voltage at the second pore to capture and partially or completely translocate the target polynucleotide from the first fluid volume through the first pore; iii) applying a third voltage at the first pore and a third voltage at the second pore such that at least a portion of the target polynucleotide is captured by the second pore but remains in the first pore; iv) applying a fourth voltage at the first pore and a fourth voltage at the second pore to control the direction of migration of the target polynucleotide.v) detecting a first set of polynucleotide features at a first sensor current as each feature passes through the first pore, and again detecting at a second sensor current as each feature passes through the second pore; vi) applying a fifth voltage at the first pore and a fifth voltage at the second pore to reverse the direction of migration of the target polynucleotide; vii) detecting a second set of polynucleotide features at a second sensor current as each feature passes through the second pore, and again detecting at the first sensor current as each feature passes through the first pore.

[0288] In some embodiments, after step i), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore, scanning to detect a first set of features as the target polynucleotide passes through the first pore.

[0289] In some embodiments, after step ii), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore.

[0290] In some embodiments, after step iv), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the first pore, and to scan again each feature as it passes through the second pore.

[0291] In some embodiments, after step vii), the method further comprises instructions to cause the processor to scan one or more features of the target polynucleotide as at least a portion of the target polynucleotide passes through the second pore, and to scan again each feature as it passes through the first pore.

[0292] In some embodiments, applying the voltage in step ii) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore.

[0293] In some embodiments, adjusting the voltage in step iii) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore.

[0294] In some embodiments, applying the voltage of step vii) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through a first pore and when at least a portion of the target polynucleotide passes again through a second pore.

[0295] In some embodiments, the method further comprises instructions to cause the processor to repeat steps iii) to vii).

[0296] In some embodiments, the method further comprises instructions to cause the processor to detect one or more features of the target polynucleotide as each feature passes through the first pore, and to detect again those not yet detected as each feature passes through the second pore in step e).

[0297] In some embodiments, steps iii) to vii) comprise a first cycle of feature detection in both directions.

[0298] In some embodiments, the method further comprises repeating steps iii) through vii) to detect a third and fourth set of features in a second cycle of detecting features in both directions.

[0299] In some embodiments, the instructions further cause iii) through vii) to be repeated until the target polynucleotide has exited the device.

[0300] In some embodiments, the device includes at least one electrode disposed in the first fluid channel and at least one electrode disposed in the second fluid channel.

[0301] In some embodiments, the one or more sensors include dual amplifying electronics configured for voltage control and current measurement at the first pore and the second pore.

[0302] In some embodiments, the first voltage at the first pore and the second voltage at the second pore are independently controlled, and the first voltage and the second voltage range from 0 mV to 1000 mV.

[0303] In some embodiments, the first pore and the second pore are spaced from each other by about 10 nm to 2 μm.

[0304] In some embodiments, the device is a dual nanopore chip, which has a length of 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, or 8 mm or more, and a width of 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, or 8 mm or more.

[0305] In some embodiments, the pore diameter ranges from about 2 nm to about 50 nm.

[0306] In some embodiments, the pore diameter is about 20 nm.

[0307] In some embodiments, the first pore and the second pore are about 500 nm apart.

[0308] In some embodiments, the first pore has a depth of at least about 0.3 nm and separates the first channel and the chamber, and the second pore has a depth of at least about 0.3 nm and separates the chamber and the second channel.

[0309] In some embodiments, the chambers are connected to a common ground associated with a first voltage at the first pore and a second voltage at the second pore.

[0310] Any of the above claimed devices further includes a controller.

[0311] In some embodiments, the controller is configured to scan varying numbers of polynucleotide features.

[0312] In some embodiments, the controller is configured to vary the number of scans.

[0313] In some embodiments, the controller is configured to control the position of the polynucleotide that is scanned.

[0314] In some embodiments, the controller is configured to vary the region of the polynucleotide that is scanned.

[0315] In some embodiments, the controller is configured to control a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0316] In some embodiments, the processor comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0317] In some embodiments, the controller comprises a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0318] In some embodiments, the controller comprises a microcontroller.

[0319] In some embodiments, the FPGA and ASIC execute control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0320] In some embodiments, the controller is configured to control the direction of movement of the polynucleotide.

[0321] In some embodiments, the method further includes instructions that cause the processor to construct a local map of regions of the polynucleotide that contain one or more features based on the scan.

[0322] In some embodiments, the method further includes instructions that cause the processor to construct a consensus map of regions of the polynucleotide that contain one or more features based on the scan.

[0323] In some embodiments, the constructing comprises a machine learning algorithm trained to detect one or more features based on training data and a probabilistic model.

[0324] In some embodiments, the method further includes instructions to cause the processor to calculate a velocity of the feature of the target polynucleotide from the time difference between detection of the feature at the first pore and detection of the feature at the second pore and the known distance between Pore 1 and Pore 2.

[0325] In some embodiments, the processor further includes instructions to calculate the distance between the features from the velocity of the features of the target polynucleotide, i.e., from the time between the features detected in the current signal from the first pore, the second pore, or both.

[0326] In some embodiments, the method further comprises instructions to cause the processor to calculate, for every scan, the velocities of features of the target polynucleotide, and to calculate statistics of the feature velocities using the distribution of velocities.

[0327] In some embodiments, the method further includes instructions that cause the processor to combine all of the velocities of the features and calculate a time history of the velocity of the polynucleotide for a given scan and a given scanning direction.

[0328] In some embodiments, the method further comprises instructions to cause the processor to perform a frequency sweep of the polynucleotide in a first direction, a second direction, or both.

[0329] In some embodiments, the method further includes instructions that cause the processor to perform an amplitude sweep of the polynucleotide in a first direction, a second direction, or both.

[0330] In some embodiments, the method further comprises instructions that cause the processor to regulate the rate of the polynucleotide.

[0331] In some embodiments, the rate ranges from 1 base pair / millisecond to 10 base pairs / millisecond.

[0332] In some embodiments, the method further includes instructions to cause the processor to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple speeds.

[0333] In some embodiments, performing multiple scans of the polynucleotide at said multiple speeds improves accuracy of detection of one or more features.

[0334] In some embodiments, the method further comprises instructions that cause the processor to perform multiple scans of the polynucleotide at multiple speeds.

[0335] In some embodiments, the method further comprises instructions that cause the processor to control a range of velocity of the polynucleotide in a first direction, a second direction, or both.

[0336] In some embodiments, the processor further includes instructions to control a voltage range for the first and second pores as the polynucleotide translocates through the first and second pores in the first direction, the second direction, or both.

[0337] In some embodiments, the method further includes instructions for determining an optimal velocity range for the polynucleotide in a first direction, a second direction, or both, that reduces the effects of Brownian motion of the polynucleotide.

[0338] In some embodiments, controlling the speed range of the polynucleotide comprises determining an optimal speed range of the polynucleotide for sequencing.

[0339] Aspects of the present disclosure include systems for carrying out the methods described herein. An embodiment of the system includes: a) a dual pore, dual amplification device for mapping one or more features of a polynucleotide sequence of a target polynucleotide through a first and a second pore, the device comprising: (i) electrodes connected to a power source configured to provide a first voltage between a first fluidic channel and a chamber of the device, and a second voltage between the chamber and a second fluidic channel of the device; (ii) a first pore; (iii) a second pore, wherein the first pore and the second pore are configured to allow the target polynucleotide to translocate simultaneously across both pores in a first direction or a second direction in a controlled manner; and (iv) identifying a first set of features from the target polynucleotide in a first cycle during translocation of the target polynucleotide through the first pore and the second pore in the first direction, and identifying a second set of features from the target polynucleotide in a first cycle during translocation of the target polynucleotide through the second pore and the first pore in the second direction. and (d) a non-transitory computer readable medium comprising instructions for causing the processor to: i) determine from the sensors the simultaneous presence of the target polynucleotide in both pores; ii) scan one or more features of the target polynucleotide; iii) count a first set of features in a first cycle in a first direction and, in response to the counts, adjust one or both of the first and second voltages to produce a first force and an opposite second force acting on the target polynucleotide, the first and second forces causing a change in direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second direction; and iv) repeating steps i) through iii) and detecting a third and fourth set of features in a second cycle.

[0340] Aspects of the present disclosure include a device for capturing polynucleotides with a nanopore device and then recapturing the polynucleotides partially or completely within the nanopore device after an extended distance and / or a predetermined period of time, the device comprising: (i) a first pore disposed between a chamber and a first fluid volume, the first pore being fluidically connected to the chamber and to the first fluid volume, the first fluid volume being a geometrically constrained housing on an opposite side of the first pore; (ii) a housing for the first fluid volume including an inlet and an outlet for filling with fluid and accessing an electrode, the first pore being connected to the geometrically constrained housing of the first fluid volume at a location between the inlet and the outlet; (iii) at least one electrode disposed within the first fluid volume and at least one electrode disposed within the chamber; (iv) a voltage between the electrode in the first fluid volume and the electrode in the chamber, and a voltage between the electrode in the first pore and the electrode in the chamber; and v) a processor; and vi) a non-transitory computer readable medium containing instructions causing the processor to: a) apply a first voltage to capture and transfer the polynucleotide from the chamber in a first direction through the first pore into the first fluid volume; b) detect a first sensor current as the polynucleotide translocates through the first pore in the first direction; c) apply a second voltage equal to zero for a predetermined period of time while the polynucleotide is contained within the first fluid volume; d) apply a third voltage to capture and partially or completely translocate the polynucleotide from the first fluid volume through the first pore into the chamber; and e) detect the first sensor current as the polynucleotide translocates partially or completely through the first pore.

[0341] Disclosed aspects include a system for capturing polynucleotides with a nanopore device and then partially or completely recapturing the polynucleotides after an extended distance and / or a predetermined period of time, the system comprising: (a) a nanopore device comprising: (i) a first pore disposed between a chamber and a first fluid volume, the first pore being fluidically connected to the chamber and to the first fluid volume, the first fluid volume being a geometrically constrained housing on an opposite side of the first pore; (ii) a housing for the first fluid volume, the housing including an inlet and an outlet for filling with fluid and accessing an electrode, the first pore being connected to the geometrically constrained housing of the first fluid volume at a location between the inlet and the outlet; (iii) a nanopore device comprising at least one electrode disposed within the first fluid volume and at least one electrode disposed within the chamber; (b) a nanopore device comprising a first electrode disposed within the first fluid volume and at least one electrode disposed within the chamber; and (d) a non-transitory computer readable medium comprising instructions for causing the processor to: i) apply a first voltage to capture and transfer the polynucleotide from the chamber in a first direction through the first pore into the first fluid volume; ii) detect a first sensor current as the polynucleotide translocates through the first pore in the first direction; iii) apply a second voltage equal to zero for a predetermined period of time while the polynucleotide is contained within the first fluid volume; iv) apply a third voltage to capture and partially or completely translocate the polynucleotide from the first fluid volume through the first pore into the chamber; and v) detect the first sensor current as the polynucleotide translocates partially or completely through the first pore.

[0342] Nanopore Devices In some embodiments, the dual pore device includes at least one nanopore (shown in FIG. 8) that forms an opening in a structure that separates the interior space of the nanopore device into two volumes. The nanopore device also includes at least a sensor in electrical communication with the opening and configured to identify an object passing through the nanopore (e.g., by detecting a change in an electrical signal parameter indicative of the object). Nanopore devices that may be used in the methods and systems described herein are also disclosed in International Publication No. WO / 2013 / 012881, International Publication No. WO / 2018 / 236673, US Application Publication No. 2017 / 0145481, and US Patent No. 9,863,912, which are incorporated herein by reference in their entireties. Amplifiers and circuits in nanopore devices that may be used in the methods and systems are also disclosed in US Application Publication No. 2017 / 0145481, which is incorporated herein by reference in its entirety.

[0343] In some embodiments, the nanopore(s) of the nanopore device(s) are nanoscale or microscale in relation to the size of the unique features. In one aspect, each pore has a size that allows the passage of small or large molecules (e.g., nucleic acid molecules or fragments) or microorganisms. In the examples, the nanopores have diameters of 1 nm to 100 nm, although in variations of the examples, the nanopores may have diameters less than 1 nm or greater than 100 nm. In some embodiments, the diameter of the pores ranges from about 2 nm to about 50 nm. In some embodiments, the diameter of the pores is about 20 nm. In variations, the pores have depths in the range of 1-10,000 nm, although in other variations, the nanopores may have depths less than 1 nm or greater than 10,000 nm. Furthermore, during the conduct of an experiment, the diameter of the nanopores may vary (within appropriate ranges), as described in more detail below.

[0344] Each of the pores of the dual pore device independently has a depth. In one embodiment, each pore has a depth that is at least about 0.3 nm. In some embodiments, each pore is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm. In some embodiments, each pore has a depth that is about 100 nm or less. Alternatively, the depth is about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm or less. In some embodiments, the pores have a depth that is about 1 nm to about 100 nm, or about 2 nm to about 80 nm, or about 3 nm to about 70 nm, or about 4 nm to about 60 nm, or about 5 nm to about 50 nm, or about 10 nm to about 40 nm, or about 15 nm to about 30 nm. In some embodiments, the first pore has a depth of at least about 0.3 nm and separates the first fluidic channel and the chamber, and the second pore has a depth of at least about 0.3 nm and separates the chamber and the second fluidic channel.

[0345] In some aspects, each of the pores of the dual pore device has a size that allows small or large molecules or microorganisms to pass through. In some embodiments, each pore is at least about 1 nm in diameter. Alternatively, each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.

[0346] In some embodiments, the pores have a diameter of about 1 nm to about 100 nm, or about 2 nm to about 80 nm, or about 3 nm to about 70 nm, or about 4 nm to about 60 nm, or about 5 nm to about 50 nm, or about 10 nm to about 40 nm, or about 15 nm to about 30 nm.

[0347] In some embodiments, the nanopores of the nanopore device have a substantially round shape. As used herein, "substantially round" refers to a shape that is at least about 80% or 90% in the form of a cylinder. However, in alternative embodiments, the nanopore device can include nanopores that are square, rectangular, triangular, oval, hexagonal, or other shapes.

[0348] In some embodiments, the nanopore extends through the membrane. For example, the pore may be protein inserted into a lipid bilayer membrane, or may be machined by drilling, etching, or other means to form a pore through a solid-state substrate, such as silicon dioxide, silicon nitride, graphene, or layers formed in combination with these or other materials.

[0349] In some embodiments, the nanopores of the device can be separated by a distance ranging from 5 to 15,000 nm. In some embodiments, the nanopores of the device can be separated by a distance ranging from 10 to 1000 nm. However, in other variations, the nanopores can be less than 5 nm or more than 15,000 nm apart. Furthermore, the nanopores can be arranged in any position as long as they can fluidly communicate between the chambers and they have a defined size and distance between them. In some embodiments, the first pore and the second pore are about 10 nm to 500 nm apart from each other. In some embodiments, the first pore and the second pore are about 500 nm apart from each other. In one variation, the nanopores are arranged such that they have no direct blockage between them. Furthermore, in one form, the pores are substantially coaxial.

[0350] In some cases, the diameter of the pore is in the range of about 2 nm to about 50 nm. In some cases, the diameter of the pore is about 20 nm. In some cases, the diameter of the first and / or second pore is in the range of about 2 nm to about 50 nm. In some cases, the diameter of the first and / or second pore is in the range of about 2 nm to about 8 nm. In some cases, the diameter of the first and / or second pore is in the range of about 10 nm to about 20 nm. In some cases, the diameter of the first and / or second pore is in the range of about 20 nm to about 30 nm. In some cases, the diameter of the first and / or second pore is in the range of about 30 nm to about 40 nm. In some cases, the diameter of the first and / or second pore is in the range of about 40 nm to about 50 nm. In some cases, the diameter of the first and / or second pore is about 2 nm, about 4 nm, about 6 nm, about 8 nm, about 10 nm, about 12 nm, about 14 nm, about 16 nm, about 18 nm, about 20 nm, about 22 nm, about 24 nm, about 26 nm, about 28 nm, about 30 nm, about 32 nm, about 34 nm, about 36 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm, or about 50 nm. In some cases, the diameter of the first and / or second pore is about 19 nm. In some cases, the first pore and the second pore have the same diameter. In some cases, the diameter of the first pore and / or the second pore is about 21 nm. In some cases, the diameter of the first pore and / or the second pore is about 22 nm. In some cases, the diameter of the first pore and / or the second pore is about 23 nm. In some cases, the diameter of the first pore and / or the second pore is about 24 nm. In some cases, the diameter of the first pore and / or the second pore is about 25 nm. In some cases, the diameter of the first pore and / or the second pore is about 27 nm. In some cases, the diameter of the first pore and / or the second pore is about 29 nm. The first pore and the second pore have different diameters. In some cases, the diameter of the pore is about 20 nm.

[0351] In some embodiments, the device includes a geometrically constrained fluid volume. In some cases, the geometrically constrained fluid volume is a fluidic channel. In some cases, the device includes a first fluidic channel. As used herein, the term "upper chamber" is used interchangeably with the terms "fluidic channel" and "geometrically constrained fluid volume", e.g., a first fluidic channel. In some embodiments, the device includes a middle chamber. As used herein, the term "middle chamber" is used interchangeably with the term "chamber". In some embodiments, the device includes a first pore connecting the upper chamber and the middle chamber. In some embodiments, the device includes a second pore connecting the middle chamber and a bottom chamber. As used herein, the term "lower chamber" is used interchangeably with the terms "fluidic channel" and "geometrically constrained fluid volume", e.g., a second fluidic channel. In some embodiments, the device includes a bottom chamber. In some embodiments, the device includes a second fluidic channel. In some embodiments, the first fluid volume, the second fluid volume, the first fluid channel, the second fluid channel, and / or the chamber contain one or more electrodes for connection to a power source such that separate voltages can be established across each of the pores between the chambers. In some embodiments, the device includes electrodes connected to a power source to provide a first voltage between the first fluid channel and the chamber of the device, and to provide a second voltage between the chamber and the second fluid channel of the device. In some embodiments, the chamber is disposed above the first and second pores. In some embodiments, the chamber is disposed above the first and second fluid channels. In some embodiments, the chamber is disposed below the first and second pores. In some embodiments, the chamber is disposed between the first and second pores. In some embodiments, the chamber is disposed between the first and second fluid channels.

[0352] In some cases, the shape of the first and / or second fluidic channel can be circular, square, rectangular, hexagonal, triangular, oval, polygonal, V-shaped, U-shaped, or any other suitable shape. In some cases, the first and second fluidic channels each have a V-shape, each with an opening at both ends of the V-shape, the V-shapes of the first and second fluidic channels are arranged on the chip opposite each other at a predetermined point of the V-shape adjacent to each other, and the first nanopore is located at a predetermined point of the V-shape of the first fluidic channel and the second nanopore is located at a predetermined point of the V-shape of the second fluidic channel. In some embodiments, each of the fluidic channels is of a different shape. The fluidic channels are not limited to the shapes and / or sizes described herein and can be any shape and / or size required by the conditions specified for their intended use.

[0353] In some cases, the fluidic channel of the nanopore device comprises one or more openings on opposite sides of the first and / or second pore, In some cases, the fluidic channel of the nanopore device comprises two openings on opposite sides of the first and / or second pore.

[0354] In some embodiments, the nanopore device has electrodes disposed in a fluidic channel, geometrically constrained volume, or chamber, and coupled to one or more power sources for applying a voltage across the nanopore(s). In some aspects, the power source comprises a voltage clamp or patch clamp that can provide a voltage across each pore and measure the current through each pore independently. In this regard, the power source and electrode configuration establishes the chamber as a common ground for both power sources. In this case, each nanopore can have its own voltage applied to it.

[0355] In some embodiments, the first voltage V1 and the second voltage V2 of different nanopores of the nanopore device are independently adjustable. In one embodiment, when multiple nanopores are connected by a chamber, the chamber can be adjusted to be ground related to the two voltages. In one embodiment, the chamber includes a medium for providing a conductance between each pore and an electrode in the chamber. In one embodiment, the chamber includes a medium for providing a resistance between each nanopore and an electrode in the chamber. Maintaining such resistance sufficiently small compared to the resistance of the nanopore is effective to separate the two voltages and currents across the pore, which aids in independent adjustment of the voltages.

[0356] Adjustment of the voltages can be used to control the movement of charged particles within the chamber. For example, if both voltages are set to the same polarity, appropriately charged particles can be moved from a first fluidic channel to the chamber and to a second fluidic channel, or can subsequently move in the opposite direction. In some embodiments, if the two voltages are set to opposite polarities, charged particles can be moved from either the first or second fluidic channel to the chamber and retained there.

[0357] Modulation of the voltage of the device is particularly useful for controlling the movement of large molecules, such as charged polymers, that are long enough to cross both pores simultaneously. In such embodiments, the direction and rate of molecular movement can be controlled by the relative magnitude and polarity of the voltages as described below.

[0358] In some cases, the first initial voltage is in the range of 0 mV to 1000 mV. In some cases, the first initial voltage is in the range of 100-200 mV, 200-300 mV, 300-400 mV, 400-500 mV, 500-600 mV, 600-700 mV, 700-800 mV, 800-900 mV, 900-1000 mV, or 1000 mV or more. In some cases, the first initial voltage is 100 mV, 200 mV, 300 mV, 400 mV, 500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or 1000 mV. In some cases, the second initial voltage is in the range of 0 mV to 1000 mV. In some cases, the second initial voltage is in the range of 100-200mV, 200-300mV, 300-400mV, 400-500mV, 500-600mV, 600-700mV, 700-800mV, 800-900mV, 900-1000mV, or 1000mV or more. In some cases, the second initial voltage is 100mV, 200mV, 300mV, 400mV, 500mV, 600mV, 700mV, 800mV, 900mV, or 1000mV.

[0359] In some cases, the method of the present disclosure includes adjusting a first and / or second voltage to control the movement of the target polynucleotide in the first pore, the first fluidic channel, the second pore, the second fluidic channel, and / or the chamber of the device. In some cases, the first voltage is adjusted to 0 mV after the target polynucleotide moves from the chamber through the first pore to the first fluidic channel. In some cases, the first voltage is adjusted to 0 mV before the transfer through the first pore, when at least a portion of the target polynucleotide is disposed in the chamber and when at least a portion of the target polynucleotide is disposed in the first fluidic channel. In some cases, the second voltage at the second pore is adjusted to 500 mV when at least a portion of the target polynucleotide is disposed in the chamber and when at least a portion of the target polynucleotide is disposed in the chamber. In some cases, the first voltage is adjusted to 0 mV, 50 mV, 100 mV, 150 mV, 200 mV, 250 mV, 300 mV, 350 mV, 400 mV, 450 mV, 500 mV, 550 mV, or 600 mV in the first, second, third, and / or fourth orientation. In some cases, the second voltage is adjusted to 0 mV, 50 mV, 100 mV, 150 mV, 200 mV, 250 mV, 300 mV, 350 mV, 400 mV, 450 mV, 500 mV, 550 mV, or 600 mV in the first, second, third, and / or fourth orientation. In some cases, the first voltage is adjusted to an intermediate voltage of 0 mV, and the second voltage is adjusted to 500 mV in the third direction (e.g., when at least a portion of the target polynucleotide is co-captured by the first pore and the second pore). In some cases, the first voltage is adjusted to 400 mV, and the second voltage is adjusted to 500 mV in the third direction (e.g., when at least a portion of the target polynucleotide is co-captured by the first pore and the second pore). In some cases, the first voltage is adjusted to 200 mV, and the second voltage is adjusted to 500 mV in the third direction (e.g., when at least a portion of the target polynucleotide is co-captured by the first pore and the second pore).

[0360] In some embodiments, the charged polymer, such as a polynucleotide, has a length that is longer than the combined distance including the depth of both pores plus the distance between both pores. For example, a 1000 bp dsDNA is ∼340 nm long, which is substantially longer than 40 nm across two pores of 10 nm length, 20 nm apart. In a first step, the polynucleotide is introduced into either the first fluidic channel or the second fluidic channel. In a first step, the polynucleotide is introduced into a chamber (e.g., the middle chamber or the common chamber) of the device. Due to its negative charge under physiological conditions (∼pH 7.4), the polynucleotide can be moved across the pore to which a voltage is applied. Thus, in a second step, two voltages are applied to the pores in the same direction and with the same or similar magnitude, which subsequently induces the movement of the polynucleotide across both pores. One or both voltages can be changed when the polynucleotide is approximately at the second pore. Because the polynucleotide is longer than the distance spanning both pores, it will also be present in the first pore when it reaches the second pore, so rapidly changing the direction of the voltage at the first pore will create a force that repels the polynucleotide away from the second pore.

[0361] In some embodiments, the dual pore devices of the present disclosure may be used to perform analysis of molecules or particles that are moved through or held within the device by controlled voltages applied across the pores. In one aspect, analysis is performed in either or both of the pores. Each voltage clamp or patch clamp system measures the ionic current through each pore, and the measured ionic current may be used to detect one or more characteristics of the passing charged particle or molecule, or any characteristic associated with the passing charged particle or molecule.

[0362] As provided above, a polynucleotide can be introduced into both pores with two voltages having the same direction. In this example, if the direction of the voltage applied to the first pore is reversed and the force induced by the new voltage is slightly smaller in magnitude than the force induced by the voltage applied to the second pore, the polynucleotide will continue to move in the same direction, but at an apparently slower speed. At this point, the amplifier that provides the voltage across the second pore also measures the current through the second pore, and the ionic current then determines the identity of the nucleotide passing through the pore, since each different nucleotide gives a different current signature (e.g., based on the shift in the magnitude of the ionic current). Thus, the identity of each nucleotide in a polynucleotide reveals the sequence of the polynucleotide.

[0363] In some embodiments, the first and second voltages adjusted in steps are about 10 to 10,000 times higher in magnitude than the difference between the two voltages. For example, the two voltages are 90 mV and 100 mV, respectively. In some embodiments, the magnitude of the voltages (~100 mV) is about 10 times the difference between them, 10 mV. In some embodiments, the magnitude of the voltages is at least about 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 times higher than the difference between them. In some embodiments, the magnitude of the voltage is less than about 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 500, 400, 300, 200, or 100 times the difference therebetween.

[0364] In some embodiments, repeated controlled delivery to resequence a polynucleotide improves the quality of the sequencing, with each voltage being alternated for longer for the controlled delivery in each direction.

[0365] The device may contain materials suitable for holding liquid samples, particularly biological samples, and / or materials suitable for nanofabrication. In one embodiment, such materials include dielectric materials, such as, but not limited to, silicon, silicon nitride, silicon dioxide, graphene, carbon nanotubes, TiO2, HfO2, Al2O3, or other metal layers, or combinations of any of these materials. In some embodiments, for example, a single layer of graphene film about 0.3 nm thick may be used as the pore-bearing membrane.

[0366] Microfluidic nanofabricated devices can be made by various means and methods. A focused electron or ion beam can be used to drill pores through the membrane and naturally align them. The pores can also be carved (reduced) to smaller sizes by applying a focused beam to each layer. Any method that drills a single hole (single nanopore) can also be used to drill pairs of pores in two membranes, taking into account the possible drilling depth for a given method and membrane thickness. Pre-drilling a micropore to a defined depth and then threading the nanopore through the remainder of the membrane allows for more precise membrane thickness. In one example, a single beam can be used to form one or more nanopores (e.g., coaxial nanopores) in the membrane of a nanopore device. Alternatively, in another example, different beams can be applied to each side of the membrane to generate aligned or misaligned nanopores.

[0367] More specifically, nanopore-bearing membranes are fabricated using transmission electron microscope (TEM) grids with 5-100 nm thick silicon, silicon nitride, or silicon dioxide windows. Spacers are used to separate the membranes, which may be insulators such as SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or evaporated metallic materials such as Ag, Au, or Pt, occupying a small volume within the otherwise aqueous portion of the intermediate chamber (e.g., chamber).

[0368] A voltage present at the Debye pore may cause the charged molecule to move between the chambers and through the pore. The speed and direction of movement may be controlled by the magnitude and polarity of the voltage. Furthermore, since each of the two voltages may be independently adjusted, the direction and speed of movement of the charged molecule may be finely controlled in each chamber. For example, when a first set of features is detected in a first cycle in a first direction, the first voltage and / or the second voltage may be adjusted for the first and second pores to change the direction of the target molecule moving from the second pore to the first pore in the second direction.

[0369] In some aspects, the nanopore device includes a polymer translocation means across the pore and / or a means for identifying an object passing through the pore. In some embodiments, the polymer is a polynucleotide or a polypeptide. In some aspects, the polymer is a polynucleotide. Non-limiting examples of polynucleotides include double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and DNA-RNA hybrids.

[0370] In some aspects, the dual pore device may be used to identify one or more features of a polymer. In some embodiments, the one or more features are 1 feature, 2 features, 3 features, 4 features, or 5 features. In some embodiments, the one or more features are 2 or more features, 3 or more features, 4 or more features, 5 or more features, 6 or more features, 7 or more features, 8 or more features, 9 or more features, or 10 or more features. In some embodiments, the one or more features are in the range of 1-5 features, 5-10 features, 10-15 features, 15-20 features, 20-25 features, 25-30 features, 30-35 features, 35-40 features, 40-45 features, or 45-50 features. In some embodiments, the one or more features range from 50 features to 100 features, 100 features to 1,000 features, 1,000 features to 10,000 features, 10,000 features to 100,000 features, 100,000 features to 200,000 features, In some embodiments, the one or more features range from 50 features or more, 100 features or more, 1,000 features or more, 10,000 features or more, 100,000 features or more, or 200,000 features or more.

[0371] Aspects of the disclosure include one or more features, each of which is spaced apart from one another by about 100 base pairs, 300 base pairs, 500 base pairs, 1 kilobase pairs, 5 kilobase pairs, 10 kilobase pairs, 20 kilobase pairs, or combinations thereof. In some embodiments, each of the features is spaced apart from one another by about 25 base pairs or more, about 50 base pairs or more, about 100 base pairs or more, about 300 base pairs or more, about 500 base pairs or more, about 1 kilobase pairs or more, about 5 kilobase pairs or more, about 10 kilobase pairs or more, about 20 kilobase pairs or more, or combinations thereof. In some embodiments, each of the features is spaced apart from one another by about 25 base pairs or less, about 50 base pairs or less, about 100 base pairs or less, about 300 base pairs or less, about 500 base pairs or less, about 1 kilobase pairs or less, about 5 kilobase pairs or less, about 10 kilobase pairs or less, about 20 kilobase pairs or less, or combinations thereof.

[0372] In some aspects, the dual pore device may be used to identify a first set of features, a second set of features, a third set of features, a fourth set of features, a fifth set of features, a sixth set of features, a seventh set of features, an eighth set of features, a ninth set of features, and / or a tenth set of features. In some cases, each set of features includes one or more features in the range of 1-5 features, 5-10 features, 10-15 features, 15-20 features, 20-25 features, 25-30 features, 30-35 features, 35-40 features, 40-45 features, or 45-50 features. In some embodiments, the first set of features overlaps with the second set of features. In some embodiments, the third set of features overlaps with the fourth set of features. In some embodiments, the first set of features partially overlaps with the second set of features. In some embodiments, the third set of features partially overlaps with the fourth set of features. In some embodiments, the first set of features is the same as the second set of features. In some embodiments, the third set of features is the same as the fourth set of features. In some embodiments, the first set of features is different from the second set of features. In some embodiments, the third set of features is different from the fourth set of features.

[0373] In some embodiments, the sets of features (e.g., the first set, the second set, the third set, the fourth set, the fifth set, the sixth set, the seventh set, the eighth set, the ninth set, and / or the tenth set) are associated with the first cycle, the second cycle, the third cycle, the fourth cycle, the fifth cycle, the sixth cycle, the seventh cycle, the eighth cycle, the ninth cycle, and / or the tenth cycle, respectively. In some cases, the first cycle includes one or more scans performed by the processor to detect the first set of features. In some cases, the first cycle includes two or more scans, three or more scans, four or more scans, five or more scans, six or more scans, seven or more scans, eight or more scans, nine or more scans, or ten or more scans. In some cases, the first cycle includes two or more scans, four or more scans, six or more scans, eight or more scans, ten or more scans, twelve or more scans, fourteen or more scans, sixteen or more scans, eighteen or more scans, or twenty or more scans. In some cases, the first cycle includes 5 or more scans, 10 or more scans, 15 or more scans, 20 or more scans, 25 or more scans, 30 or more scans, 35 or more scans, 40 or more scans, 45 or more scans, or 50 or more scans.

[0374] In some cases, the second cycle includes one or more scans performed by the processor to detect the third set of features. In some cases, the second cycle includes 2 or more scans, 3 or more scans, 4 or more scans, 5 or more scans, 6 or more scans, 7 or more scans, 8 or more scans, 9 or more scans, or 10 or more scans. In some cases, the second cycle includes 2 or more scans, 4 or more scans, 6 or more scans, 8 or more scans, 10 or more scans, 12 or more scans, 14 or more scans, 16 or more scans, 18 or more scans, or 20 or more scans. In some cases, the second cycle includes 5 or more scans, 10 or more scans, 15 or more scans, 20 or more scans, 25 or more scans, 30 or more scans, 35 or more scans, 40 or more scans, 45 or more scans, or 50 or more scans. In some cases, the first cycle and the second cycle together comprise 50 or more scans, 100 or more scans, 150 or more scans, 200 or more scans, 250 or more scans, 300 or more scans, 350 or more scans, 400 or more scans, or 500 or more scans. In some cases, the first cycle, second cycle, third cycle, fourth cycle, and fifth cycle together comprise 50 or more scans, 100 or more scans, 150 or more scans, 200 or more scans, 250 or more scans, 300 or more scans, 350 or more scans, 400 or more scans, or 500 or more scans.

[0375] Aspects of the disclosure include a processor and a computer readable medium, the computer readable medium including instructions to cause the processor to repeat determining the presence of the target polynucleotide in both pores, scanning one or more features, and varying the voltage to control movement of the polynucleotide (e.g., in either direction) for a third cycle, a fourth cycle, and a fifth cycle, or as the polynucleotide exits the device.

[0376] In some aspects, the dual pore device is used to identify one or more characteristics of a polymer. In some embodiments, the polymer is a polynucleotide. In some embodiments, the one or more characteristics of the polynucleotide include one or more characteristics associated with the polynucleotide. Non-limiting examples of the one or more characteristics associated with the polynucleotide include, but are not limited to, modifications to transcription factors, nucleosomes, or characteristics including modifications to histone tails. In some embodiments, the one or more characteristics of the polynucleotide are one or more sequence or structural variations.

[0377] In some embodiments, the one or more features of the polynucleotide include one or more payload molecules attached to the polynucleotide. In some embodiments, the one or more features of the polynucleotide include one or more payload molecules hybridized to the polynucleotide. In some embodiments, the one or more features of the polynucleotide include one or more payload molecules incorporated into the genome of the polynucleotide. In some embodiments, the one or more features of the polynucleotide include a molecular motif of the polynucleotide sequence of the target polynucleotide. In some embodiments, the one or more features include the following positions: one or more CpG's, or one or more methylation sites and CpG's, of the polynucleotide sequence of the target polynucleotide. In some embodiments, the one or more features include one or more histone positions of the target polynucleotide. In some embodiments, the one or more features include a molecule selected from the group consisting of a nucleic acid, a TALEN, a CRISPR, a peptide nucleic acid, and a chemical compound. In some embodiments, the one or more features include a DNA binding protein, a polypeptide, an anti-DNA antibody, a streptavidin, a transcription factor, a histone, a peptide nucleic acid (PNA), a DNA-heparin, a DNA molecule, an aptamer, or a combination thereof.

[0378] Non-limiting examples of payload molecules attached to polynucleotides can be found in US Patent Publication No. 2018 / 0023115, which is incorporated herein by reference in its entirety. For example, the payload molecule can include a dendrimer, double-stranded DNA, single-stranded RNA, DNA aptamer, fluorophore, protein, polypeptide, nanorod, nanotube, fullerene, PEG molecule, liposome, or cholesterol-DNA hybrid. In some embodiments, the polynucleotide and payload are directly or indirectly connected via covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, cation-π interactions, planar stacking interactions, or metal bonds. The payload adds size to the target polynucleotide or amplicon, facilitating detection by having an amplicon attached to the payload have a current signature significantly different from background molecules when passing through the nanopore. In some embodiments, the payload molecule includes an azide chemical handle for attachment to the primer. In some embodiments, the primer is conjugated to a biotin molecule. In some embodiments, the payload molecule is conjugated to another molecule, affecting the bulkiness of the molecule, which can increase the sensitivity of the detection of the amplicon at the nanopore. In some embodiments, the primer is conjugated to a biotin molecule or includes a binding site for binding to a biotin molecule. In some embodiments, the biotin is further conjugated by streptavidin, increasing the size of the payload molecule to enhance its detection at the nanopore over background molecules. The added bulkiness can result in a more distinct signature difference between the amplicon containing the target sequence and background molecules.

[0379] In some embodiments, attachment of the payload to the primer or amplicon can be accomplished by a variety of methods, for example, the primer can be a dibenzocyclooctyne (DBCO) modified primer, which effectively labels all amplicons with a DBCO chemical group and is used for the purpose of conjugation to azide tagged amplicons or primers via copper-free "click" chemistry.

[0380] In some aspects, the primer contains chemical modifications that induce and facilitate recognition and binding of the payload molecule. For example, methylated DNA sequences can be recognized by transcription factors, DNA methyltransferases, or methylation repair enzymes. In other embodiments, biotin can be incorporated into and recognized by avidin family members. In such embodiments, biotin forms the fusion binding domain, and avidin or an avidin family member becomes the scaffold-binding domain of the polymer upon fusion. Due to the complementarity of these bonds, the payload molecule binding domain of the primer / amplicon and primer binding domain of the payload molecule can be inverted such that the payload binding domain becomes the primer binding domain and vice versa.

[0381] Molecules, particularly proteins, that can specifically recognize nucleotide binding motifs are known in the art. For example, protein domains such as helix-turn-helix, zinc finger, leucine zipper, winged helix, winged helix-turn-helix, helix-loop-helix, and HMG-box are known to be capable of binding to nucleotide sequences. Any of these molecules can function as payload molecules that bind to amplicons or primers.

[0382] In some embodiments, the payload binding domain can be locked nucleic acids (LNAs), bridged nucleic acids (BNAs), protein nucleic acids of all types (e.g., bisPNAs, gamma-PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), or aptamers (e.g., DNA, RNA, protein, or combinations thereof).

[0383] In some embodiments, the payload binding domain is one or more DNA binding proteins (e.g., zinc finger proteins), antibody fragments (Fabs), chemically synthesized binders (e.g., PNA, LNA, TALENS, or CRISPR), or chemical modifications (i.e., reactive moieties) on a synthetic polymer scaffold (e.g., thiolate, biotin, amine, carboxylate).

[0384] In some embodiments, the one or more features include one or more features in a polynucleotide. In some embodiments, the one or more features in a polynucleotide include one or more modifications to a polynucleotide. In some embodiments, the one or more modifications include DNA methylation (e.g., 5mC, 5hmC, 5mA, etc., at CpG dinucleotides). In some embodiments, the one or more features in a polynucleotide include sequence variation, mutation, or large structural variation. In some embodiments, the one or more features of a polynucleotide include rearrangements, deletions, insertions, and / or translocations to the polynucleotide sequence.

[0385] In some embodiments, the one or more features include one or more features of a polynucleotide. In some embodiments, the one or more features of a polynucleotide include a modification to the polynucleotide. In some embodiments, the modification includes a molecule attached to a monomer. In some embodiments, the one or more features of a polynucleotide include one or more molecules attached to the polynucleotide. In some embodiments, the modification includes attachment of a molecule to the polynucleotide. For example, for a DNA molecule, the attached molecule can be a DNA-binding protein such as RecA, NF-κB, and p53. In some embodiments, the modification is a particle that binds to a specific monomer or fragment. For example, quantum dots or fluorescent labels attached to specific DNA sites for genotyping or DNA mapping can be detected by the device.

[0386] In some embodiments, the polynucleotide sequence includes one or more nick sites. As a non-limiting example, a nicking restriction endonuclease introduces a nick in a recognition sequence for barcoding. This sequence appears many times in the genome. A single azido-azido-N3 labeled nucleotide is introduced at the nick site. The reaction is filtered to remove unincorporated nucleotides. A DNA molecule labeled with DCBO at either the 5' or 3' end or a DNA molecule labeled in the body is added to the reaction. The DNA molecule is covalently attached to the nick site by copper-free click chemistry. A 1000-10000 fold excess of DNA molecules may be used. In another non-limiting example, Cas9D10A nickase can be used for site-specific labeling. Cas9-D10A is targeted to a specific site and a single stranded nick is introduced. Cas9D10A is removed. A single azido-N3 nucleotide is introduced at the nick site by nick translation. The reaction is filtered to remove unincorporated nucleotides. DNA molecules either 5', 3' or body-labeled with DCBO are added to the reaction. The DNA molecules are covalently attached to the nick sites by copper-free click chemistry. A 1000-10000-fold excess of DNA molecules may be used.

[0387] In some embodiments, the nanopore device comprises multiple chambers, each chamber communicating with an adjacent chamber through at least one pore.

[0388] In some embodiments, the nanopore device can be a multi-pore device having more than one pore. In some embodiments, the nanopore device includes two nanopores, with the first nanopore positioned relative to the second nanopore in such a manner that at least a portion of the target polynucleotide can move out of the first pore and into the second nanopore. In some embodiments, the nanopore device includes one or more sensors in each nanopore, each sensor capable of identifying the target polynucleotide while it moves across at least one sensor. In some embodiments, the identification involves identifying individual components of the target polynucleotide. In some embodiments, the identification involves identifying a payload molecule bound to the target polynucleotide. When a single sensor is used, the single sensor includes two electrodes positioned at either end of the pore and can measure the ionic current across the pore. In another embodiment, the single sensor includes elements other than electrodes.

[0389] In some embodiments, the nanopore device includes three chambers connected through two pores. Devices with more than three chambers can be readily designed to include one or more additional chambers on either side of the three-chamber device, or between any two of the three chambers. Similarly, more than two pores can be included in the device to connect the chambers. In some embodiments, the chambers are connected to a common ground associated with two voltages.

[0390] In one embodiment, there are two or more pores between two adjacent chambers, allowing multiple polymer scaffolds to move simultaneously from one chamber to the next. Such multi-pore design can increase the throughput of target polynucleotide analysis in the device. For multiplexing, one chamber can have one type of target nucleotide, and another chamber can have another type of target polynucleotide.

[0391] In some embodiments, the device further comprises a means for transferring a target polynucleotide from one chamber to the other. In one embodiment, the transfer occurs by introducing a target polynucleotide (e.g., an amplification product or amplicon containing a target sequence) simultaneously across both the first pore and the second pore. In another embodiment, the means further enables transfer of the target polynucleotide in the same direction through both pores.

[0392] Although several variations of nanopore devices are described above, the nanopore device(s) may be configured as described in US Application Publication. No. 2013-0233709 (Patent Document 6), U.S. Patent No. 9,863,912 (Patent Document 4), and PCT Application Publication No. WO2018 / 236673 (Patent Document 2), which are incorporated by reference in their entireties herein.

[0393] Systems and Devices - Sensors As discussed above, in various embodiments, the nanopore device further comprises one or more sensors that produce an electrical signal corresponding to a substance passing through the nanopore.

[0394] The sensor used in the nanopore device may include any sensor suitable for identifying a target polynucleotide amplicon bound or unbound to a payload molecule. For example, the sensor may be configured to identify a target polynucleotide by measuring a current, a voltage, a pH value, an optical characteristic, or a residence time associated with the polymer. In other embodiments, the sensor may be configured to identify one or more individual components of a target polynucleotide, or one or more components bound or attached to a target polynucleotide. The sensor may be formed of any element configured to detect a change in a measurable parameter, which is indicative of a target polynucleotide, a component of a target polynucleotide, or, in some cases, a component bound or attached to a target polynucleotide. In one embodiment, the sensor includes a pair of electrodes disposed on two sides of the pore that measure an ionic current across the pore as a molecule or other pore entity, particularly a target polynucleotide, moves through the pore. In some embodiments, the ionic current across the pore is measurably changed when a target polynucleotide segment passing through the pore is bound to a payload molecule. Such changes in current flow can, for example, vary in a predictable and measurable manner corresponding to the presence, absence, and / or size of target polynucleotide molecules present.

[0395] In one embodiment, the sensor includes electrodes that are used to apply a voltage and measure the current across the nanopore. The translocation of a molecule through the nanopore imparts an electrical impedance (Z) that affects the current through the nanopore according to Ohm's law, V=IZ, where V is the voltage, I is the current through the nanopore, and Z is the impedance. Conversely, the conductance G=1 / Z is monitored to signal and quantify nanopore events. The result when a molecule translocates through the nanopore in an electric field (e.g., under an applied voltage) is a current signature that can be correlated to the molecule passing through the nanopore upon further analysis of the current signal.

[0396] When using residence time measurements from the current signature, the magnitude of a component can be correlated to a particular component based on the length of time it takes for that component to pass through the sensing device.

[0397] In one embodiment, the nanopore device may be provided with sensors that measure optical characteristics of the polymer, its components (or units), or components bound or attached to the polymer. One example of such a measurement includes the identification of absorption bands unique to particular units by infrared (or ultraviolet) spectroscopy.

[0398] In some embodiments, the sensor is an electrical sensor. In some embodiments, the sensor detects a fluorescent signature. The signature can be detected using a radiation source at the exit of the pore. Non-limiting examples of nanopore device sensor circuits can be found in PCT Application Publication No. WO2018 / 236673, which is incorporated herein by reference in its entirety.

[0399] Systems and Devices - Processors, Controllers, and Other Components As described above, systems of embodiments of the present disclosure are configured to interface with a set of one or more nanopore devices and include an electronics subsystem for receiving electrical signals from sensors of the set of nanopore devices, and for generating local, global, and / or consensus maps of one or more features of a molecule, e.g., in real time (e.g., by manipulating voltages applied across the nanopores and scanning and probing different regions across the molecule). The electrical subsystem can include signal processing elements (e.g., amplifiers, filters, signal pre-conditioning elements, etc.) and / or elements for controlling the voltages applied across different nanopores to enable automated detection and mapping of one or more features across different regions of a molecule within the nanopore device.

[0400] Aspects of the disclosure include a device including a processor. In some embodiments, the device includes a non-transitory computer readable medium, the medium including instructions for causing the processor to detect the simultaneous presence of a target polynucleotide in both pores from one or more sensors. In some embodiments, the instructions cause the processor to scan one or more features of the target polynucleotide. In some embodiments, the instructions cause the processor to count a first set of features in a first cycle in a first direction and adjust one or both of the first and second voltages in response to the count to produce a first force and an opposite second force acting on the target polynucleotide. In some embodiments, the first and second forces change the direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in a second direction. In some embodiments, the process is repeated to detect the second set of features in a second cycle. In some embodiments, the process detects a third and fourth set of features in a second cycle. In some embodiments, these steps are repeated until the polynucleotides exit the dual pore device.

[0401] In some embodiments, the computer readable medium further comprises instructions that cause the processor to construct a local map of regions of the polynucleotide that contain one or more features based on the scan. In some embodiments, the computer readable medium further comprises instructions that cause the processor to construct a consensus map of regions of the polynucleotide that contain one or more features based on the scan. In some embodiments, the construction comprises a machine learning algorithm trained to detect the one or more features based on the training data and probabilistic models, as described in more detail below.

[0402] Aspects of the present disclosure include a device that includes a controller. In some embodiments, the controller is a field programmable gate array (FPGA). In some embodiments, the controller is configured to control the number of features to scan. In some embodiments, the controller is configured to control the number of features to rescan. In some embodiments, the controller is configured to control the movement of the target polynucleotide. In some embodiments, the controller is configured to control the direction of the target polynucleotide. In some embodiments, the controller determines which of one or more features to perform an additional scan. In some embodiments, the controller determines when one or more features already detected move away. In some embodiments, the controller determines when to scan an area of ​​the polynucleotide that has not yet been scanned. In some embodiments, the FPGA executes control logic to change a) the number of features to scan, b) the number of features to rescan, c) the movement or direction of the target polynucleotide, d) the direction of the target polynucleotide, or e) a combination thereof.

[0403] In some embodiments, the processor and computer readable medium containing instructions cause the processor to perform functions directed by the controller (e.g., number of features to scan, number of features to rescan, movement of the target polynucleotide, orientation of the target polynucleotide, and / or combinations thereof). In some embodiments, the processor is a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0404] In some embodiments, the computer readable medium includes a controller, a processor, and instructions to the processor to change the orientation of the target polynucleotide when the first set of probes is detected. In some embodiments, the first voltage and the second voltage are adjusted in real time, the adjustment being performed by an active feedback controller using hardware and software. In some embodiments, the controller is configured to control the first and second voltages based on feedback of measurements of the first or second or both ionic currents.

[0405] Embodiments of the device and system include a processor having a first operational mode for measuring and evaluating a set of metrics derived from the received electrical signals related to one or more features of the molecule, a second operational mode for generating a determination of the one or more features upon processing the values ​​of the set of metrics, and a third operational mode for performing one or more operations to continue scanning the same region of the molecule for additional features, to continue scanning the same region of the molecule for rescanning the same probes already detected, to scan the same region with a varying number of probes, or to move to a different region of the molecule for scanning based on the determination, where the system may include structure for performing embodiments of the method as described in more detail below.

[0406] The devices and systems may also generate notifications for provision to an operator of the system. The notifications may include content describing one or more of the status of the system, the status of one or more nanopore devices interfaced with a control element of the system, the status of one or more nanopores, instructions for adjusting the operation of the system, instructions for proceeding with an experimental protocol in relation to the status of the nanopore / nanopore device, and other content. Notifications may be provided by the system in a visual format (e.g., using a display), in an audio format (e.g., using a speaker), tactilely (e.g., using a haptic device), and / or in other suitable formats.

[0407] The devices and systems also generate computer readable instructions related to the state of the nanopore / nanopore device to move between different system operation modes (e.g., to an idle mode, to an "experiment stopped" mode, to an "experiment restart" mode, to a calibration mode, to a mode involving the use of a subset of the nanopores until they have suitable quality, etc.). The computer readable instructions can be communicated to a controller of the system to move the system between operational modes.

[0408] An embodiment of a machine learning architecture associated with an embodiment of the described system and method "learns" when to move from one location to another in a target polynucleotide, when to sequentially scan one or more features, when to change the number of features to scan, and when to divert from sequentially scanning one or more features to a location that has not yet been explored / scanned, further away from one or more features already scanned. The goal of the automated operation is to generate enough useful data set to build a consensus map for each molecule (i.e., polynucleotide). For example, a machine learning architecture with control logic can result in scanning a region of a molecule for a period of time, building a local map of that region in real time, and then moving to a different location that has not yet been scanned to build a consensus map of the molecule. In one example, Bayesian Optimization is used, which is operable on hardware with limited processing power that is required to react in real time / near real time. Although Bayesian Optimization is described, other statistical and / or machine learning approaches can be used for automated exploration or exploration to generate molecular maps. In variations, such models may implement learning styles including unsupervised learning (e.g., K-means clustering), supervised learning (e.g., using regression methods, using backpropagation networks), semi-supervised learning, reinforcement learning, or any other suitable learning style.

[0409] The devices and systems may additionally or alternatively use regression algorithms (e.g., least squares, logistic, stepwise, multivariate adaptive, etc.), instance-based methods (e.g., K-nearest neighbors, learning vector quantization, self-organizing maps, etc.), regularization methods (e.g., ridge regression, lasso regression, least absolute shrinkage and selection operator, elastic net, etc.), decision tree learning methods, kernel methods (e.g., support vector machines, radial basis functions, linear discriminant analysis, etc.), clustering methods (e.g., K-means, expectation maximization, etc.), association rule learning algorithms (e.g., Eclat algorithm, etc.), neural networks, deep learning algorithms, dimensionality reduction methods (e.g., principal component analysis, partial least squares regression, etc.), ensemble methods (e.g., boosting, bootstrap aggregation, etc.), and / or other methods to learn the ensemble. Any one or more of the following algorithms may be implemented: linear aggregation, AdaBoost, stacked generalization, gradient boosting machine methods, random forests, etc., and any suitable form of algorithm.

[0410] The application of such algorithms for automated exploration and probing for molecular map generation is described in more detail below.

[0411] In some aspects, devices and systems of the disclosure include a non-transitory computer readable medium comprising instructions to cause a processor to: i) determine from the sensor that a target polynucleotide is simultaneously present in both pores; ii) scan one or more features of the target polynucleotide; iii) count a first set of features in a first cycle in a first direction and, in response to the count, adjust one or both of the first and second voltages to produce a first force and an opposite second force acting on the target polynucleotide, where the first and second forces change the direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second direction; and repeat steps i) through iii) to detect a third and fourth set of features in a second cycle.

[0412] In some aspects, the devices and systems of the present disclosure include a controller, hi some embodiments, the controller is a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0413] Aspects of the disclosure include devices for performing the functions of the methods described herein. The disclosure includes a device for mapping one or more features of a polynucleotide sequence of a target polynucleotide through a first and a second pore, the device comprising: (i) electrodes configured and connected to provide a first voltage at the first pore of the device and a second voltage at the second pore of the device; (ii) a first pore; (iii) a second pore, wherein the first and second pores are configured to allow the target polynucleotide to translocate simultaneously across both pores in a first direction or a second direction, and in a controlled manner; (iv) a first set of features from the target polynucleotide during translocation of the target polynucleotide through the first pore and the second pore in a first direction in a first cycle, and a second set of features from the target polynucleotide during translocation of the target polynucleotide through the second pore and the first pore in a second direction in a first cycle. The method includes: (a) determining from the one or more sensors the simultaneous presence of a target polynucleotide in both pores; (b) scanning one or more features of the target polynucleotide; (c) counting a first set of features in a first cycle in a first direction and adjusting one or both of the first and second voltages in response to the counts to produce a first force and an oppositely oriented second force acting on the target polynucleotide, wherein the first and second forces change the direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second direction; and (d) repeating steps a) through c) and detecting a third and fourth set of features in a second cycle.

[0414] In some cases, the instructions cause the processor to further repeat c) until the target polynucleotide exits the device. In some cases, the first pore and the second pore are separated from each other by about 10 nm to about 2 μm.

[0415] In some cases, the diameter of the pores is in the range of about 2 nm to about 50 nm. In some cases, the diameter of the pores is about 20 nm. In some cases, the diameter of the first and / or second pores is in the range of about 2 nm to about 50 nm. In some cases, the diameter of the first and / or second pores is in the range of about 2 nm to about 8 nm. In some cases, the diameter of the first and / or second pores is in the range of about 10 nm to about 20 nm. In some cases, the diameter of the pores is in the range of about 20 nm to about 30 nm. In some cases, the diameter of the first and / or second pores is in the range of about 30 nm to about 40 nm. In some cases, the diameter of the first and / or second pores is in the range of about 40 nm to about 50 nm. In some cases, the diameter of the first and / or second pore is about 2 nm, about 4 nm, about 6 nm, about 8 nm, about 10 nm, about 12 nm, about 14 nm, about 16 nm, about 18 nm, about 20 nm, about 22 nm, about 24 nm, about 26 nm, about 28 nm, about 30 nm, about 32 nm, about 34 nm, about 36 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm, or about 50 nm. In some cases, the diameter of the first and / or second pore is about 19 nm. In some cases, the first pore and the second pore have the same diameter. In some cases, the diameter of the first pore and / or the second pore is about 21 nm. In some cases, the diameter of the first pore and / or the second pore is about 22 nm. In some cases, the diameter of the first pore and / or the second pore is about 23 nm. In some cases, the diameter of the first pore and / or the second pore is about 24 nm. In some cases, the diameter of the first pore and / or the second pore is about 25 nm. In some cases, the diameter of the first pore and / or the second pore is about 27 nm. In some cases, the diameter of the first pore and / or the second pore is about 29 nm. The first pore and the second pore have different diameters. In some cases, the diameter of the pore is about 20 nm.

[0416] In some cases, the first pore and the second pore are about 500 nm apart from each other. In some cases, the first pore has a depth of at least about 0.3 nm and separates the first channel and the chamber, and the second pore has a depth of at least about 0.3 nm and separates the chamber and the second channel. In some cases, the chamber is connected to a common ground associated with two voltages.

[0417] In some cases, the device further comprises a controller. In some cases, the controller is configured to scan varying numbers of features of the polynucleotide. In some cases, the controller is configured to vary the number of scans. In some cases, the controller is configured to control the position of the polynucleotide that is scanned. In some cases, the controller is configured to vary the area of ​​the polynucleotide that is scanned. In some cases, the controller is configured to control a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, or h) a combination thereof.

[0418] In some cases, the processor includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller is a microcontroller.

[0419] In some cases, the FPGA or ASIC executes control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0420] In some cases, the controller is configured to control the direction of movement of the polynucleotide. In some cases, the device further includes instructions to cause the processor to construct a local map of the region of the polynucleotide containing one or more features based on the scan. In some cases, the device further includes instructions to cause the processor to construct a consensus map of the region of the polynucleotide containing one or more features based on the scan. In some cases, the constructing includes a machine learning algorithm trained to detect the one or more features based on training data and a probabilistic model. In some cases, the device further includes instructions to cause the processor to calculate a velocity of the feature of the target polynucleotide from the time difference between detection of the feature at the first pore and detection of the feature at the second pore and the known distance between pore 1 and pore 2.

[0421] In some cases, the device further comprises instructions to cause the processor to calculate the distance between features from the velocities of the features of the target polynucleotide, i.e., from the time between the features detected in the current signal from the first pore, the second pore, or both. In some cases, the device further comprises instructions to cause the processor to calculate the velocities of the features of the target polynucleotide for every scan, and to calculate statistics of the velocities of the features using the distribution of velocities. In some cases, the device further comprises instructions to cause the processor to combine all the velocities of the features and calculate the time history of the velocity of the polynucleotide for a given scan and a given scanning direction.

[0422] In some cases, the device further includes instructions to cause the processor to perform a frequency sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the device further includes instructions to cause the processor to perform an amplitude sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the device further includes instructions to cause the processor to adjust the velocity of the polynucleotide. In some cases, the velocity ranges from 1 base pair / millisecond to 10 base pairs / millisecond.

[0423] In some cases, the device further includes instructions to cause the processor to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple velocities. In some cases, performing multiple scans of the polynucleotide at the multiple velocities improves accuracy of detection of one or more features. In some cases, the device further includes instructions to cause the processor to perform multiple scans of the polynucleotide at multiple velocities. In some cases, the device further includes instructions to cause the processor to control a velocity range of the polynucleotide in a first direction, a second direction, or both. In some cases, the device further includes instructions to cause the processor to control a voltage range of the first and second pores as the polynucleotide moves through the first and second pores in the first direction, the second direction, or both. In some cases, the device further includes instructions to determine an optimal velocity range of the polynucleotide in the first direction, the second direction, or both that reduces the effects of Brownian motion of the polynucleotide.

[0424] In some cases, controlling the speed range of the polynucleotide includes determining an optimal speed range of the polynucleotide for sequencing.

[0425] In some cases, the target polynucleotide is substantially linearized. In some cases, the target polynucleotide is substantially linearized by the effect of adjustment to the first voltage, the second voltage, or both.

[0426] Aspects of the present disclosure include a system for carrying out the methods described herein, comprising: a) a dual pore, dual amplification device for mapping one or more features of a polynucleotide sequence of a target polynucleotide through a first and a second pore, the device comprising: (i) electrodes connected to a power source configured to provide a first voltage at the first pore of the device and a second voltage at the second pore of the device, (ii) a first pore, (iii) a second pore, wherein the first and second pores are configured to allow the target polynucleotide to translocate simultaneously across both pores in a first direction or a second direction, and in a controlled manner, and (iv) identifying a first set of features from the target polynucleotide during translocation of the target polynucleotide through the first pore and the second pore in a first direction in a first cycle, and a second set of features from the target polynucleotide during translocation of the target polynucleotide through the second pore and the first pore in a second direction in a first cycle. a device including one or more sensors enabling the detection of a target polynucleotide in both pores; c) a processor; and d) a non-transitory computer readable medium including instructions that cause the processor to: i) determine from the sensors the simultaneous presence of a target polynucleotide in both pores; ii) scan one or more features of the target polynucleotide; iii) count a first set of features in a first cycle in a first direction and adjust one or both of the first and second voltages in response to the counts to produce a first force and an oppositely directed second force acting on the target polynucleotide, wherein the first and second forces change the direction and rate of movement of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second direction; and iv) repeat steps i) through iii) and detect a third and fourth set of features in a second cycle.

[0427] In some cases, the device includes a controller. In some cases, the controller is configured to vary the number of features of the polynucleotide to scan. In some cases, the controller is configured to vary the number of scans. In some embodiments, the controller is configured to control the position of the molecule being scanned. In some cases, the controller is configured to control a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0428] In some cases, the processor includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller is a microcontroller. In some cases, the FPGA or ASIC executes control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltage of the first and second pores, or i) a combination thereof.

[0429] In some cases, the controller is configured to change the orientation of the molecule being scanned.

[0430] In some cases, the system further comprises instructions that cause the processor to construct a local map of regions of the polynucleotide that contain one or more features based on the scan. In some embodiments, the system further comprises instructions that cause the processor to construct a consensus map of regions of the polynucleotide that contain one or more features based on the scan. In some embodiments, the construction comprises a machine learning algorithm trained to detect the one or more features based on the training data and probabilistic models.

[0431] In some cases, the system further includes instructions to cause the processor to calculate a velocity of the target polynucleotide feature from the time difference between detection of the feature at the first pore and detection of the feature at the second pore and the known distance between pore 1 and pore 2. In some cases, the system further includes instructions to cause the processor to calculate the distance between the features from the velocity of the target polynucleotide feature, i.e., from the time between the features detected in the current signal from the first pore, the second pore, or both. In some cases, the system further includes instructions to cause the processor to calculate the velocity of the target polynucleotide feature for every scan and to calculate statistics of the feature velocities using the distribution of velocities. In some cases, the system further includes instructions to cause the processor to perform a frequency sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the system further includes instructions to cause the processor to perform an amplitude sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the system further includes instructions to cause the processor to adjust the velocity of the polynucleotide.

[0432] In some cases, the rate ranges from 1 base pair / millisecond to 10 base pairs / millisecond.

[0433] In some cases, the system further includes instructions to cause the processor to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple speeds, in some cases, performing multiple scans of the polynucleotide at multiple speeds improves accuracy of detection of one or more features.

[0434] In some cases, the system further includes instructions to cause the processor to perform multiple scans of the polynucleotide at multiple velocities. In some cases, the system further includes instructions to cause the processor to control a range of velocities of the polynucleotide in a first direction, a second direction, or both. In some cases, the system further includes instructions to cause the processor to control a range of voltages of the first and second pores as the polynucleotide moves through the first and second pores in the first direction, the second direction, or both.

[0435] In some cases, the system further includes instructions to determine an optimal velocity range for the polynucleotide in a first direction, a second direction, or both, that reduces the effects of Brownian motion of the polynucleotide. In some cases, adjusting the voltage to perform multiple scans at multiple different velocities improves the comprehensiveness of the data of which features are mapped. For example, at higher velocities (i.e., when the voltage difference is larger), the molecules (e.g., polynucleotides, payload molecules, etc.) are more likely to be deterministic and the molecules are less affected by Brownian motion (e.g., Brownian motion will be less likely to "pollute" the scanning data). In some cases, the system determines an optimal velocity at which one or more features can be detected before the molecule escapes (escapes) the device or reverses direction. In some cases, the system further includes instructions to cause the processor to determine a maximum velocity at which Brownian motion has the least effect on the molecule (e.g., a maximum velocity at which Brownian motion is reduced). In some cases, one or more features are charged such that they perturb the force and therefore the movement of the polynucleotide as it passes through the pore.

[0436] In some cases, controlling the speed range of the polynucleotide includes determining an optimal speed range of the polynucleotide for sequencing.

[0437] In some cases, the system further includes instructions that cause the processor to combine all of the velocities of the features and calculate a time history of the velocity of the polynucleotide for a given scan and a given scanning direction.

[0438] In some cases, the target polynucleotide is substantially linearized. In some cases, the target polynucleotide is substantially linearized by the effect of an adjustment to the first voltage, the second voltage, or both.

[0439] Aspects of the present disclosure include a dual pore, dual amplification device for sequencing a polynucleotide sequence of a target polynucleotide through a first and a second pore, the device comprising: (i) electrodes configured and connected to provide a first voltage at the first pore of the device and to provide a second voltage at the second pore of the device; (ii) the first pore; and (iii) a second pore, wherein the first and second pores are configured to allow the target polynucleotide to move simultaneously across both pores in a first direction or a second direction, and in a controlled manner.(iv) one or more sensors that enable identification of a first set of primers from the target polynucleotide during movement of the target polynucleotide through the first pore and the second pore in a first direction in a first cycle, and a second set of primers from the target polynucleotide during movement of the target polynucleotide through the second pore and the first pore in a second direction in a first cycle; (v) a processor; and (vi) a non-transitory computer readable medium, comprising: a) determining from the one or more sensors the simultaneous presence of the target polynucleotide in both pores; b) scanning the one or more primers hybridized to the target polynucleotide; c) detecting the first set of primers when the target polynucleotide is in both pores in the first direction in the first cycle; d) detecting the one or more primers hybridized to the target polynucleotide. e) adjusting the first voltage, the second voltage, or both for the first and second pores when the first set of primers is detected in the first cycle in the first orientation to change the orientation of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second orientation; f) detecting the presence of the second set of primers in the first cycle when the target polynucleotide is in both pores simultaneously in the second orientation; g) identifying each nucleotide of the polynucleotide that passes through one of the pores by measuring an ionic current across the pore as the nucleotide passes through that pore; and h) repeating steps a)-g) and detecting a third and fourth set of primers in the second cycle.

[0440] Aspects of the present disclosure include a system, the system comprising: a) a dual pore, dual amplification device for mapping one or more features of a polynucleotide sequence of a target polynucleotide through a first and a second pore, the device comprising: (i) electrodes configured and connected to provide a first voltage at the first pore of the device and to provide a second voltage at the second pore of the device; (ii) the first pore; and (iii) a second pore, wherein the first and second pores are configured to allow the target polynucleotide to move simultaneously across both pores in a first direction or a second direction, and in a controlled manner.(iv) one or more sensors that enable identification of a first set of primers from the target polynucleotide during movement of the target polynucleotide through the first pore and the second pore in a first direction in a first cycle, and a second set of primers from the target polynucleotide during movement of the target polynucleotide through the second pore and the first pore in a second direction in a first cycle; c) a processor; and d) a non-transitory computer readable medium, comprising: i) determining from the one or more sensors the simultaneous presence of the target polynucleotide in both pores; ii) scanning the one or more primers hybridized to the target polynucleotide; iii) detecting the first set of primers when the target polynucleotide is in both pores in the first direction in the first cycle; iv) detecting the one or more primers hybridized to the target polynucleotide when the target polynucleotide is in both pores in the first direction; v) adjusting a first voltage, a second voltage, or both for the first and second pores when the first set of primers is detected in the first cycle in the first orientation to change the orientation of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in the second orientation; vi) detecting the presence of the second set of primers in the first cycle when the target polynucleotide is in both pores simultaneously in the second orientation; vii) identifying each nucleotide of the polynucleotide that passes through one of the pores by measuring an ionic current across the pore as the nucleotide passes through that pore; and viii) repeating steps i)-vii) and detecting a third and fourth set of primers in the second cycle.

[0441] Generate a map of one or more features in a molecule In the context of adaptive theory, the goal is to identify one or more features of a target polynucleotide and create a local and / or global consensus map of one or more features of the target polynucleotide. The adaptive controller can vary the number of features to look for which molecular features and positions to rescan (e.g., bidirectional scanning) and / or when to move to different positions not yet explored, in near real-time. The adaptive controller can create real-time local maps of one or more features and generate a data set for creating a consensus map of the entire molecule. When implementing machine learning algorithms, the model can be trained with training data related to the following criteria: IRMS, baseline drift, baseline up-down motion, LFNP, and other criteria. The model can then be used to determine a combination of criteria that indicate the probability of when to change the direction of molecular transition in response to detecting the passage of a molecular feature and when to automatically take the best intervening action for different types of characteristic deviations. Using active logic based on field programmable gate arrays (FPGAs) or application specific integrated circuits (ASICs), the direction of molecular translocation can be changed in response to detecting the passage of molecular features. This allows for back and forth rescanning of local DNA regions, which can be used to increase accuracy through averaging.

[0442] The system uses machine learning techniques to generate prior distributions of "events" of certain types for experiments / reagents, generate features for all types of molecular detections (e.g., in relation to the value of the evaluation criteria), and generate features for other distributions (e.g., distributions of properties of reagents over time). Training data can be derived from real-time feedback of event signatures for each molecular motif, model accuracy, false positive / false negative molecule calls, fraction of CpGs correctly called per molecule, predictions of CpG distance / mapping, performance, predictions of fraction of 5hmC vs. 5mC, predictions of genomic distance, distance between molecular motifs, and density of molecular motifs. Models can be developed in a supervised learning approach that uses algorithms to process the distribution of tagging or other aspects of the training data and put these into types of excursions. The system can also collect data on which interventions were appropriate for which characteristic distributions / events.

[0443] When a molecular motif (e.g., one or more features of a polynucleotide and / or one or more features within a polynucleotide) event is detected using the trained model, the system performs one or more correction actions, including one or more of: reversing the polarity of the voltage, applying an electrical stimulus at a specific voltage (and / or by a specific number or percentage of zapping instances), changing the voltage to change the orientation of the molecule for rescanning, changing the position of the molecule where probing / interrogation is performed, changing the number of molecular motifs for probing / interrogation, or performing no action (e.g., by notifying an operating entity).

[0444] Consensus Mapping Using Deep Learning Another approach is to use a signal processing convolutional neural network to electronically identify distinct molecular motifs and the locations and spacing between them, and feed this into a deep Q-learning algorithm to develop adaptive logic and continuous improvements in the consensus mapping. This technique improves future decisions made by the system for automated molecular exploration and exploration to generate maps.

[0445] Further considerations The above description of embodiments of the invention is provided for purposes of illustration and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Those skilled in the relevant art will recognize that many modifications and variations are possible in light of the above disclosure.

[0446] Some portions of this description describe embodiments of the invention in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are commonly used by those skilled in the art of data processing to effectively convey the substance of their work to others skilled in the art. These operations, while described functionally, computationally, or logically, will be understood to be implemented by computer programs or equivalent electrical circuits, microcode, or the like. Further, without loss of generality, it has proven often convenient to refer to these arrangements of operations as modules. The described operations and their associated modules may be embodied in software, firmware, hardware, or any combination thereof.

[0447] The steps, operations, or processes described herein may be implemented or performed by one or more hardware or software modules, alone or in combination with other devices. In one embodiment, the software modules are implemented by a computer program product that includes a computer-readable non-transitory medium containing computer program code that is executed by a computer processor to perform any or all of the described steps, operations, or processes.

[0448] Embodiments of the invention also relate to products resulting from the computing processes described herein. Such products include information resulting from the computing processes, which information is stored on a tangible, non-transitory computer-readable medium, and may include any of the embodiments of a computer program product or combination of other data described herein.

[0449] Methods - Extended recapture of polynucleotides in nanopore devices The present disclosure provides methods for recapturing a polynucleotide in a nanopore device.The systems and devices of the present disclosure perform methods for recapturing a target polynucleotide.

[0450] Aspects of the present disclosure include a method for recapturing a polynucleotide in a nanopore, the method comprising providing a nanopore device for controlling movement of a polynucleotide through one or more pores. In some cases, the device comprises at least a first pore disposed between a chamber and a first fluid volume, the first pore being in fluid communication with the chamber and the first liquid volume, the first fluid volume being a geometrically constrained enclosure having an opening on an opposite side of the first pore. In some cases, the device comprises at least one electrode disposed within the first fluid volume, the one electrode configured to provide a first voltage at the first pore. In some cases, the device comprises one or more sensors (e.g., amplifiers) configured for voltage control and current measurement at the first pore. In some cases, the method comprises introducing a polynucleotide into a chamber of the device. In some cases, the method comprises introducing a polynucleotide into a geometrically constrained fluid volume of the device. In some cases, the method includes applying a first voltage across the first pore to displace the polynucleotide from the chamber in a first direction, capture the polynucleotide in the first pore, and allow the polynucleotide to pass through the first pore into the first fluid volume. In some cases, the method includes detecting a first ionic current in the first pore as the polynucleotide passes through the first pore in the first direction. In some cases, the method includes adjusting the voltage to zero for a predetermined time while the polynucleotide is contained within the first fluid volume. In some cases, the method includes applying a second voltage and reversing the direction of the polynucleotide located in the first fluid volume, where the second voltage displaces at least a portion of the polynucleotide from the first fluid volume in the second direction and recaptures a portion of the polynucleotide in the first pore such that the polynucleotide is maintained within the first pore. In some cases, the method includes detecting a second ionic current in the first pore when the polynucleotide is recaptured in the first pore.In some cases, the device is configured to pass the polynucleotide from the chamber through the first pore and from the first fluidic channel through the first pore. In some cases, the method further includes repeating the steps described herein until the polynucleotide exits the device.

[0451] In some cases, the nanopore device further comprises a second pore, a third pore, a fourth pore, a fifth pore, a sixth pore, a seventh pore, an eighth pore, a ninth pore, or a tenth pore. In some cases, the nanopore device further comprises 2 or more pores, 3 or more pores, 4 or more pores, 5 or more pores, 6 or more pores, 7 or more pores, 8 or more pores, 9 or more pores, or 10 or more pores.

[0452] In some cases, the nanopore device includes a geometrically constrained first fluid volume and a geometrically constrained second fluid volume. In some cases, the first and second geometrically constrained volumes are first and second fluid channels. In some cases, the nanopore device includes 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more geometrically constrained volumes. In some embodiments, the geometric constrain of the first fluid channel is increased while the first voltage applied at the first pore is maintained. In some embodiments, the geometric constrain of the first fluid channel is increased while the first voltage applied at the first pore is maintained with respect to the geometric constrain of the cavity. In some embodiments, a first voltage applied at a first pore to capture and translocate polynucleotides passing through the first pore (e.g., from a chamber of the device through the first pore) is maintained for a period in the range of 0-1000 milliseconds, 1-20 milliseconds, 20-60 milliseconds, 60-120 milliseconds, 120-150 milliseconds, 150-300 milliseconds, 300-500 milliseconds, 500-1000 milliseconds, 1-20 seconds, 20-60 seconds, 60-120 seconds, 120-150 seconds, 150-300 seconds, 300-500 seconds, or 500-1000 seconds. In some embodiments, the first voltage applied at the first pore that captures and transfers the polynucleotide through the first pore (e.g., from the chamber of the device through the first pore) is maintained for 20 milliseconds or more, 60 milliseconds or more, 120 milliseconds or more, 150 milliseconds or more, 300 milliseconds or more, 500 milliseconds or more, 1000 milliseconds or more, 20 seconds or more, 60 seconds or more, 120 seconds or more, 150 seconds or more, 300 seconds or more, 500 seconds or more, or 1000 seconds or more. In some embodiments, the first voltage is maintained for a period described herein after detecting capture and transfer of the polynucleotide through the first pore. In some embodiments, the first voltage is maintained for a period described herein before setting the second voltage at the first pore to zero mV.

[0453] In some cases, the first fluid volume is a first fluid channel. In some cases, the second fluid volume is a second fluid channel. In some cases, the first and / or second fluid channels can be circular, square, rectangular, hexagonal, triangular, oval, polygonal, V-shaped, U-shaped, or any other suitable shape. In some cases, the first and / or second fluid channels each have a V-shape, each with an opening at both ends of the V-shape, the V-shapes of the first and second fluid channels arranged on the chip opposite each other at a predetermined point of each V-shape are adjacent to each other, the first nanopore is located at a predetermined point of the V-shape of the first fluid channel, and the second nanopore is located at a predetermined point of the V-shape of the second fluid channel. In some embodiments, each of the fluid channels is of a different shape. The fluid channels are not limited to the shapes and / or sizes described herein, but can be any shape and / or size required by the conditions specified for their intended use. In some cases, the first fluid channel and / or the second fluid channel include a geometrically constrained volume.

[0454] In some cases, the first fluid channel and / or the second fluid channel have a length in the range of about 0.05 mm to about 5 mm. In some cases, the first fluid channel and / or the second fluid channel have a length in the range of about 0.05 mm to about 4 mm. In some cases, the first fluid channel and / or the second fluid channel have a length in the range of about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0455] In some cases, the first fluid channel and / or the second fluid channel have a width in the range of 50-500 μm. In some cases, the first fluid channel and / or the second fluid channel have a width in the range of 50-100 μm, 100-150 μm, 150-200 μm, 200-250 μm, 250-300 μm, 300-350 μm, 350-400 μm, 400-450 μm, or 450-500 μm. In some cases, the first fluid channel and / or the second fluid channel have a width of about 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, 300 μm or more, 350 μm or more, 400 μm or more, 450 μm or more, or 500 μm or more.

[0456] In some cases, the first fluidic channel and / or the second fluidic channel have a depth in the range of 0.5 μm to about 2 μm. In some cases, the first fluidic channel and / or the second fluidic channel have a depth in the range of 0.5-1 μm, 1-1.5 μm, or 1.5-2 μm. In some cases, the first fluidic channel and / or the second fluidic channel have a depth in the range of about 0.5 μm or more, 1 μm or more, 1.5 μm or more, 2 μm or more, 2.5 μm or more, or 3 μm or more.

[0457] In some cases, the first fluid volume and the second fluid volume each include an opening on the opposite side of the first pore and / or the second pore. In some cases, the second pore is in fluid communication with the second fluid volume, and the second fluid volume is a geometrically constrained housing with an opening on the opposite side of the second pore. In some cases, the first fluid volume and / or the second fluid volume includes two openings on the opposite side of the first pore and / or the second pore. In some cases, the two openings of the first fluid channel are on the same plane and parallel to each other. In some cases, the two openings of the second fluid channel are on the same plane and parallel to each other. In some cases, one or more openings of the first fluid volume and / or the second fluid volume are configured to allow polynucleotides to exit the device through the one or more openings. In some cases, the first fluid volume is configured to allow polynucleotides to exit the device through the openings. In some cases, the second fluid volume is configured to allow polynucleotides to exit the device through an opening.

[0458] In some cases, the polynucleotide travels from the chamber through at least the first pore to the first fluid volume in a range of 30 ms to 500 ms or more. In some cases, the polynucleotide travels from the first fluid volume through at least the first pore to the chamber in a range of 30 ms to 500 ms or more. In some cases, the first end of the polynucleotide is positioned away from at least the first pore by a distance ranging from 5 microns to 5 millimeters or more. In some cases, the second end of the polynucleotide is positioned away from at least the first pore by a distance ranging from 5 microns to 5 millimeters or more.

[0459] In some cases, the polynucleotide is retained in the first fluid volume when the first voltage is adjusted to zero for a range of 0 to 1000 milliseconds or 0 to 1000 seconds. In some cases, adjusting the first voltage to an intermediate voltage of 0 mV results in relaxation of the polynucleotide to its equilibrium conformation.

[0460] In some cases, the method prevents the polynucleotide from exiting the device for a period ranging from 5 ms to 5 minutes. In some cases, the method prevents the polynucleotide from exiting a chamber of the device for a period ranging from 5 ms to 5 minutes. In some cases, the method prevents the polynucleotide from exiting a first fluid volume and / or a second fluid volume of the device for a period ranging from 5 ms to 5 minutes.

[0461] In some cases, the chamber is disposed over the first pore. In some cases, the first pore is disposed between the chamber and the first fluid volume. In some cases, the second pore is disposed between the chamber and the second fluid volume. In some cases, the second pore is connected to the chamber and the second fluid volume. In some cases, the chamber is disposed over the first and second pores. In some cases, the chamber is connected to a common ground for the first voltage.

[0462] In some cases, the first pore has a diameter in the range of about 2 nm to about 50 nm. In some cases, the first pore has a diameter in the range of about 15 nm to about 30 nm. In some cases, the second pore has a diameter in the range of about 2 nm to about 50 nm. In some cases, the second pore has a diameter in the range of about 15 nm to about 30 nm.

[0463] In some cases, a first voltage is applied between the first fluid volume and the chamber. In some cases, the nanopore device includes at least one electrode disposed in the second fluid volume, the at least one electrode configured to apply a third voltage at the second pore.

[0464] In some cases, the nanopore device includes dual-amplifier electronics configured to control the voltage and measure the current at the first pore and the second pore.

[0465] In some cases, the method further includes, after detecting the second ionic current, adjusting a first voltage at the first pore and setting a third voltage at the second pore such that at least a portion of the polynucleotide moves through the first pore and the second pore in a third direction (wherein the third direction is from the first pore to the second pore).

[0466] In some cases, the first voltage is 0 mV. In some cases, the first voltage is in the range of 0 to 1000 mV. In some cases, the first voltage, the second voltage, the third voltage range, and / or the fourth voltage are each independently in the range of 0 mV to 1000 mV. In some cases, the adjustments of the first voltage, the second voltage, the third voltage, and / or the fourth voltage are each independently in the range of 0 to 1000 mV.

[0467] In some cases, the polynucleotide is substantially linearized. In some cases, the polynucleotide is substantially linearized by the effect of adjusting the first voltage, the second voltage, the third voltage, or a combination thereof. In some cases, the polynucleotide is substantially linearized.

[0468] In some cases, the method further comprises adjusting a first voltage to the first pore, adjusting a third voltage to the second pore, or both, to change the orientation of the polypeptide such that at least a portion of the polynucleotide moves in a fourth direction (the fourth direction is from the second pore to the first pore) from the second pore through the first pore. In some cases, adjusting the first voltage to the first pore, adjusting the third voltage to the second pore, or both, to at least a portion of the polynucleotide moves in the third direction and / or the fourth direction is repeated until the polynucleotide exits the device. In some cases, the third voltage is applied between the chamber and the second fluid volume of the device. In some cases, the third voltage is applied for a period ranging from 5 ms to 5 minutes. In some cases, the method further comprises detecting a first set of characteristics of the polynucleotide when the polynucleotide is in both pores in the third direction. In some cases, the method further comprises detecting a second set of characteristics of the polynucleotide when the polynucleotide is in both pores simultaneously in a fourth orientation.

[0469] In some cases, the method includes adjusting the first voltage such that the polynucleotide translocates through the first pore in a range of 5 ms to 5 minutes. In some cases, the polynucleotide passes through the first pore, the chamber, and the second pore. In some cases, the polynucleotide partially passes through the first pore, the chamber, and the second pore.

[0470] In some cases, the method further comprises detecting a third ionic current at the first pore and a fourth ionic current at the second pore when the polynucleotide is in both pores in a third orientation, In some cases, the method further comprises detecting a fifth ionic current at the first pore and a sixth ionic current at the second pore when the polynucleotide is in both pores in a fourth orientation.

[0471] In some cases, the fifth voltage creates a voltage gradient across the first pore and along the length of the first fluid volume. In some cases, the third voltage creates a voltage gradient across the second pore and along the length of the second fluid volume.

[0472] In some cases, the resistance of the first fluid channel is inversely proportional to the width of the first fluid channel. In some cases, the resistance of the second fluid channel is inversely proportional to the width of the second fluid channel. In some cases, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel. In some cases, the resistance of the first fluid channel and / or the second fluid channel is proportional to the volume of the first fluid channel and / or the second fluid channel. In some cases, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of the first fluid channel and / or the second fluid channel. In some cases, the resistance of the first fluid channel and / or the second fluid channel is proportional to the radius of the cross-section of the first fluid channel and / or the second fluid channel.

[0473] In some cases, the polynucleotide is entropically trapped within the first fluid volume and / or the second fluid volume. In some cases, the polynucleotide is entropically trapped within the first fluid volume and / or the second fluid volume.

[0474] The method further includes controlling, by the controller, when the polynucleotide requires rescanning of one or more features of the polynucleotide at a second or third time. In some cases, the controller determines which of the one or more features of the polynucleotide to perform additional recapture of the one or more features in the first orientation and / or the second orientation.

[0475] In some cases, the method further comprises moving away from one or more features of the already recaptured polynucleotide.

[0476] In some cases, the first voltage, the second voltage, and the third voltage are in the range of 0 to 1000 mV. In some cases, the first voltage, the second voltage, and the third voltage are in the range of 0 to 100 mV. In some cases, the first voltage, the second voltage, and the third voltage are in the range of 100 to 200 mV. In some cases, the first voltage, the second voltage, and the third voltage are in the range of 200 to 300 mV. In some cases, the first voltage, the second voltage, and the third voltage are in the range of 300 to 400 mV. In some cases, the first voltage, the second voltage, and the third voltage are in the range of 400 to 500 mV. In some cases, the first voltage to the first pore is lower than the second voltage. In some cases, the first voltage to the first pore is higher than the second voltage. In some cases, the first voltage to the first pore and the second voltage to the second pore are the same. In some cases, the first voltage to the first pore and the second voltage to the second pore are the same in a first direction. In some cases, the first voltage to the first pore is lower than the second voltage to the first pore in a second direction. In some cases, the first voltage to the first pore is lower than the third voltage to the second pore in a third direction. In some cases, the first voltage to the first pore is higher than the third voltage to the second pore in a fourth direction.

[0477] In some cases, the method further includes controlling a direction of the polynucleotide through the first and / or second pores by a controller, a processor, and a non-transitory computer readable medium including instructions to the processor to change the direction of the polynucleotide. In some cases, the processor includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller is a microcontroller.

[0478] In some cases, the recapture provides for detection of polynucleotides including polynucleotide sequences having lengths ranging from 5 base pairs to about 3,000,000 base pairs.

[0479] Method - Flossing and recapture for one or more features The present disclosure provides a method for mapping one or more features of a polynucleotide sequence of a target polynucleotide bound to a plurality of probes through a pore.The present disclosure further provides a method for differential detection of 5mC and 5hmC regions in a mixed sample containing one or more polynucleotide sequences that migrate separately through first and second pores of a nanopore device.

[0480] The present disclosure provides an automated method for mapping one or more features of a target polynucleotide. The present disclosure also provides an automated method for sequencing a polynucleotide sequence. The present disclosure also provides a method for differential detection of methylated and unmethylated regions in a polynucleotide sequence. The present disclosure also provides devices and systems for carrying out the methods of the present disclosure.

[0481] Aspects of the invention include a method for mapping one or more characteristics of a target polynucleotide, the method comprising: a) providing a dual pore, dual amplification device for controlling movement of a target polynucleotide simultaneously through a first and second pore, the device comprising: (i) a first pore, (ii) a second pore, (iii) a power source configured to provide a first voltage at the first pore and a second voltage at the second pore, each voltage being independently controllable; and (v) dual amplification electronics configured to independently control the voltage and measure the current at each pore, the first and second pores being configured to allow the target polynucleotide to simultaneously move across both pores in a controlled manner in a first direction or a second direction; b) introducing a target polynucleotide into the device; c) detecting that at least a portion of the target polynucleotide is amplified in a first direction (wherein the first direction is from the first pore to the second pore) by a first amplification device; setting a first initial voltage at the first pore and a second initial voltage at the second pore such that the target polynucleotide migrates through the first pore and the second pore; d) scanning at least one feature of the target polynucleotide; e) detecting a first set of features in a first cycle when the target polynucleotide is in both pores in the first orientation; f) adjusting the first voltage, the second voltage, or both for the first and second pores to change the orientation of the target polynucleotide when the first set of features is detected in the first cycle in the first orientation such that at least a portion of the target polynucleotide migrates from the second pore to the first pore in a second direction (wherein the second direction is from the second pore to the first pore); g) detecting the presence of the second set of features when the target polynucleotide is in both pores simultaneously in the second orientation; and h) repeating steps c) through g) and detecting a third and fourth set of features in a second cycle.

[0482] In some cases, the first pore is connected to a first channel and chamber of the device.

[0483] In some cases, the second pore is connected to a chamber and a second channel of the device.

[0484] In some cases, a first voltage is applied between a first channel and the chamber, and a second voltage is applied between the chamber and a second channel of the device.

[0485] In some cases, detecting the characteristic of the target polynucleotide is accomplished by measuring current from the first pore, or the second pore, or both.

[0486] In some cases, the method further includes calculating, by the processor, a velocity of one or more features of the target polynucleotide from the time difference between detection of the feature at the first pore and the second pore and the known distance between the first pore and the second pore.

[0487] In some cases, the method further includes calculating, by the processor, a distance between one or more features by using the calculated velocity of the feature of the target polynucleotide from the time between one or more features detected in the current signal from the first pore, the second pore, or both.

[0488] In some cases, the method further includes calculating, by the processor, a velocity of the characteristic of the target polynucleotide for every scan.

[0489] In some cases, the method further includes calculating, by the processor, statistics of the velocities of the features using the distribution of velocities for every scan.

[0490] In some cases, the method further includes calculating, by the processor, a time history of the velocity of the polynucleotide using all of the velocities of the features in a given scan and in a given direction of the scan.

[0491] In some cases, the target polynucleotide is substantially linearized.

[0492] In some cases, the target polynucleotide is substantially linearized by the action of adjusting the first voltage, the second voltage, or both.

[0493] In some cases, the method further comprises controlling, with a controller, a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, or h) a combination thereof.

[0494] In some cases, the method further includes controlling the number of features to scan. In some cases, the controller determines which of the one or more features to perform additional scans on. In some cases, the method further includes moving away from one or more features already scanned. In some cases, the method further includes scanning an area of ​​the polynucleotide that has not yet been scanned.

[0495] In some cases, the method further includes constructing, by the processor, a consensus map for each polynucleotide. In some cases, the constructing includes a machine learning algorithm trained to detect one or more features based on training data and probabilistic models. In some cases, the method further includes constructing, by the processor, a local map for each polynucleotide in real time. In some cases, the method further includes repeating steps c) through g) until the target polynucleotide exits the pore device.

[0496] In some cases, the one or more features include: a) one or more payload molecules bound to the polynucleotide; b) one or more payload molecules hybridized to the polynucleotide; c) one or more payload molecules incorporated into the genome of the polynucleotide; d) a molecular motif of a polynucleotide sequence of the target polynucleotide; or e) a combination thereof.

[0497] In some cases, the one or more payload molecules are enzymatically incorporated into the genome of the polynucleotide. In some cases, the one or more payload molecules are chemically incorporated into the genome of the polynucleotide. In some cases, the one or more payload molecules are chemically incorporated into the genome of the polynucleotide using click chemistry. In some cases, the method further comprises determining a position of one or more features of the target polynucleotide. In some cases, the method further comprises determining a distance between each of the one or more features of the target polynucleotide. In some cases, the method further comprises determining a distance between each feature in a first set of features of the target polynucleotide. In some cases, the method further comprises determining a distance between each feature in a second set of features of the target polynucleotide.

[0498] In some cases, the one or more characteristics include the position of one or more methylation sites, one or more CpG's, or one or more methylation sites and CpG's of the polynucleotide sequence of the target polynucleotide. In some cases, the one or more characteristics include the position of one or more nucleosomes of the target polynucleotide. In some cases, the one or more characteristics include the position of one or more nucleosomes bound to the target polynucleotide from its original chromatin state without or with chemical fixation. In some cases, the one or more characteristics include the position of one or more histones contained within one or more nucleosomes of the target polynucleotide, or the modification state and position of one or more histones contained within one or more nucleosomes of the target polynucleotide.

[0499] In some cases, the modification state of the histone is contained within the tail of the histone, which includes lysine residues that can be acetylated, methylated, or coupled to ubiquitin (a large polypeptide chain), arginine residues that can be methylated, serine residues that can be phosphorylated, or other modifications to the tail contained within the histone contained within the nucleosome.

[0500] In some cases, the method further includes identifying one or more characteristics of the target polynucleotide by measuring an ionic current across one of the multiple probes as the probes pass through the pore.

[0501] In some cases, the first cycle includes one or more scans performed by the processor to detect the first set of features, in some cases the first cycle includes two or more scans, three or more scans, four or more scans, five or more scans, six or more scans, seven or more scans, eight or more scans, nine or more scans, or ten or more scans.

[0502] In some cases, the first cycle includes 2 or more scans, 4 or more scans, 6 or more scans, 8 or more scans, 10 or more scans, 12 or more scans, 14 or more scans, 16 or more scans, 18 or more scans, or 20 or more scans. In some cases, the first cycle includes 5 or more scans, 10 or more scans, 15 or more scans, 20 or more scans, 25 or more scans, 30 or more scans, 35 or more scans, 40 or more scans, 45 or more scans, or 50 or more scans. In some cases, the second cycle includes 1 or more scans performed by the processor to detect a third set of features. In some cases, the second cycle includes 2 or more scans, 3 or more scans, 4 or more scans, 5 or more scans, 6 or more scans, 7 or more scans, 8 or more scans, 9 or more scans, or 10 or more scans. In some cases, the second cycle includes 2 or more scans, 4 or more scans, 6 or more scans, 8 or more scans, 10 or more scans, 12 or more scans, 14 or more scans, 16 or more scans, 18 or more scans, or 20 or more scans. In some cases, the second cycle includes 5 or more scans, 10 or more scans, 15 or more scans, 20 or more scans, 25 or more scans, 30 or more scans, 35 or more scans, 40 or more scans, 45 or more scans, or 50 or more scans. In some cases, the first cycle and the second cycle include 50 or more scans, 100 or more scans, 150 or more scans, 200 or more scans, 250 or more scans, 300 or more scans, 350 or more scans, 400 or more scans, or 500 or more scans.

[0503] In some cases, steps c) through g) are repeated for the third, fourth, and fifth cycles, or when the polynucleotides exit the device. In some cases, the first, second, third, fourth, and fifth cycles together comprise 50 or more scans, 100 or more scans, 150 or more scans, 200 or more scans, 250 or more scans, 300 or more scans, 350 or more scans, 400 or more scans, or 500 or more scans.

[0504] In some cases, the first set of features is 1 or more features, 2 or more features, 3 or more features, 4 or more features, 5 or more features, 6 or more features, 7 or more features, 8 or more features, 9 or more features, or 10 or more features. In some cases, the second set of features is 2 features, 3 features, 4 features, or 5 features. In some cases, the second set of features is larger than the first set of features. In some cases, the sets of features across the set of scans are combined to generate a map of location and distance between features for each target polynucleotide.

[0505] In some cases, the one or more features include a DNA-binding protein.

[0506] In some cases, the method further comprises labeling the polynucleotide with a payload molecule at one or more 5-methylcytosine (5mC) regions, one or more 5-hydroxymethylcytosine (5hmC) regions, or one or more 5mC and 5hmC regions of the target polynucleotide. In some cases, the one or more features comprise a DNA-binding protein, a polypeptide, an anti-DNA antibody, streptavidin, a transcription factor, a histone, a peptide nucleic acid (PNA), a DNA-hairpin, a DNA molecule, an aptamer, a 5-methylcytosine (5mC) region, a 5-hydroxymethylcytosine (5hmC) region, a nucleotide base, two or more nucleotide bases, or a combination thereof.

[0507] In some cases, the method further comprises determining the location of one or more methylcytosines (5mC) of the target polynucleotide. In some cases, the method further comprises determining the location of one or more 5-hydroxymethylcytosines (5hmC) of the target polynucleotide. In some cases, the method further comprises determining the location of one or more methylcytosines (5mC) and one or more 5-hydroxymethylcytosines (5hmC) of the target polypeptide. In some cases, the method further comprises determining one or more CpG's of the target polynucleotide. In some cases, the polynucleotide sequence has a length ranging from 5 base pairs to about 2,000,000 base pairs. In some cases, the polynucleotide sequence has a length ranging from 100 base pairs to 500 base pairs. In some cases, the polynucleotide sequence has a length ranging from 1 megabase pair (Mb) to about 2 Mb. In some cases, the DNA molecule is labeled with DCBO. In some cases, the DNA molecule is covalently attached at the nick site of the target polynucleotide. In some cases, the polypeptide is monostreptavidin (MS) incorporated at the nick site of the polynucleotide sequence.

[0508] In some cases, the method further comprises distinguishing between 5mC and 5hmC regions of the polynucleotide. In some cases, the one or more features comprises a molecule selected from the group consisting of a nucleic acid, a TALEN, a CRISPR, a peptide nucleic acid, and a chemical compound. In some cases, the one or more features comprises a molecule selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA / RNA hybrid, a polypeptide, or any chemically derived polymer.

[0509] In some cases, the target polynucleotide is selected from the group consisting of double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and a DNA-RNA hybrid.

[0510] In some cases, the method further includes controlling a direction of the target polynucleotide through the first and second pores by a controller, a processor, and a non-transitory computer readable medium comprising instructions to the processor to change a direction of the target polynucleotide when the first set of probes is detected. In some cases, adjusting the first and second voltages occurs in real time, and the adjusting is performed by an active feedback controller using hardware and software.

[0511] In some cases, the method further includes controlling the first or second voltages by a feedback controller based on feedback of the first or second, or both, ion current measurements. In some cases, the processor includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller includes a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In some cases, the controller is a microcontroller. In some cases, the FPGA or ASIC executes control logic to vary a) the number of features to scan, b) the number of features to rescan, c) the type of features to scan or rescan, d) the number of cycles to scan or rescan, e) the movement of the target polynucleotide, f) the direction of the target polynucleotide, g) the velocity of the target polynucleotide, h) the voltages of the first and second pores, or i) a combination thereof.

[0512] In some cases, the first set of features and the second set of features are the same. In some cases, the first set of features and the second set of features are different. Non-limiting examples of when the first set of features and the second set of features are different include when one or more additional features are found in the second set of features that are not in the first set of features. In some cases, the third set of features and the fourth set of features are the same. In some cases, the third set of features and the fourth set of features are different. In some cases, the second set of features is larger than the second set of features.

[0513] In some cases, each feature is spaced apart from one another by about 100 base pairs, 300 base pairs, 500 base pairs, 1 kilobase pairs, 5 kilobase pairs, 10 kilobase pairs, 20 kilobase pairs, or a combination thereof. In some cases, the polynucleotide sequence comprises one or more nicked sites. In some cases, each feature is spaced apart from one another by about 1 base pair or more, about 2 base pairs or more, about 3 base pairs or more, about 4 base pairs or more, about 5 base pairs or more, about 6 base pairs or more, about 7 base pairs or more, about 8 kilobase pairs or more, about 9 kilobase pairs or more, about 10 base pairs or more, about 25 base pairs or more, about 50 base pairs or more, about 100 base pairs or more, about 300 base pairs or more, about 500 base pairs or more, about 1 kilobase pairs or more, about 5 kilobase pairs or more, about 10 kilobase pairs or more, about 20 kilobase pairs or more, or a combination thereof. In some cases, each feature is spaced apart from each other by about 25 base pairs or less, about 50 base pairs or less, about 100 base pairs or less, about 300 base pairs or less, about 500 base pairs or less, about 1 kilobase pairs or less, about 5 kilobase pairs or less, about 10 kilobase pairs or less, about 20 kilobase pairs or less, or a combination thereof.

[0514] In some cases, the method further comprises identifying each nucleotide of the polynucleotide that passes through one of the pores by measuring the ionic current across the pore as the nucleotide passes through that pore.

[0515] In some cases, the method further comprises sequencing the polynucleotide.

[0516] In some cases, the controller is configured to perform a control voltage frequency sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the controller is configured to perform a control voltage amplitude sweep of the polynucleotide in a first direction, a second direction, or both. In some cases, the controller is configured to adjust the velocity of the polynucleotide. In some cases, the velocity ranges from 0.1 base pairs / millisecond to 10 base pairs / millisecond. In some cases, the controller is configured to adjust the first and second voltages to perform multiple scans of the polynucleotide at multiple rates. In some cases, performing multiple scans of the polynucleotide at multiple rates improves accuracy of detection of one or more features. In some cases, the method includes performing multiple scans of the polynucleotide at multiple rates. In some cases, the controller is configured to control a range of velocities of the polynucleotide in a first direction, a second direction, or both. In some cases, the controller is configured to control a voltage range for the first and second pores as the polynucleotide moves through the first and second pores in a first direction, a second direction, or both. In some cases, the controller is configured to determine an optimal velocity range for the polynucleotide in the first direction, the second direction, or both, the optimal velocity range for the polynucleotide reducing effects of Brownian motion of the polynucleotide. In some cases, the velocity range for the polynucleotide includes determining an optimal velocity for the polynucleotide for sequencing.

[0517] Aspects of the disclosure include a method for differential detection of 5mC and 5hmC in a mixed sample containing one or more polynucleotide sequences that migrate separately through first and second pores, the method comprising: a) labeling 5mC and 5hmC regions of one or more target polynucleotide sequences of one or more target polynucleotides with binding molecules; b) contacting the one or more target polynucleotides with a payload molecule configured to bind to a binding site of the one or more target polynucleotides; c) providing a dual pore dual amplification device for controlling the movement of the target polynucleotides bound to the payload molecules through the first and second pores simultaneously, the device comprising: (i) a first channel, a chamber and a second channel; (ii) a first pore; (iii) a second pore; (iv) a power source configured to provide a first voltage at the first pore and a second voltage at the second pore, both voltages being independently regulated; and (v) dual amplification electronics configured to independently control the voltage and measure the current at each pore, the first and second pores being configured to detect when the target polynucleotide is in both pores. the device being configured to simultaneously migrate across both pores in a directional and controlled manner; d) introducing a sample containing one or more target polynucleotides into the device; e) applying a first initial voltage and a second initial voltage such that at least a portion of the one or more target polynucleotides migrate through the first pore and the second pore, wherein the first and second voltages comprise translocating the one or more target polynucleotides through the first and second pores separately for each of the labeled 5mC regions of the polynucleotide sequence and the labeled 5hmC regions of the one or more target polynucleotides; f) generating a plurality of events resulting from the translocation of the one or more target polynucleotides through the first and second pores; g) identifying an amount of a first event signature associated with the labeled 5mC region of the one or more polynucleotide sequences and an amount of a second event signature associated with the labeled 5hmC region of the one or more polynucleotide sequences, and distinguishing between the 5mC region and the 5hmC region of the one or more polynucleotide sequences.

[0518] In some embodiments, the disclosed methods include detecting (capturing) an ionic current of one or more features of at least a portion of a target polynucleotide in a first direction (the first direction is from the chamber through the first pore to the first fluidic channel). In some cases, the disclosed methods include redetecting (recapturing) an ionic current of one or more features of at least a portion of a target polynucleotide in a second direction (the second direction is from the first fluidic channel through the second pore to the chamber). In some cases, the method includes detecting (recapturing) a third ionic current and a fourth ionic current (e.g., simultaneously, a third ionic current as the one or more features pass through the first pore and a fourth ionic current as the one or more features pass through the second pore) of one or more features of at least a portion of the target polynucleotide in a third direction (the third direction is from the first pore to the second pore) (e.g., at least a portion of the target polynucleotide migrating from the first pore through the chamber to the second pore). In some cases, the method includes detecting (recapturing) a fourth ionic current of one or more features of at least a portion of the target polynucleotide in a fourth direction (the fourth direction is from the second fluidic channel through the second pore to the chamber).

[0519] In some cases, at least a portion of the target polynucleotide moves through the second pore in a range of 0.01 ms to 200 ms. In some cases, at least a portion of the target polynucleotide moves across the second pore in about 165 ms. In some embodiments, the first voltage is then adjusted to about 400 mV to pull the other end of the target polynucleotide in the opposite direction (e.g., the second direction, the third direction, or the fourth direction) and reach a tug-of-war state between the first and second pores. In some cases, the target polynucleotide comprises an increased force toward the second pore relative to the force of the first pore. In some cases, the target polynucleotide forms a tug-of-war state between the first and second pores for a range of 0.01 ms to 1000 ms. In some cases, the molecule exits the device in a range of 200 ms to 1000 ms.

[0520] In some embodiments, the target polynucleotide comprises a diffusion coefficient. In some cases, the diffusion coefficient of the target polynucleotide is greater than or equal to 0.01 mkm 2 / s to 3mkm 2 In some cases, the diffusion coefficient of the target polynucleotide is in the range of about 0.10, about 0.15, about 0.20, about 0.25, about 0.5, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1.0, about 1.5, or about 2.0 mkm 2 In some cases, the diffusion coefficient of the target polynucleotide is about 0.47 mkm 2 / s. In some cases, the diffusion coefficient is derived by A. Einstein's equation: D = κT / 6πηr, where κ is the Boltzmann constant, T is the thermodynamic temperature, η is the dynamic viscosity, and r is the radius of the particle.

[0521] In some cases, the target polynucleotide is introduced into the nanopore device. In some cases, the target polynucleotide is introduced into a chamber of the device. In some cases, the target polynucleotide is introduced into a first fluid volume of the device. In some cases, the target polynucleotide is introduced into a second fluid volume of the device. In some cases, introducing a sample containing the target polynucleotide into the chamber results in an exchange of different reagents. In some cases, scanning and / or detecting one or more features of the polynucleotide in a first direction (e.g., from the chamber through the first pore to the first fluidic channel) and / or in a second direction (e.g., from the first fluidic channel through the first pore to the chamber) screens out short fragments of the polynucleotide sequence. In some cases, movement of the target polynucleotide in the first direction for an extended period of time increases the efficiency of detection of one or more features of the target polynucleotide in the first direction.

[0522] In some embodiments, the tug-of-war state comprises a target molecule moving back and forth between the first and second pores. In some cases, the target polynucleotide is co-captured by the first and second pores. In some cases, when co-capture occurs, the control logic turns on constant competing voltages at the first and second pores, leading to a tug-of-war. In some cases, opposing forces are applied to the region of the target molecule passing through the pore. In some cases, the forces exert conformational and kinetic control on the co-captured target polynucleotide. In some cases, the forces unfold the target polynucleotide and reduce the translocation rate. In some cases, when the first and second voltages are adjusted such that the electrophoretic forces applied to the first and second pores are balanced, the lifetime of the tug-of-war state is limited by the diffusive sliding of the target polynucleotide between the first and second pores. In some cases, the ionic currents of each of the first and second pores provide independent sensors that synchronously measure different regions of the same polynucleotide, allowing for sequential detection of one or more features. In some cases, the control logic is a controller. In some cases, the controller includes an FPGA or ASIC circuit and performs active feedback control. In some cases, the control logic is configured to facilitate co-capture of the target polynucleotide in both the first and second pores. In some cases, the control logic engages in a competing voltage tug of war of the co-captured target polynucleotide until the target polynucleotide exits the first and second pores. In some cases, the detection of one or more features and the exit of the target polynucleotide into the first pore only affects the first ionic current detected at the first pore. In some cases, the detection of one or more features and the exit of the target polynucleotide into the second pore only affects the second ionic current detected at the second pore.

[0523] Aspects of the present disclosure include a method of mapping one or more characteristics of a target polynucleotide, the method comprising: a) providing a dual pore, dual amplification device for controlling the movement of a target polynucleotide simultaneously through first and second pores, the device comprising: (i) a first pore disposed between a chamber and a first fluid channel; (ii) a second pore disposed between the chamber and a second fluid channel; and (iii) one or more electrodes disposed in the first and second fluid channels to provide a first voltage at the first pore and a second voltage at the second pore. (i) providing a dual pore dual amplification device comprising: (a) one or more electrodes configured to provide a second voltage at each pore, wherein each voltage is independently regulated; (ii) one or more sensors configured to independently control the voltage at each pore and measure a current, wherein the first and second pores are configured to allow a target polynucleotide to migrate across the first pore, across the second pore, and simultaneously in both directions across both pores in a controlled manner; (b) introducing a target polynucleotide into a chamber of the device; (c) providing a second voltage at each pore, wherein each voltage is independently regulated; and (iv) one or more sensors configured to independently control the voltage at each pore and measure a current, wherein the first and second pores are configured to allow a target polynucleotide to migrate across the first pore, across the second pore, and simultaneously in both directions across both pores in a controlled manner; applying a first voltage at the first pore and a second voltage at the second pore such that at least a portion of the target polynucleotide migrates from the chamber in a first direction, capturing the target polynucleotide at the first pore and allowing the target polynucleotide to pass from at least the first pore to a first fluid channel; d) scanning one or more features of the target polynucleotide; e) detecting a first set of features in a first cycle as the target polynucleotide passes through the first pore; f) detecting the first set of features in the first cycle in the first direction. adjusting a first voltage, a second voltage, or both, for the first and second pores to change the orientation of the target polynucleotide when the target polynucleotide is released, such that at least a portion of the target polynucleotide moves out of the first fluidic channel in a second orientation, and recapturing the target polynucleotide in the first pore such that the target polynucleotide is maintained within at least the first pore; g) repeating step d); h) detecting a first set of characteristics in a first cycle when at least a portion of the target polynucleotide is recaptured in the first pore;i) adjusting the first voltage, the second voltage, or both for the first and second pores such that when the first set of features are recaptured in the first cycle in the second direction, at least a portion of the target polynucleotide simultaneously translocates across the first pore and the second pore in a third direction (the third direction being from the first pore to the second pore); j) repeating step d); k) detecting the first set of features in the first cycle when at least a portion of the target polynucleotide is in both pores; l) detecting the first and second voltages when the first set of features are detected in the third direction. adjusting the first voltage, the second voltage, or both, for the second pore to change the orientation of the target polynucleotide such that at least a portion of the target polynucleotide simultaneously migrates in a fourth direction across the second pore and the first pore (the fourth direction being from the second pore to the first pore); m) detecting the presence of the second set of features in a first cycle when at least a portion of the target polynucleotide is in both pores simultaneously in the fourth direction; n) repeating steps c) through m) and detecting the third and fourth sets of features in a second cycle.

[0524] Aspects of the present disclosure include a method for capturing a target polynucleotide in a nanopore device, the method comprising: a) providing a nanopore device for controlling movement of a target polynucleotide through one or more pores, the nanopore device including: (i) at least a first pore disposed between a chamber and a first fluid volume, the first pore being in fluid communication with the chamber and the first fluid volume, the first fluid volume being a geometrically constrained housing having an opening on an opposite side to the first pore; (ii) at least one electrode disposed within the first fluid volume, the one electrode configured to provide a first voltage at the first pore; and (iii) an amplifier configured for voltage control and current measurement at the first pore; b) introducing the target polynucleotide into a chamber of the device; c) translocating the target polynucleotide from the chamber in a first direction and detecting the first voltage at the first pore; The method includes applying a first voltage across the first pore to capture the target polynucleotide within the pore and allow the target polynucleotide to pass through the first pore into the first fluid volume; d) detecting a first ionic current in the first pore as the target polynucleotide passes through the first pore in a first direction; e) adjusting the voltage to zero for a predetermined time while the target polynucleotide is contained within the first fluid volume; f) applying a second voltage to reverse the direction of the target polynucleotide disposed in the first fluid volume, the second voltage displacing at least a portion of the target polynucleotide from the first fluid volume in the second direction such that the target polynucleotide is maintained within the first pore and recapturing the portion of the target polynucleotide in the first pore; and g) detecting a second ionic current in the first pore as the target polynucleotide is recaptured in the first pore.

[0525] Aspects of the present disclosure include devices and systems for mapping one or more features of a polynucleotide sequence of a target polynucleotide through first and second pores, the device comprising: (i) a first pore fluidically disposed between a chamber and a first fluid channel; (ii) a second pore disposed between the chamber and a second fluid channel, the first pore and the second pore being positioned such that the first pore and the second pore can be moved across the first pore, across the second pore, and both pores can be moved simultaneously, in respective directions and in a controlled manner; (iii) a first pore of the device. (iv) one or more electrodes configured to provide a first voltage at the first pore and a second voltage at a second pore of the device; (v) one or more sensors configured to independently control the voltage at each pore and measure a current from the target polynucleotide in a first cycle during translocation of at least a portion of the target polynucleotide from the chamber through the first pore in a first direction, from the first fluidic channel through the first pore in a second direction, from the first pore through the second pore in a third direction, and from the second pore through the first pore in a fourth direction. (v) one or more sensors capable of identifying a first set of features; (v) a processor; and (vi) a non-transitory computer readable medium, causing the processor to: a) apply a first voltage at the first pore and a second voltage at the second pore to cause at least a portion of the target polynucleotide to migrate from the chamber in a first direction, trapping the target polynucleotide in the first pore and allowing the target to pass through the first pore and into a first fluid channel; b) scan the one or more features of the target polynucleotide; c) detect the first set of features from the one or more sensors; detecting the presence of one or more features of the target polynucleotide in the first pore in the first direction; d) counting a first set of features in a first cycle in the first direction and, in response to the counting, adjusting one or both of the first and second voltages such that at least a portion of the target polynucleotide is displaced from the first fluidic channel in the second direction and recaptures said portion of the target polynucleotide in the first pore, thereby maintaining the target polynucleotide in the first pore; e) repeating step b); f) detecting from the one or more sensors in the second direction,determining the presence of one or more features of the target polynucleotide in the first pore; g) counting a first set of features in the first cycle in the second direction and adjusting one or both of the first and second voltages in response to the counts such that at least a portion of the target polynucleotide simultaneously translocates across the first pore and the second pore in a third direction; h) repeating step b); i) simultaneously detecting the presence of one or more features of the target polynucleotide in the first pore and the second pore in a third direction from one or more sensors; j) counting the features in the first cycle in the third direction. and adjusting one or both of the first and second voltages in response to the counts such that at least a portion of the target polynucleotide simultaneously translocates across the second pore and the first pore in a fourth direction; k) repeating step b); l) simultaneously detecting the presence of one or more features of the target polynucleotide in the first pore and the second pore in a fourth direction from one or more sensors; and m) repeating steps a) through l) and detecting a third and fourth of the features in a second cycle.

[0526] Aspects of the disclosure include a method for determining a sequence of a target polynucleotide, the method comprising: a) providing a dual pore, dual amplification device for controlling the movement of a target polynucleotide simultaneously through a first and a second pore, the device comprising: (i) a first pore; (ii) a second pore; (iii) a power supply configured to provide a first voltage at the first pore and a second voltage at the second pore, each voltage being independently regulated; and (v) one or more dual amplification voltage sources configured to independently control the voltage and measure current at each pore. a) providing a dual pore dual amplification device, the dual pore dual amplification device including dual amplification electronics configured such that a first and second pore can simultaneously migrate a target polynucleotide in a first direction or in a second direction and across both pores in a controlled manner; b) introducing a target polynucleotide into the device; c) applying a first initial voltage to the first pore and a second initial voltage to the second pore such that at least a portion of the target polynucleotide migrates through the first pore and the second pore in a first direction (the first direction being from the first pore to the second pore); setting a first voltage; d) scanning one or more primers hybridized to the target polynucleotide; e) detecting a first set of primers in a first cycle when the target polynucleotide is in both pores in a first orientation; f) rescanning the one or more primers hybridized to the target polynucleotide; g) adjusting the first voltage, the second voltage, or both for the first and second pores when the first set of primers is detected in the first cycle in the first orientation to detect at least a portion of the target polynucleotide being hybridized to the first pore. h) changing the orientation of the target polynucleotide to move from the second pore to the first pore in the second orientation; i) detecting the presence of a second set of primers in a first cycle when the target polynucleotide is in both pores simultaneously in the second orientation; i) identifying each nucleotide of the polynucleotide that passes through one of the pores by measuring the ionic current across the pore as the nucleotide passes through that pore; and j) repeating steps c) to i) and detecting a third and fourth set of primers in a second cycle.

[0527] Finally, the terminology used in the specification has been selected primarily for purposes of readability and instruction, and not to delineate or delineate the boundaries of the subject matter of the invention. Accordingly, it is intended that the scope of the invention be limited not by this detailed description, but rather by any claims issued on an application based thereon. Accordingly, the disclosure of embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the following claims.

[0528] Examples of Non-Limiting Aspects of the Disclosure Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the above, certain non-limiting aspects of the disclosure numbered 1 to 171 are provided below. As would be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect can be combined with or with any of the individually numbered aspects above or below. This is intended to provide support for all such combinations of aspects and not be limited to the combinations of aspects explicitly provided below. EXAMPLES

[0529] The following examples are submitted to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to imply that the following examples are all or the only ones performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation are included. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp base pairs, kb kilobases, pl picoliters, s or sec seconds, min minutes, h or hr hours, aa amino acids, kb kilobases, bp base pairs, nt nucleotides, im intramuscular, ip intraperitoneal, sc subcutaneous, etc.

[0530] Example 1: Nanopores flossing DNA in a dual pore device Here, an active control technique, termed "flossing", was presented that uses a dual nanopore device to trap protein-tagged DNA molecules and performs up to 100 back and forth electrical scans of the molecules within seconds. Protein motifs bound to 48 kb λ DNA are used as detectable features to actively trigger bidirectional control. Molecular noise is suppressed by averaging data from multiple scans, resulting in an estimate of the averaged inter-tag distance that can be compared to known values. Since mapping applications of nanopore features require linearization of DNA as it passes through the pore, a key advantage of flossing was that the second scan increased trans-pore linearization to >98%, compared to 35% for the passage of the same set of molecules through a single nanopore. In cooperation with barcoding methods, the dual pore flossing technique enables applications of genome mapping and structural variation, or mapping of loci of epigenetic relevance.

[0531] Highly accurate spatial information correlating with motif binding can be obtained from a "single" labeled carrier dsDNA strand by repeated scanning back and forth of the trapped molecule in the nanopore device. Using a novel active control technique called "flossing", the inventors were able to perform up to 100 scans of a given trapped molecule within a few seconds. Flossing is specifically demonstrated here using a model system consisting of 48.5 kbp double-stranded λ-DNA with a set of chemically incorporated sequence-specific protein tags. This testing approach captures existing carrier-strand DNA nanopore technology by enhancing the quality of information extracted from a single trapped molecule. By taking a large number of statistically equivalent scans, stochastic fluctuations are reduced by scan averaging, and even allowing for missed tags in a subset of scans, the average tag spatial estimates are comparable to known inter-tag distances.

[0532] The disclosed device used a dual-pore architecture. By having the dual pores close enough, the DNA was captured by both pores simultaneously and existed in a "tug-of-war" state, where competing electrophoretic voltage forces were applied to each pore. During the tug-of-war, the molecular orientation and identity were maintained, the molecular velocity was adjusted to facilitate the sensing of the tag, and the likelihood of the molecule finding a conformation linearized through the pore was increased by 70%2 compared to 30% for the single-pore data. The advantage of flossing was that linearization through the pore was further increased to >98% by a second scan, which then increased the throughput of the application of nanopore feature-mapping.

[0533] Another distinct feature of the presented approach is that an active controller periodically adjusted the voltage at one pore by feeding back in real time to the sensing current of the other pore. In particular, during control, repeated application of unbalanced competing voltage forces was used to drive the movement of molecules in one direction and then in the reverse direction after real-time detection of a set number of tags, thereby using a DNA "flossing" concept. Binding DNA to a predefined state provided both rate control and minable data generation during the movement of molecules through a solid-state pore, at the cost of complex instrumentation, high sensing noise, and low throughput. In the presented flossing control method, the interrogated DNA molecule is expelled from the pore and new DNA is captured without the required molecular tethering, with the same throughput and ease of any single nanopore-based assay.

[0534] Results and Discussion DNA Flossing Concept FIG. 9 introduces the general flossing concept and shows repeated bidirectional scanning of co-trapped molecules in a dual pore device, pictorially and with actual recorded data. The dual pore device was fabricated using known methods and had voltages V1 and V2 applied independently at pores 1 and 2, respectively. Two currents (I1 and I2) were also measured independently at the pores. The tagged reagent features monovalent streptavidin (MS) bound along the DNA.

[0535] Figure 9a illustrates each step in multiple scan cycles. Since the control of movement is two-way, a single transit is defined in a fixed direction as a "scan", and two consecutive scans of opposite polarities are defined as a "cycle". By convention, the left pore was defined as pore 1 and the right pore as pore 2. During the sequence of multiple scan control logic, V2 across pore 2 is maintained constant while V1 is adjusted in a stepwise manner. Signal I2 was monitored in real time for tag-related events as a logic trigger for changes in V1, as described next.

[0536] Flushing control logic starts by first co-trapping DNA tagged at both pores to reach a netting state. When co-trapping occurs, a voltage lower than that of pore 2 is applied across pore 1 (V1 < V2), inducing movement of the molecule towards pore 2 (from left to right or L to R). While monitoring I2, after a set number N of tags, V1 > V2 is detected, and the control logic re-adjusts the voltage at pore 1 to transfer through pore 2. The re-adjustment induces a return of the molecular movement towards pore 1 (R to L). After detecting the transfer of the same set number N of tags through pore 2, the logic resets V1 < V2 to initiate another L to R scan, thereby starting a new cycle. Figure 9b shows a recorded example of the first two cycles with the tag number setting of N = 2 and the last cycle of the signal of I2. In this example, the multiple scan cycle was initiated with molecules moving from L to R for the first 9 ms and continued until 172 ms when DNA escaped (the escape mode is discussed in detail below and in the SI) immediately after the V1 adjustment. Details regarding the set of dual-pore chips and voltage settings used in this experiment are provided in Tables S1–S2 (Figs. 18–19). Although the general concept of flushing was described, the method is next shown in more detail and with the results obtained using the method.

[0537] Initializing netting and specifying scan voltages The control logic that automated the flossing process shown in Figure 9 is now described in detail. The flossing method first used active control to automate the initialization of single molecule co-capture and tug-of-war. The control logic was implemented in real time (MHz clock speed) on a field programmable gate array (FPGA). The designed tug-of-war control process was modified to allow the introduction of reagents into a common fluid chamber above the two nanopores and screen out short fragments (SI section 2, Figure 16). Once co-capture is achieved, competing voltage forces at V1 and V2 lead to a tug-of-war, slowing down the DNA velocity during detection. The average duration of all co-capture events was calculated at each of the different sets of V1 while holding V2 constant, showing a bell-shaped curve with a peak indicating the force-balancing voltages that maximize the duration of co-capture. Figure 16c shows an example device with a peak average duration of 110 ms with both V1 and V2 set to 500 mV. This data was generated with bare DNA without tags.

[0538] During molecular capture with the velocities and conformational control induced by the tug-of-war of the dual pores, bare double-stranded DNA presented no detectable features to monitor the translocation of molecules. To enable in-situ feature monitoring, MS-tagged DNA with up to seven sites tagged on the DNA was developed as a model reagent (SI Section 3, Figure 21). The tag features were used to provide interferences for the speed and direction of each scan, which allowed the scanning voltages to be identified as described next.

[0539] The scan voltage is found after the force-balance voltage that maximizes the duration of co-capture is identified for a given chip. In particular, scan voltage values ​​for V1 are selected above and below its force-balance value while holding V2 at its force-balance value to promote the movement of unperturbed DNA toward pore 1 and pore 2, respectively. The V1 scanning value is heuristically selected using the following guidance: The working scan speed should allow robust sensing of tag blockades at the bandwidth of the recording (i.e., not too fast) while ensuring a high enough pecklet number so that the distribution of transition times is well defined (i.e., not too slow). That is, a wide distribution of transition times increases the likelihood of variability, which may undermine the goal of mapping time versus distance between tags, as described in a later section. In practice, achieving a scan speed that is neither too fast nor too slow is achievable for a wide range of scanning V1 voltages. For the data in this paper, the range was only 150 mV and as high as 500 mV away from the force equilibrium voltage.

[0540] Representative flossing event with protein-tagged DNA Once the scan voltage values ​​have been identified, the complete flossing multiple scan logic can be applied. A detailed description of the logic implemented in the FPGA was provided in the supplementary material (SI section 4, Fig. 17). The output of the logic was informed here by representative flossing data

[10] . Figure 10a showed the complete signal traces of I1, V1, I2, and V2 from a typical flossing event. tV2 was held unchanged at V2 = 300 mV during the event. V1 was set to V1 = 100 mV for the L to R transfer and V1 = 850 mV for the R to L transfer. Note that the baseline of I2 changes when V1 is adjusted, even though V2 is constant. This effect was attributed to the crosstalk between pore 1 and pore 2, which was characterized earlier. The detectability of the tags relative to the DNA baseline of I2 was still robust, despite the baseline change. As detailed in the previous section, a sufficiently large voltage difference is selected between V1 and V2 to facilitate controlled movement in each direction during sensing.

[0541] Figure 10b, c gives a close-up view of the signal for the first cycle, including the 1st and 2nd scans, and including the 81st and 82nd scans. By convention, the multiple scan logic starts in the L to R direction, so that odd scans correspond to L to R transfers and even scans correspond to R to L transfers. The event in Figure 10a includes 41 cycles and a total of 82 scans, all in less than 0.66 seconds. As shown in Figure 1, the FPGA logic was designed to switch V1 when two tags were detected in I2. The FPGA detects tags when I2 drops at least 70 pA below the untagged DNA baseline for at least 0.012 ms. The data in Figure 2 showed two tags, A and B, in both I1 and I2 when the molecule made an L to R transfer in 9 to 19 ms. Following a 1.5 ms delay, after detecting tag B, the FPGA sets V1 = 850 mV and drives the molecule to move from R to L in 19 to 25 ms. The same tags, B and A, are detected in both I1 and I2 in the reverse order. The same logic continues until the FPGA no longer detects tag B in the last cycle (this is due to the tag appearing too close to the voltage change that causes the FPGA to detect it) (Figure 10c).

[0542] The distribution of total flossing times and number of scans per event is shown in Figure 10d for all 309 flossing events in the experiment, including the 82 scan events in 10a. Although the total flossing time increases with more scans, all events were completed in less than a few seconds, even considering the largest scan count for this data was 157. During the last 5-15 ms of each individual scan (Figures 9b and 10b-c), the total time data showed that using the present flossing method significantly increased (up to more than 100-fold) the time required to interrogate each molecule.

[0543] From the probability plot in Figure 10d, 37% of the events were less than 5 scans, and events with more scan counts were unlikely. The probability P(n) of seeing a specific total number of scans n was examined, and the probability of any intermediate scan has a correct detection probability p and a missed probability (1-p). An event of total scans n indicates a system that successfully captures the first (n-1) scans, but fails to capture the nth scan. The probability P(n) is therefore P(n)=p n-1 (1-p) (1) It is.

[0544] Fitting the data to equation (1) gives p=0.89 for this particular data set (Figure 10d). To determine why molecules escape co-capture, the final cycle was examined, finding four common cases: tag escape on the nth scan (Figure 18a), a false positive spike detected on the (n-1)th scan (Figure 18b), a false negative spike detected on the nth scan (Figure 18c), and a molecule escaping the pore during the FPGA delay state (Figure 18c). A discussion of how to increase the number of total scans per event can be found in SI section 5. Naturally, varying the voltage settings will affect the event duration between tags, which will in turn affect the probability of tag detection.

[0545] The dependence of tag size on scan direction is observed in Figure 10a-c, where the MS tag shows relatively shallow and fast spikes at the two higher voltages as it passes through the pore. The finding of fast and shallow tag events at higher voltages is consistent with the single-pore results and is in part an artifact of the low-pass filter (10 kHz bandwidth) preventing the tag events from hitting full depth (i.e., the faster the events, the shallower they are).

[0546] Multiple scan experiments using a three tag triggering setup were also performed (Figure 19). It was more difficult to obtain many scans using three tag triggers than using two. In part, this occurred because few molecules (<20%) displayed more than three tags. Also, even if the system co-captured one molecule with three tags in both pores, the probability of correctly detecting all three tags was low (Figure 19c), and failure to detect any one of the tags would move the molecule to a new region, thereby lowering the scan count for the three tag region originally scanned. Therefore, we focused our experiments on this initially presented two-tag triggering to generate more data with more scan counts.

[0547] Flossing increases the fraction of DNA that has mappable data. The application of nanopore feature mapping requires linearization of DNA as it passes through the sensing pore. In this case, the fraction of DNA that can be linearized with the flossing technique is considered, where linearization refers to the removal of the DNA folds that are initially in the pore when co-capture is initialized. As an example, the molecule in Figure 10a-c is partially folded in about 10 ms in the first (1st) cycle (Figure 10b), which is removed in the second (2nd) cycle, showing the tendency of tug-of-war control to induce and maintain DNA linearization thereafter. The probability of complete linearization over the scanning cycles through pore 2 was tested as a function of scan number to see if the trend in Figure 10a-c was a representative distribution. Indeed, Figure 11 showed that the probability of linearization increased to 98% by the second scan. In the data, folding events were identified where the blockage of current was 1.5 times higher than the unfolded blockage amount and persisted for more than 180 μs. Figure 11a, b shows representative single-hole and multi-scan events with examples of observable folding.

[0548] A quantitative mechanism for the progression of unfolding during flossing in Figure 11c was proposed. Going from L to R aligns the migration and field forces at pore 2, which ultimately promotes the refolding to migrate through pore 2 to channel 2. Subsequently, migration from R to L pulls only the region of DNA under tension by the tug back through pore 2, despite the opposing field force at pore 2, while the folds not under the tension of the tug only experience the field force and are therefore maintained in channel 2 and move away from pore 2. Figure 11d showed the percentage of unfolded events versus the progression of the type of transition that each of the 309 events went through. Thus, the statistics for each pillar were indeed from the same group of molecules, the same data as in Figure 10. The 35% unfolding probability from the initial single-hole capture (pore 1 of the device) is consistent with the other single-hole tests. Following co-trapping, 66% of the first (1st) scan was unfolded, consistent with the tug-of-war data without flossing.

[28] By the second (2nd) scan, only 6 molecules (2%) out of 309 remained folded, and only 5 remained folded by the final scan. Thus, the flossing process effectively linearizes the DNA molecules through the nanopore sensor with a high probability.

[0549] Tag data analysis: A single scan view The multiple scan data set (consisting of L to R and R to L for each pore) contains rich information about the basic tag binding profile and translocation physics. Each individual scan taken at the two pores provides a snapshot of the translocation process for the portion of DNA being scanned. There are two ways to estimate the translocation rate from the tag. The first approach is to quantify the rate of the tag moving through a single pore ("dwell time estimation"), which is the first step in determining whether tag blockade occurs. The second approach is specific to the dual pore technology and involves assessing the velocity of the tag as it travels from the first pore (entrance) to the second pore (exit). This entry-to-exit time refers to the time the tag remains in the common chamber above both pores, also referred to as the pore-to-pore time. In the pore-to-pore velocity assessment, the pore-to-pore time was classified as the measured distance between the pores for each chip. By definition, the pore-to-pore approach exploits the relationship between the two current signals I1 and I2, since the time begins when the tag exits the entry pore and the tag-bearing end enters the exit pore. At the applied voltage, the DNA in the common reservoir was expected to be fully stretched between the pores (0.34 nm / bp).

[0550] FIG. 12 shows an example of adjacent pairs of scans in a multiple scan event, illustrating how to estimate the separation distance between tags from each scan. For the L to R scan (FIG. 12a, c), tag A then tag B moves through pore 1, and about 1 ms later they move through pore 2. The signal pattern shows that tags A and B were spatially closer than the distance between the pores (0.64 μm). The FPGA monitored I2 for the two tags during the control logic. During the wait time after detecting A and B in I1, a third tag C passes through pore 1. Visually, it was clear from the pore-pore passing time of A and B that tags B and C were roughly twice as far apart as the distance between the pores. When V1 was changed to facilitate the reverse direction, R to L movement (FIG. 12b, d), the first observable tag in I1 was C returning through pore 1. Again, this logic detects A and B at I2 and at this time both tags pass pore 1 before the logic activates V1, facilitating the transfer of L to R. Seeing the three tags in I1 was a result of the separation of the three physical tags being close enough to accommodate the 3-tag pore 1 transit, within the window of the 2-tag pore 2 detection time of I2, as implemented in the FPGA. A further generalization is to reliably see two tags in both pores, as detailed in the next section.

[0551] For the L to R scan, the tag blockade at I2 corresponds to a tag leaving the common reservoir and exiting pore 2 from L to R, whereas the R to L movement means that the tag at I2 enters the common reservoir. While I2 shows two tags, the presence of a third tag at I1 for both scan directions gives the opportunity to quantify the separation distance of the two tag-pairs. For the signal shown, the number of tags between the pores was plotted, as well as the tag velocity based on the dwell-time and pore-to-pore velocity. These velocity values ​​versus time provide an indication of how the molecules are moving along the diagram (Fig. 12a, 12b) during the co-capture tug-of-war, which has been added for visualization. The pore-to-pore velocity was moderately faster in the R to L direction (0.8 vs. 0.6 μm / ms), which corresponded to a larger voltage difference for R to L migration than for L to R migration (V1 = 250 mV, V2 = 400 mV).

[0552] The tag-to-tag separation distance (12c, d lower) was estimated by multiplying the average pore-to-pore velocity within a scan by the tag-to-tag time recorded within that scan and adding the membrane thickness. Since the tag-tag time is calculated from the ascending end of a detected tag blockade to the descending end of the next detected tag blockade, the membrane thickness is added to calculate an additional spatial separation equal to any tag passing through the length of the pore. The tag-to-tag distance estimates were shown for both L to R and R to L scans. The results suggest that the prediction of each separation will vary across the two different scan directions. The prediction of tag-pair separation distance will also vary with the difference between the tag-to-tag velocity and the actual velocity profile during the tag-to-tag time for a given pore. That is, the assumption that the velocity between tags is constant and equal to the pore-to-pore velocity is not actually correct. This assumption seems better when the velocity is shorter than the tag-to-tag time (which is the case for tags A and B, but not for tags B and C in FIG. 12). If the assumption that in any case the DNA was moving at a constant pore-to-pore velocity was correct on average, then an average of many rescans was expected to predict the exact separation distance between any two consecutive tags. Multiple scan averages were then calculated and tested to see how well they prepared predictions with known spacing (separation) of the tagged DNA from the model reagent.

[0553] Combining scans to improve tag-to-tag distance predictions The quality of error reduction of averaging distances obtained from individual scans within a multiple scan event was tested. Five different multiple scan events containing at least 30 cycles were reported in Table 1. For each event, averaged distance estimates were shown for each scan direction, using the estimates of pore 1 and pore 2 alone and in combination with the estimate of pore 2. The table reports the number of cycles equal to the number of scans in each direction, and the number of tag-pairs contributing to the estimate of each separation distance. In all cases, there were fewer distance estimates than scans. For example, for event (v) with 65 scans in each direction, and for the R to L direction, 57 scans resulted in detectable tag-pairs in I2, while 62 tag-pairs were detected in I1 for all 119 separation (separation) estimates. Friction is the reason why the probability of missing a tag within any one scan range increases with cycle count, as explained in equation (1).

[0554] The correspondence between the averaged spacing distances (separation distances) in Table 1 and the known tag-to-tag spacings (separation) possible from the position map was evaluated. The known distances are calculated using the conventional 0.34 nm / bp, assuming DNA in a common reservoir fully stretched between the pores. In event (i), the L to R data showed sufficient change in pore-to-pore velocity (as described in SI section 11) that only the R to L scan direction was combined and reported for event (i). Note that the two scans shown in Figure 4 were from event (i) that pathologically generated two tag-pair estimates in the current of pore 1, for reasons indicated in that figure. If the assumption is that tag 6 is not present in the molecule and tags 4, 5, and 7 are present, then the adjacent tag-pair separation for event (i) is the most consistent of all of the possible adjacent tag-pair combinations possible according to the position map. In particular, the maps showed adjacent distances of 0.1 and 1.5 µm between tags 4–5 and 5–7, which was reasonable to assume that a tag (i.e., tag 6) was absent.

[0555] Events (ii-v) in Table 1 show good agreement across both scan directions and between both pores when comparing the pore 2 results to the combined results. For these events, only a single separation distance estimate was generated, which was most typical for control logic with N=2 tag detection at I2 triggering a change of direction. By comparing the averaged separation distance (separation distance) with the known inter-tag spacing (separation), events (ii) and (iii) correspond most closely to the 1.5 μm and 1.3 μm distances between 5-7 and 6-7, respectively, with a value of 1.5 μm possible if the tag 6 position is assumed to be empty for the molecule in event (ii). Also, events (iv) and (vi) correspond most closely to the 0.3 μm and 0.2 μm distances between 4-6 and 5-6, respectively, with a value of 0.3 μm possible if the tag 5 position is assumed to be empty for the molecule in event (iv).

[0556] The correspondence between distance estimates and mappable combinations was generally not as clear for individual scans (e.g., FIG. 12) as it was for averaged scans and not possible for scans in which tags are missed (representative examples are shown in FIGS. 30, 32, and 33). This illustrates the reduction in error values ​​by averaging across the set of multiple scan data generated for each molecule. Additional data on velocities for events (i-v) in Table 1, as well as data for four additional multiple scan events, are reported in Table S3 (FIG. 21). The error of the estimate for each interval (separation) is taken as the error for the average over the group of estimates, where a sufficient reduction in error was achieved through averaging.

[0557] The data in Table 1 (FIG. 13) shows the power of the flossing approach when events have tag-to-tag times in I2 that can be clearly attributed to the same physical set of tags (representative scans in both I1 and I2 signals are provided in FIGS. 26-33). However, in other data, when a tag is missed in I2 within the scan range, the two tag scan logic will eject the molecule and subsequently shift to a new physical tag pairing of the same molecule resulting in a resistance shift in the tag-to-tag time data. An example of this was event (vi) in Table S3 (FIG. 21) with a register-shift scan signal. While this complexity can be visually observed and manually accommodated in the data, we next sought to develop an alternative approach that can detect and automatically analyze such resistance shifts.

[0558] An alternative method presented next is based on aligning signals in the time domain based on a tag blockade approximation with the goal of automating the binning of tag-pair times (especially since there is significant ambiguity in assigning such times across a scan).

[0559] In time-based signal alignment methods, the temporal position of each tag relative to the start time of each scan is first calculated. To facilitate alignment, the method must tolerate potentially large differences in the shape of tag events, which modifies the tag analysis procedure, which is based on fitting a model function to each peak, based on box convolution with a Gaussian function (SI section 10). This model characterizes tag blockades that are both broad / rectangular or narrow / Gaussian-like in nature.

[0560] To align the scans in a systematic way, the algorithm automatically groups tags and removes translational offsets across the scans. Fig. 14c, f shows an example of an aligned event, and the SI section entitled "Tag Alignment Procedure" provides a detailed description of the approach. Essentially, the algorithm works by assuming that at least two tags are shared between two consecutive scans. To identify one of the shared or "common" tags, the algorithm aligns each possible tag pair in the two scans by shifting one scan with respect to the other. Note that only the translational offset is applied, i.e., there is no global expansion of the time scale. For each of these possible trial alignments, the algorithm calculates a measure of alignment error based on the summed squared difference between distinct tag pairs in the trial alignment. The algorithm identifies the pairing that gives the smallest error as a genuine common tag and performs a translational shift to align this pair. This approach gives both the translational offset between scans and the correspondence of tags shared between scans. By iterating across all scans in a set, tags observed across scans can be grouped together and the translational offset removed. The tag group with the largest set of scans is identified as the origin tag, each scan is shifted, and this origin is set to zero. The algorithm outputs a final barcode or set of averaged relative tag positions for each set of scans of a single molecule.

[0561] The output backcode is in units of time. To correct the scans to units of distance, the aggregate translocation velocity corresponding to the scan set was calculated as the average of the subset of pore-to-pore velocities measured within multiple scan events, using only those velocities where the scans displayed a preserved number of tags in both pores. The barcodes are then corrected to units of distance by multiplying by the average pore-to-pore velocity. Finally, the reported error in the corrected tag positions is incorporated into the error in velocity and in separation due to the standard population. Note that the sharpening of the plateau indicates the accuracy achieved through the averaging of multiple scans. In particular, it was found that when the extracted tag separations were sorted by magnitude, the data showed a clear plateau that corresponded to the expected separations of the maps. Spacing away from the expected spacing (separation) may result from non-specific binding (e.g., tags attached non-specifically to random nicks present in λ-DNA) or from offsets caused by imperfections in the tag blockage analysis algorithm.

[0562] In single-pore operation, sub-4 nm diameter membrane-based pores harboring PNA-based motifs (15 bp footprint) showed resolution of 100 bp inter-tag distance, whereas pipette-based pores had resolution as low as 200 bp. It was observed that peaks showed good resolution for separations of 300 bp (e.g., visible spacing between tags in the traces in Figure 11), and a lower limit of less than 300 bp is achievable, as slower passage and larger bandwidth are the knobs to be turne...

Claims

1. 1. A method for differential detection of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a mixed sample containing one or more polynucleotide sequences that migrate separately through first and second pores, comprising: a) labeling the 5mC and 5hmC regions of one or more target polynucleotides with a binding molecule; b) contacting one or more target polynucleotides with a payload molecule configured to bind to a binding site of the one or more target polynucleotides; c) providing a dual pore dual amplification device for controlling the movement of a target polynucleotide bound to a payload molecule through a first and a second pore simultaneously, said device comprising: (i) a first channel, a chamber, and a second channel; (ii) First pore (iii) Second pore (iv) a power supply configured to provide a first voltage at the first pore and a second voltage at the second pore, both voltages being independently regulated; and (v) dual amplification electronics configured to control the voltage and measure the current at each pore independently; the first and second pores are configured to allow a target polynucleotide to translocate simultaneously across both pores in both directions and in a controlled manner; d) introducing the sample containing one or more target polynucleotides into the device; e) applying a first initial voltage and a second initial voltage such that at least a portion of the one or more target polynucleotides migrate through the first pore and the second pore, the first and second voltages being: one or more tagged 5mC regions of a polynucleotide sequence; A tagged 5hmC region of one or more polynucleotide sequences; inducing translocation of the one or more target polynucleotides through the first and second pores separately for each of the first and second pores; f) generating a plurality of event signatures resulting from translocation of one or more target polynucleotides through said first and second pores; g) identifying the amount of a first event signature associated with the labeled 5mC region of the one or more polynucleotide sequences and the amount of a second event signature associated with the labeled 5hmC region of the one or more polynucleotide sequences to distinguish between the 5mC region and the 5hmC region of the one or more polynucleotide sequences; A method comprising:

2. 2. The method of claim 1, wherein the second pore is fluidly connected to the second channel, the second channel being a geometrically constrained enclosure with a second inlet and a second outlet, and the second pore is fluidly connected to the second channel between the inlet and the outlet.

3. 2. The method of claim 1, comprising detecting a first set of event signatures of the polynucleotide when the polynucleotide is in both pores.

4. The method of claim 1 , wherein the first initial voltage creates a voltage gradient across the first pore and along the length of the first channel.

5. The method of claim 1 , wherein a resistance of the first channel is inversely proportional to a width of the first channel.

6. 2. The method of claim 1, wherein the resistance of the first channel and / or the second channel is inversely proportional to one or more of the volume of the first channel and / or the second channel, the radius of the first channel and / or the second channel, and the radius of the cross-section of the first fluid channel and / or the second channel.

7. 10. The method of claim 1, further comprising controlling, by a controller, when the polynucleotide requires rescanning of one or more features of the polynucleotide for a second or third time.

8. 8. The method of claim 7, wherein the controller determines any of one or more features of the polynucleotide and performs additional recapture of the one or more features in the first orientation and / or the second orientation.

9. 2. The method of claim 1, wherein the length of the polynucleotide is at least 2 times, at least 3 times, or at least 5 times the distance between the first pore and the second pore, between the chamber and the first channel, and / or between the chamber and the second channel.

10. 2. The method of claim 1, wherein the first voltage is maintained for a period in the range of 0 to 1000 milliseconds, 0 to 20 milliseconds, 20 to 50 seconds, 50 to 100 seconds, 100 to 500 seconds, or 500 to 1000 seconds.

11. 2. The method of claim 1, wherein the first voltage is maintained for a period of time after a target polynucleotide is captured and translocated through the first pore.

12. The method of claim 1, further comprising determining the location of one or more 5mC or 5hmC on the one or more target polynucleotides.

13. The method of claim 1 , wherein the binding molecule is a 5mC or 5hmC binding molecule.

14. 1. A method for determining a sequence of a target nucleotide, comprising: a) providing a dual pore dual amplification device for controlling the movement of said target polynucleotide through first and second pores simultaneously, said device comprising: (i) First pore (ii) Second pore (iii) a power supply configured to provide a first voltage at the first pore and a second voltage at the second pore, each voltage being independently regulated; and (iv) dual amplification electronics configured to control the voltage and measure the current independently at each pore, the first and second pores being configured such that the target polynucleotide can translocate simultaneously across both pores in a first direction or a second direction and in a controlled manner; and b) introducing the target polynucleotide into the device; c) setting a first initial voltage of the first pore and a second initial voltage of the second pore such that at least a portion of the target polynucleotide migrates through the first pore and the second pore in the first direction, the first direction being from the first pore to the second pore; d) scanning one or more primers for hybridization to said target polynucleotide; e) detecting a first set of primers in a first cycle when the target polynucleotide is in both pores in the first orientation; f) rescanning one or more primers hybridized to said target polynucleotide; g) adjusting the first voltage, the second voltage, or both for the first and second pores when the first set of primers is detected in a first cycle in a first orientation to change the orientation of the target polynucleotide such that at least a portion of the target polynucleotide moves from the second pore to the first pore in a second orientation; h) detecting the presence of a second set of primers in the first cycle when the target polynucleotide is in both pores simultaneously in the second orientation; i) identifying each nucleotide of said polynucleotide passing through one of said pores by measuring the ionic current across said pore as the nucleotide passes through that pore; j) repeating step c) through step i) and detecting the third and fourth sets of primers in the second cycle; A method comprising:

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