Forced Polynucleotide Scanning for Mapping Features Within Nanopore Devices

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. Also provided herein is an automated method for sequencing a polynucleotide sequence. Also provided herein is a method for enhanced recapture of a polynucleotide within a nanopore device. Also provided herein are devices and systems for carrying out the methods of 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 applications are incorporated herein by reference in their entireties. [Background technology]

[0003] Precise mapping of binding locations of molecular motifs along long individual dsDNA strands within 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 occurs when double-stranded DNA (dsDNA) is electrically driven 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] U.S. Chartered Bid Publication No. 2018 / 0023115 [License 6] US Chartered Petition Publication No. 2013-0233709 [Non-licensed literature]

[0005] [Non-licensed Document 1] Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), Features, Chapter 11, and Table 11.1 [Non-licensed Document 2] Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) [Non-licensed Document 3] Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001) [Non-licensed Document 4] Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999) [Non-licensed Document 5] Protein Methods (Bollag et al., John Wiley & Sons 1996) [Non-licensed Document 6] Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999) [Non-Patent Document 7] Viral Vectors (Kaplift & Loewy eds., Academic Press 1995) [Non-patent document 8] Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997) [Non-Patent Document 9] Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998) Summary of the Invention [Problem to be solved by the invention]

[0006] There are (1) the need for consistent linearization of transferred molecules, (2) the need to reduce the effects of molecular variation that introduces random errors, and (3) the need to develop strategies for accurate genomic distance calibration. [Means for solving the problem]

[0007] Aspects of the present disclosure disclose a method for partially or completely recapturing a polynucleotide in a nanopore device that has been previously captured, the method comprising: 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; and (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; and c) applying a first voltage to the first pore. d) capturing and translocating the polynucleotide from the chamber through the first pore into the first fluid volume in a first direction; 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 translocated through the first pore prior to performing step (e), indicating that the polynucleotide exceeds 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 duration is less than a threshold.

[0012] In some embodiments, the detecting step of step (d), in which the polynucleotide is captured by the first pore and translocated from the first pore at a first voltage, maintains the first voltage for a time period ranging 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 of 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 at least 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 the 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 connects the chamber and the 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 includes at least one electrode disposed within the second fluid volume, the at least one electrode configured to provide a voltage at the second pore that is controllable independently of the voltage at the first pore.

[0027] In some embodiments, the nanopore device comprises dual-amplifier electronics configured to control voltage and measure 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, and 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 higher 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 enclosure, 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 moves 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 the third voltage at the first pore, the first voltage at the second pore, or both to change the direction of the polynucleotide such that at least a portion of the polynucleotide translocates from the second pore through the first pore in the first direction.

[0043] In some embodiments, adjusting the third voltage at the first pore, adjusting the first voltage at the second pore, or both, to cause at least a portion of the polynucleotide to move 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 in both pores in the first orientation.

[0047] In some embodiments, the method includes 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 time in the range of 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 the 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 any of one or more characteristics of the polynucleotide and performs additional recapture of the one or more characteristics 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 range from 0 mV to 1000 mV.

[0067] In some embodiments, the first voltage, the second voltage, and the third voltage range from 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 across the first pore is less than the second voltage.

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

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

[0075] In some embodiments, the first voltage across the first pore and the second voltage across 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 across the first pore is lower than the third voltage across the second pore in the third direction.

[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 the 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 the 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 has a length from inlet to outlet in the range of 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 to 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 that detects capture and translocation 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 present 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 simultaneously through first and second pores, 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 the 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 pore being a geometrically constrained enclosure, each filled with fluid and having 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) the electrode in the first fluid volume and the chamber; a) introducing a target polynucleotide into the chamber of the device; b) providing a voltage between the electrode within the first fluid volume, a voltage between a second electrode in the second fluid volume and the electrode in the chamber, and c) providing a current measurement that detects capture and partial or complete translocation of polynucleotides into and through the first pore, and d) providing a current measurement that detects capture and partial or complete translocation of polynucleotides into and through the second pore; and c) applying a first 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; 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 movement 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 enclosure, 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 of step (d) is partially translocated from the first fluid volume through the first pore.

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

[0100] In some embodiments, the method includes repeating steps (c) and (d) before 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 translocation 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 before each detection step.

[0112] In some embodiments, the method further comprises detecting one or more features of the polynucleotide that each pass through the second pore in step g) and that have not yet been 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 a second aperture of feature detection 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, the 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 fluid channel a distance ranging from 5 microns to 5 millimeters or more away from the first pore.

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

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

[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 time 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 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 between steps c) and d) for a time 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.

[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 using a processor to calculate 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, if the minimum duration is greater than a threshold, the polynucleotide feature is identified as a target, and if the duration is less than the threshold, the feature is identified as a non-target.

[0147] In some embodiments, the method further comprises calculating, by a 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 a 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 to 500 μm.

[0154] In some embodiments, the target polynucleotide is substantially linearized by the effect of adjusting 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 is across at least the second pore, creating a voltage gradient 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 the 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 any of one or more characteristics of the target polynucleotide and performs additional recapture of the one or more characteristics in the first direction and / or the second direction.

[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 on.

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

[0171] In some embodiments, the method further comprises scanning regions of the polynucleotide that have not already been scanned.

[0172] In some embodiments, the method further comprises constructing, by a 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 a 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 comprise: a) one or more payload molecules attached 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 each of 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, fourth, and fifth cycle, or when the polynucleotides exit the device.

[0187] In some embodiments, 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.

[0188] In some embodiments, the second set of features is larger than the first set of features.

[0189] In some embodiments, the sets of features across the 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 the 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 the direction of the target polynucleotide when the first set of features is detected.

[0194] In some embodiments, adjusting the first and second voltages 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 voltage 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 comprises 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 range 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 explanation of the drawings]

[0213] [Figure 1] Figure 1 depicts DNA-based tagging of λ-DNA molecules. The depicted tag is in the same position as mono-streptavidin. 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 GAA GAG (SEQ ID NO: 1). [Figure 2] FIG. 2 shows the modulation of nanopore impedance by varying the size and shape of DNA attachment. [Figure 3A] FIG. 3A shows an example of the evolution of ionic current over time as a detection parameter for a DNA molecule in a single pore 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 pore 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 pore 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 DNA molecules in a single pore and confirmation of tagging the DNA molecules with one or more probes. [Figure 4] 4 illustrates an example of reading the tagged molecule's region and then panning / gliding the molecule to different regions for detection. The algorithm involved in mapping one or more features of a molecule in this non-limiting example includes: (a) initializing scan count=0; (b) the dual system first detects the Ntag (Ntag=2 in this example) tag and begins flossing; (c) incrementing the scan count each time and starting 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; and (f) repeating steps (b) through (e) until the molecule exits. [Figure 5] FIG. 5 is a flowchart of the processor steps, highlighting steps 8 and 15-19 related to rescanning and gliding, with the states shown in bold text being key states that are highly relevant to rescanning and gliding. [Figure 6]6 is a diagram illustrating 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) initializing scan count = 0; (b) the dual system first detects Ntag (in this example, Ntag = 2); (c) incrementing scan count and starting a new scan each time; (d) after a certain number of flossing scans Nscan (in this example, Nscan = 4), the system increments the number of triggering tags to Ntag = Ntag + 1; resetting scan count to 0; (e) the system continues flossing based on Ntag; and (f) repeating steps (b) through (e) until the molecule is exited. [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 begins flossing; (c) Each time increment scan count and start a new scan; (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) through (e) until Ntag reaches Nmax (Nmax = 4 in this example); (g) The system continues flossing based on Nmax; (h) Stop incrementing scan count and hold flossing based on Nmax until molecules leave. [Figure 8A]Figure 8 shows a 3D schematic diagram of a dual-pore device. Two nanopores are located on the same membrane. Two "V"-shaped microchannels are attached to the glass, and the entire surface is covered with a SiN membrane. The two channels lead to a buffer center, where a micrometer bridge separates the two channels. A nanopore is drilled at the end of the channel. The same DNA molecule can span the two nanopores, achieving control of both pores. C, D, and E are top views of the device at different magnifications. C shows the entire chip with an 8 mm × 8 mm footprint. D is a zoomed view of a portion of the field showing the elbow of the v-channel. E is a focused 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 labeled Channel 1 and Pore 1, and the right side is labeled Channel 2 and Pore 2. G is the placement of electrodes to simultaneously draw the same molecules into the channel. This design facilitates separate access to the two pores, as the electrodes are positioned at the corners within the access ports and the common ground is located in the center. [Figure 8B]Figure 8 shows a 3D schematic diagram of a dual pore device. Two nanopores are located on the same membrane. Two "V"-shaped microchannels are attached to the glass, and the entire surface is covered with a SiN membrane. The two channels lead to a buffer center, where a micrometer bridge separates the two channels. A nanopore is drilled at the end of the channel. The same DNA molecule can span the two nanopores, achieving control of both pores. C, D, and E are top views of the device at different magnifications. C shows the entire chip with an 8 mm × 8 mm footprint. D is a zoomed view of a portion of the field showing the elbow of the v-channel. E is a focused 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 labeled Channel 1 and Pore 1, and the right side is labeled Channel 2 and Pore 2. G is the placement of electrodes to simultaneously draw the same molecules into the channel. This design facilitates separate access to the two pores, as the electrodes are positioned at the corners within the access ports and the common ground is located in the center. [Figure 9]FIG. 9 is a diagram showing DNA flushing by the force of competing voltages within a dual pore device. (a) After DNA is co-captured, the DNA molecules pass through from left to right (L to R) in a stitched manner 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 referred to as 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 molecules 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 outflow from pores 1→2 and 2→1 after 50+ scans, using logic where the predefined number of tags detected is 2. After this, the controller induces a change in direction. Signals between 30 - 150 ms are omitted for visualization. This example shows raw data from multiple scanning events. [Figure 10A]Figure 10 shows representative dual current signals and scan count statistics generated during a flossing experiment using MS-tagged DNA. a) Full signal traces for I1, V1, I2, and V2 are shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in on the vertical scale at low and high ranges between low and high V1 values. b) Zoomed-in on the first cycle, where the two-tag logic revealed resolvable tags A and B in both signals. c) Zoomed-in on the 41st and final cycle, where an undetected tag indicates the end of co-capture. d) Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts across all co-capture events for the device used (bin width = 4). The red line for the probability data is a fitted model (Eq. (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 using MS-tagged DNA. a) Full signal traces for I1, V1, I2, and V2 are shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in on the vertical scale at low and high ranges between low and high V1 values. b) Zoomed-in on the first cycle, where the two-tag logic revealed resolvable tags A and B in both signals. c) Zoomed-in on the 41st and final cycle, where an undetected tag indicates the end of co-capture. d) Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts across all co-capture events for the device used (bin width = 4). The red line for the probability data is a fitted model (Eq. (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 using MS-tagged DNA. a) Full signal traces for I1, V1, I2, and V2 are shown for a representative multi-scan flossing event. The break in the vertical axis of the I1 signal allows zooming in on the vertical scale at low and high ranges between low and high V1 values. b) Zoomed-in on the first cycle, where the two-tag logic revealed resolvable tags A and B in both signals. c) Zoomed-in on the 41st and final cycle, where an undetected tag indicates the end of co-capture. d) Total flossing time (mean ± standard deviation) and probability distribution vs. scan counts across all co-capture events for the device used (bin width = 4). The red line for the probability data is a fitted model (Eq. (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] Figure 11 shows that flossing increases the linearization of DNA in a dual-pore device. (a) Typical I1 trace of a single-pore event, including both unfolded and folded examples. Only single-pore events that occurred during co-trapping were included in the subsequent probability calculation (pre-1 step events in Figure 16). (b) Typical I2 trace of a multiple-scan event, where scan 1 indicates folding and subsequent scans indicate unfolding. (c) Illustration of the mechanism by which the folded portion (only initially in I2) is removed by the second 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 different transition types. A total of 309 events were performed, all in four types, sequentially. [Figure 11B]Figure 11 shows that flossing increases the linearization of DNA in the dual-pore device. (a) Typical I1 trace of a single-pore event, including both unfolded and folded examples. Only single-pore events that occurred during co-trapping were included in the subsequent probability calculation (pre-1 step events in Figure 16). (b) Typical I2 trace of a multiple-scan event, where scan 1 indicates folding and subsequent scans indicate unfolding. (c) Illustration of the mechanism by which the folded portion (only initially in I2) is removed by the second 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 different transition types. A total of 309 events were run, all in four types, sequentially. [Figure 12]Figure 12 shows the estimation of the separation distance between tags from the dual current signals generated during a multiple-scan experiment. The illustrations (a) L to R and (b) R to L help visualize the relative tag positions indicated by the scan signals. The signals (c) L to R and (d) R to L were from adjacent 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. Therefore, entry and exit relate to the direction of tag movement 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 they are in the common chamber, whereas (aii) visualizes B exiting but C before entering 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 tag pore-to-pore velocity was calculated by dividing the known distance between pores by the pore-to-pore time. The predicted separation distance between tags (separation distance) 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 voltage was set to V1 = 250 mV from L to R and 600 mV from R to L, while V2 was held constant at 400 mV. The FPGA monitored I2 for N=2 tags (exit signal L to R, entry signal R to L), and three tags were visible in 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 estimation 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 with HpaII (lanes 1-3) and Msp1 (lanes 4-6) for 0, 30, and 60 min of incubation. (b) Dual-pore rescanning data using a 100 s scan for MeCP2 and antibodies bound to the 5mC site of lambda DNA. (c) Comparison of tag signals for MS proteins (three tags spaced 301 bp and 323 bp apart) bound to biotin

[19] versus MeCP2 and antibodies bound to 5mC. Proteins of ~50 kDa in size produce similar blockade, while antibodies of 150 kDa produce deeper blockade, potentially enabling multiplexing. [Figure 16] Figure 16 shows the entire process of a tug-of-war event. (a) Represents each step. (b) Current traces from both Pore 1 and Pore 2. I1 is red, while I2 is 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. Blue boxes indicate system states, and magenta numbers represent FPGA states in (a). Black boxes indicate decisions. Green boxes indicate actions. Arrows between boxes indicate how the system flows between 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. Blue boxes indicate system states, and magenta numbers represent FPGA states in (a). Black boxes indicate decisions. Green boxes indicate actions. Arrows between boxes indicate how the system flows between different steps. [Figure 18A] Figure 18 shows four main cases where the FPGA cannot capture multiple scans. The plot shows the state signal from the FPGA and I2 of the final cycle. The state definitions are in Figure 17b. (a) The tag indicates rising 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 in the (n-1)th scan. (c) A false negative spike in 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. The plot shows the state signal from the FPGA and I2 of the final cycle. The state definitions are in Figure 17b. (a) The tag indicates rising 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 in the (n-1)th scan. (c) A false negative spike in 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. The plot shows the state signal from the FPGA and I2 of the final cycle. The state definitions are in Figure 17b. (a) The tag indicates rising 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 in the (n-1)th scan. (c) A false negative spike in 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. The plot shows the state signal from the FPGA and I2 of the final cycle. The state definitions are in Figure 17b. (a) The tag indicates rising 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 in the (n-1)th scan. (c) A false negative spike in 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 using three tag triggers. (a) Full signal trace of I1, V1, I2, and V2. V2 is set to V2 = 400 mV during the event, with V1 = 200 mV for the L to R scan and V1 = 800 mV for the R to L scan. (b) Zoomed-in plot of the second cycle. (c) Distribution of scan counts per event using theoretical fitting. [Figure 19B] Figure 19 shows a multiple scan experiment using three tag triggers. (a) Full signal trace of I1, V1, I2, and V2. V2 is set to V2 = 400 mV during the event, with V1 = 200 mV for the L to R scan and V1 = 800 mV for the R to L scan. (b) Zoomed-in plot of the second cycle. (c) Distribution of scan counts per event using theoretical fitting. [Figure 19C] Figure 19 shows a multiple scan experiment using three tag triggers. (a) Full signal trace of I1, V1, I2, and V2. V2 is set to V2 = 400 mV during the event, with V1 = 200 mV for the L to R scan and V1 = 800 mV for the R to L scan. (b) Zoomed-in plot of the second cycle. (c) Distribution of scan counts per event using theoretical fitting. [Figure 20]Figure 20 illustrates the tag alignment procedure. (a) Example of tag position versus scan number for an event with two tags (blue circles, tags measured closest to the start of the scans 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 for the event in (a) by the final average tag position (where blue circles correspond to measurements associated with tag A and red squares correspond to measurements associated with tag B). (e) Example of tag position versus scan number for an event with three tags (blue circles, tag positions closest to the start of the event, red squares at intermediate distances, magenta triangle tags observed further away from the start of the event). (f) Inter-tag distance for the event in (e). [Figure 21] Figure 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 for a nanopore device of the present disclosure. [Figure 23] FIG. 23 shows a graph plotting the relationship between channel resistance and channel width for devices with different geometries. [Figure 24] FIG. 24 shows a graph plotting the relationship between channel volume and channel width for devices with different geometries. [Figure 25] FIG. 25 shows a graph plotting the relationship between polynucleotide delivery time and channel width. [Figure 26] 20A-20C, wherein the target polynucleotide in "pre-i" includes a wait period of approximately 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 approximately 20 ms before the target polynucleotide changes direction. The total period was approximately 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 the 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 INVENTION

[0214] The figures depict embodiments of the present invention for illustrative purposes only. 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 containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-naturally occurring, or derivatized nucleotide bases.

[0216] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E. F. 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). 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-strand and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two distinct single-strand cleavage events.

[0219] The terms "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 targeted location within the genomic DNA of the cell). Examples of genome editing endonucleases include, but are not limited to, (a) Type II CRISPR / Cas proteins, such as Cas9 protein; (b) Type V CRISPR / Cas proteins, such as Cpf1 protein, C2c1 protein, C2c3 protein, etc.; and (c) Type V CRISPR / Cas proteins, such as Class 2 CRISPR / Cas endonucleases, such as 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 can be contained within a single polynucleotide strand, or the cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain can 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.) comprises 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 specific 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 binding pair member), 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, etc. "Binding pair partner" or "binding pair member" refers to one first and second moiety that have specific binding affinity for each other. 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 one another.

[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 this 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 is to be limited only by the appended claims.

[0225] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, to one-tenth of the unit of the lower limit, is included within the scope of the invention, 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 limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the invention.

[0226] Unless otherwise defined, 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, in which the publications are cited in connection with the methods and / or materials, are incorporated herein by reference to disclose and describe the methods and / or materials.

[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 the mutant dystrophin gene 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 using 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 embodiments belonging to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. 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 were 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 within a polynucleotide sequence. The present disclosure also provides a method for extended recapture of a polynucleotide within a pore device. A method for sequencing a polynucleotide within 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 characteristics of a polynucleotide sequence of a target polynucleotide passing through a first and second pore. The present disclosure also provides devices and systems for capturing a target polynucleotide. The present disclosure also provides devices and systems for sequencing the polynucleotide sequence of a target polynucleotide passing through a first and second pore.

[0232] Aspects of the present disclosure include a dual-pore, dual-amplification device for sequencing a polynucleotide sequence of a target polynucleotide passing through a first and a second pore. In some cases, the device includes: (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) the 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) 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 i) a non-transitory computer-readable medium comprising instructions that 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 opposite second force acting on the target polynucleotide, wherein 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 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 and middle chambers. In some embodiments, the device includes a second pore connecting the middle and bottom chambers. 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 fluid channel and the chamber of the device and a second voltage between the chamber and a second fluid 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 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.

[0234] In some embodiments, the first pore and the second pore are configured to allow the target polynucleotide to simultaneously translocate in a controlled manner across both pores in a first direction or a second direction. In some embodiments, the dual pore device comprises one or more sensors. In some embodiments, the one or more sensors are capable of identifying a first set of features from the target polynucleotide. In some embodiments, the sensors are capable of identifying a first set of features in a first cycle (e.g., a first cycle having one or more scans) during translocation 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 are capable of identifying a first set of features from the target polynucleotide. In some embodiments, the one or more sensors are capable of 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 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 a processor to determine, from one or more sensors, that a 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 the second direction. In some embodiments, this 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 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 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 fluid volume and the second fluid volume, each housing being filled with a fluid and including 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; the second fluid volume. (v) one or more sensors configured to independently control a voltage at each pore and measure a current; (vi) a processor; and (vii) a non-transitory computer-readable medium that controls the processor to: a) apply a first 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; 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. 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 migration 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 each feature at a second sensor current 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 movement of the target polynucleotide; g) detecting a second set of features of the target polynucleotide at a second sensor current as it passes through the second pore, and again detecting each feature at the first sensor current 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 each feature again as it passes through the second pore.

[0239] In some embodiments, after step f), the method further includes 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 in step f) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore and when at least a portion of the target polynucleotide passes again through the second pore.

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

[0244] In some embodiments, the method further includes 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 again detect any not yet detected features 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 amplification 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 spaced about 500 nm apart from each other.

[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 for a varying number 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 that cause the processor to calculate the 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 method further includes instructions that cause the processor to calculate the distance between features from the velocity of the features of the target polynucleotide, i.e., from the time between 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 use the distribution of velocities to calculate statistics of the feature 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 that 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 the multiple speeds improves the 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 velocities of the polynucleotide in a first direction, a second direction, or both.

[0284] In some embodiments, the method further includes instructions to cause the processor to control voltage ranges for the first and second pores as the polynucleotide moves 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, the system comprising: a) 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 fluidly 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 fluidly connected to the chamber and to the first fluid volume; (iii) a second pore fluidly connected to a second fluid volume, the second fluid volume being a geometrically constrained housing on an opposite side of the second pore; (iv) housings for the first and second fluid volumes, each containing 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; (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 measure a current; (b) a processor; and (c) a non-transitory computer readable medium, the device configured to cause the processor to: i) apply a first voltage at the first pore and a second 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 each feature again 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 each feature again 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 in step vii) corresponds to a number of characteristics that are detected when at least a portion of the target polynucleotide passes through the first pore and when at least a portion of the target polynucleotide passes again through the second pore.

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

[0296] In some embodiments, the method further includes 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 again detect any not yet detected features 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 amplification 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 spaced about 500 nm apart from each other.

[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 for a varying number 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 that cause the processor to calculate the 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 features from the velocity of the features of the target polynucleotide, i.e., from the time between 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 use the distribution of velocities to calculate statistics of the feature 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 that 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 the multiple speeds improves the 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 velocities of the polynucleotide in a first direction, a second direction, or both.

[0336] In some embodiments, the method further includes instructions to cause the processor to control voltage ranges for the first and second pores as the polynucleotide moves 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 passing through first and second pores, the device comprising: (i) electrodes connected to a power source configured to provide a first voltage between a first fluid channel and a chamber of the device, and a second voltage between the chamber and a second fluid 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 controlled manner in a first direction or a second direction; 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 containing instructions to cause 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 adjust one or both of the first and second voltages responsive to the counts to produce a first force and an opposite second force acting on the target polynucleotide, the first and second forces changing 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 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 fluidly 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 non-transitory computer-readable medium containing instructions that cause the processor to: a) apply a first voltage to capture and translocate 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 embodiments 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 fluidly 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) at least one electrode disposed within the first fluid volume and at least one electrode disposed within the chamber; and (b) a nanopore device comprising: a first electrode disposed within the first fluid volume and at least one electrode disposed within the chamber; and a sensor configured to provide a voltage between the first pore and a current measurement that detects capture and translocation of the polynucleotide into and through the first pore; (c) a processor; and (d) a non-transitory computer-readable medium containing instructions that cause the processor to: i) apply a first voltage to capture and translocate 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 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 to 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 Nos. WO / 2013 / 012881 (Patent Document 1), WO / 2018 / 236673 (Patent Document 2), U.S. Application Publication No. 2017 / 0145481 (Patent Document 3), and U.S. Patent No. 9,863,912 (Patent Document 4), 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 U.S. Patent Application Publication No. 2017 / 0145481 (Patent Document 3), 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, referring to the size of the unique feature. 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 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 pore diameters range from about 2 nm to about 50 nm. In some embodiments, the pore diameters are about 20 nm. In variations, the pores have depths in the range of 1 to 10,000 nm, although in other variations, the nanopores may have depths less than 1 nm or greater than 10,000 nm. Furthermore, the diameter of the nanopores may vary (within appropriate ranges) during the conduct of an experiment, 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 fluid 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 fluid channel.

[0345] In some aspects, each of the pores in 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% cylindrical in shape. 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 a 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 a layer formed from a combination of 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 separated by less than 5 nm or more than 15,000 nm. Furthermore, the nanopores can be arranged in any position, so long as they allow fluid communication between the chambers and have a defined size and distance between them. In some embodiments, the first pore and the second pore are separated by about 10 nm to 500 nm. In some embodiments, the first pore and the second pore are separated by about 500 nm. In one variation, the nanopores are positioned so that there is no direct blockage between them. Furthermore, in one form, the pores are substantially coaxial.

[0350] 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 first and / or second 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.

[0351] In some embodiments, the device includes a geometrically constrained fluid volume. In some cases, the geometrically constrained fluid volume is a fluid channel. In some cases, the device includes a first fluid 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 fluid 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 "bottom chamber" is used interchangeably with the terms "fluidic channel" and "geometrically constrained fluid volume," e.g., a second fluid channel. In some embodiments, the device includes a bottom chamber. In some embodiments, the device includes a second fluid 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 so 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 over the first and second pores. In some embodiments, the chamber is disposed over 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 fluid channel can be circular, square, rectangular, hexagonal, triangular, oval, polygonal, V-shaped, U-shaped, or any other suitable shape. In some cases, the first fluid channel and the second fluid channel 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 facing each other at predetermined points of the adjacent V-shapes, the first nanopore positioned at a predetermined point of the V-shape of the first fluid channel, and the second nanopore positioned at a predetermined point of the V-shape of the second fluid channel. In some embodiments, each of the fluid channels has a different shape. The fluid channels are not limited to the shapes and / or sizes described herein and can be any shape and / or size required by the specified conditions 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, and 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 apply 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 ground relative to the two voltages. In one embodiment, the chamber includes a medium for providing conductance between each pore in the chamber and an electrode. In one embodiment, the chamber includes a medium for providing resistance between each nanopore in the chamber and an electrode. Maintaining such resistance sufficiently small compared to the resistance of the nanopore is effective in separating the two voltages and currents across the pore, which facilitates independent adjustment of the voltages.

[0356] Adjusting 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 fluid channel to the chamber and to a second fluid 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 fluid channel or the second fluid 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 speed of the molecule's 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 to 200 mV, 200 to 300 mV, 300 to 400 mV, 400 to 500 mV, 500 to 600 mV, 600 to 700 mV, 700 to 800 mV, 800 to 900 mV, 900 to 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 100-200 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 second initial voltage is 100 mV, 200 mV, 300 mV, 400 mV, 500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or 1000 mV.

[0359] In some cases, the disclosed methods include adjusting a first and / or second voltage to control movement of a target polynucleotide within a first pore, a first fluidic channel, a second pore, a second fluidic channel, and / or a chamber of a device. In some cases, the first voltage is adjusted to 0 mV after the target polynucleotide has moved from the chamber through the first pore to the first fluidic channel. In some cases, the first voltage is adjusted to 0 mV before translocation through the first pore, and at least a portion of the target polynucleotide is disposed in the chamber and 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 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 directions. 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 directions. 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, a charged polymer, such as a polynucleotide, has a length greater than the combined distance of the depth of both pores plus the distance between the pores. For example, a 1000-bp dsDNA is approximately 340 nm long, which is substantially longer than 40 nm across two 10-nm-long pores separated by 20 nm. In the first step, the polynucleotide is introduced into either the first fluidic channel or the second fluidic channel. In the first step, the polynucleotide is introduced into a chamber (e.g., the intermediate chamber or the common chamber) of the device. Due to its negative charge under physiological conditions (approximately pH 7.4), the polynucleotide can be translocated across the pore to which a voltage is applied. Therefore, in the second step, two voltages are applied to the pores in the same direction and with the same or similar magnitude, subsequently inducing translocation of the polynucleotide across both pores. One or both voltages can be changed when the polynucleotide nearly reaches 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, and therefore, 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 can 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 pores. Each voltage clamp or patch clamp system measures the ionic current through each pore, and this measured ionic current can be used to detect one or more characteristics of, 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 a significantly slower speed. At this point, the amplifier that supplies 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 a shift in the magnitude of the ionic current). Thus, the identity of each nucleotide within a polynucleotide reveals the sequence of the polynucleotide.

[0363] In some embodiments, the magnitude of the adjusted first and second voltages in steps is about 10 to 10,000 times higher 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. In some embodiments, the magnitude of the voltage is no more 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 sequencing, with each voltage being alternated for longer periods of 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 fabricated by a variety of means and methods. A focused electron or ion beam can be used to drill pores through a membrane and naturally align them. The pores can also be sculpted (reduced) to smaller sizes by applying a focused beam to each layer. Any method for drilling a single pore (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 create 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 use insulators such as SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or evaporated metal 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 in the Debye pore can cause charged molecules to move between chambers through the pore. The speed and direction of movement can be controlled by the magnitude and polarity of the voltage. Furthermore, because each of the two voltages can be adjusted independently, the direction and speed of movement of the charged molecules can 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 second voltage can 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 a second direction.

[0369] In some aspects, the nanopore device includes a polymer transport 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 can be used to identify one or more features of a polymer. In some embodiments, the one or more features are one feature, two features, three features, four features, or five features. In some embodiments, the one or more features are two or more features, three or more features, four or more features, five or more features, six or more features, seven or more features, eight or more features, nine or more features, or ten 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, from 100 features to 1,000 features, from 1,000 features to 10,000 features, from 10,000 features to 100,000 features, or from 100,000 features to 200,000 features. In some embodiments, the one or more features range from 50 features or more, from 100 features or more, from 1,000 features or more, from 10,000 features or more, from 100,000 features or more, or from 200,000 features or more.

[0371] Aspects of the present disclosure include one or more features, each 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 embodiments, each feature 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 a combination thereof. In some embodiments, each feature 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 a combination thereof.

[0372] In some aspects, the dual pore device can 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, a set of features (e.g., a first set, a second set, a third set, a fourth set, a fifth set, a sixth set, a seventh set, an eighth set, a ninth set, and / or a tenth set) is associated with a first cycle, a second cycle, a third cycle, a fourth cycle, a fifth cycle, a sixth cycle, a seventh cycle, an eighth cycle, a ninth cycle, and / or a 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 a third set of features. In some cases, the second 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 second 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 second cycle includes five or more scans, ten or more scans, fifteen or more scans, twenty or more scans, twenty-five or more scans, thirty or more scans, thirty-five or more scans, forty or more scans, forty-five or more scans, or fifty 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 present disclosure include a processor and a computer-readable medium, the computer-readable medium comprising instructions that 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 the movement of the polynucleotide (e.g., in either direction) for a third, fourth, and 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 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, one or more features of the polynucleotide comprise one or more payload molecules attached to the polynucleotide. In some embodiments, one or more features of the polynucleotide comprise one or more payload molecules hybridized to the polynucleotide. In some embodiments, one or more features of the polynucleotide comprise one or more payload molecules incorporated into the genome of the polynucleotide. In some embodiments, one or more features of the polynucleotide comprise a molecular motif in the polynucleotide sequence of the target polynucleotide. In some embodiments, one or more features comprise the following positions: one or more CpGs, or one or more methylation sites and CpGs, in the polynucleotide sequence of the target polynucleotide. In some embodiments, one or more features comprise one or more histone positions in the target polynucleotide. In some embodiments, one or more features comprise 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, 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-heparin, a DNA molecule, an aptamer, or a combination thereof.

[0378] Non-limiting examples of payload molecules conjugated to polynucleotides can be found in U.S. Patent Publication No. 2018 / 0023115, which is incorporated herein by reference in its entirety. For example, payload molecules can include dendrimers, double-stranded DNA, single-stranded RNA, DNA aptamers, fluorophores, proteins, polypeptides, nanorods, nanotubes, fullerenes, PEG molecules, liposomes, or cholesterol-DNA hybrids. 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 metallic bonds. The payload adds size to the target polynucleotide or amplicon, facilitating detection by ensuring that amplicons conjugated to the payload have a current signature significantly different from background molecules when passed through a 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 bulk of the molecule, which can increase the sensitivity of amplicon detection 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 bulk can result in a more distinct signature between amplicons containing the target sequence and background molecules.

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

[0380] In some embodiments, primers contain chemical modifications that facilitate and facilitate recognition and binding of payload molecules. For example, methylated DNA sequences are 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 so that the payload-binding domain becomes the primer-binding domain, or 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 a locked nucleic acid (LNA), a bridged nucleic acid (BNA), any type of protein nucleic acid (e.g., bisPNAs, gamma-PNAs), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeat (CRISPR), or an aptamer (e.g., DNA, RNA, protein, or a combination 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 comprise one or more features within a polynucleotide. In some embodiments, the one or more features within a polynucleotide comprise one or more modifications to the polynucleotide. In some embodiments, the one or more modifications comprise DNA methylation (e.g., 5mC, 5hmC, 5mA, etc., at CpG dinucleotides). In some embodiments, the one or more features within a polynucleotide comprise sequence variation, mutation, or large structural variation. In some embodiments, the one or more features of a polynucleotide comprise 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 contains one or more nick sites. As a non-limiting example, a nicking restriction endonuclease introduces a nick at a barcoding recognition sequence. This sequence occurs multiple 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. DNA molecules labeled with DCBO at either the 5' or 3' end, or DNA molecules labeled in the body, are added to the reaction. The DNA molecules are covalently linked to the nick site via copper-free click chemistry. A 1000- to 10,000-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, introducing a single-stranded nick. 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 labeled with DCBO at either the 5' or 3' end, or backbone labeled, are added to the reaction. The DNA molecules are covalently linked to the nick sites via copper-free click chemistry. A 1000- to 10,000-fold excess of DNA molecules can 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 a manner that allows at least a portion of the target polynucleotide to translocate 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 capable of identifying the target polynucleotide as it translocates 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 on either side of the pore and is capable of measuring 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 easily be 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, two or more pores exist between two adjacent chambers, allowing multiple polymer scaffolds to move simultaneously from one chamber to the next. Such a multi-pore design can increase the throughput of target polynucleotide analysis in the device. For multiplexing, one chamber can contain one type of target nucleotide, and another chamber can contain 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 simultaneously introducing the target polynucleotide (e.g., an amplification product or amplicon containing the target sequence) across both the first pore and the second pore. In another embodiment, the means further enables transfer of the target polynucleotide through both pores in the same direction.

[0392] Although several variations of nanopore devices are described above, the nanopore device(s) may be configured as described in U.S. 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 herein by reference in their entireties.

[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 can include any sensor suitable for identifying a target polynucleotide amplicon, whether bound or unbound to a payload molecule. For example, the sensor can be configured to identify a target polynucleotide by measuring a current, voltage, pH value, optical characteristic, or residence time associated with the polymer. In other embodiments, the sensor can 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 can be formed of any element configured to detect a change in a measurable parameter, which change 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 positioned 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 certain embodiments, the ionic current across the pore measurably changes when a target polynucleotide segment passing through the pore is bound to a payload molecule. Such changes in current flow may, 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 used to apply a voltage and measure the current across the nanopore. Translocation of a molecule through the nanopore imparts an electrical impedance (Z), which 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. When a molecule translocates through the nanopore in an electric field (e.g., under an applied voltage), the result is a current signature that can be correlated to the molecule passing through the nanopore upon further analysis of the current signal.

[0396] 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 involves identifying 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. A radiation source at the exit of the pore can be used to detect the signature. Non-limiting examples of nanopore device sensor circuits can be found in PCT Application Publication No. WO2018 / 236673 (Patent Document 2), 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 a set of one or more nanopore devices and include an electronics subsystem for receiving electrical signals from sensors in 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 present disclosure include a device including a processor. In some embodiments, the device includes a non-transitory computer-readable medium containing 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 opposing 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 a 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 polynucleotide exits 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 containing 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 containing one or more features based on the scan. In some embodiments, the construction comprises a machine learning algorithm trained to detect one or more features based on training data and probabilistic models, as described in more detail below.

[0402] Aspects of the present disclosure include a device including 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 target polynucleotide, orientation of 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 for 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 ion currents, or both.

[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: continuing to scan the same region of the molecule for additional features, continuing to scan the same region of the molecule for rescanning the same probe already detected, scanning the same region with a varying number of probes, or moving to a different region of the molecule for scanning based on the determination, in which case the system may include structure for carrying out embodiments of the present methods as described in more detail below.

[0406] The devices and systems may also generate notifications for system operators. 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 the system's control elements, the status of one or more nanopores, instructions for adjusting the operation of the system, instructions for advancing 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), an audio format (e.g., using a speaker), tactilely (e.g., using a tactile device), and / or other suitable format.

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

[0408] An embodiment of a machine learning architecture associated with the described system and method embodiments "learns" when to move from one location to another in a target polynucleotide, when to sequentially scan one or more features, when to vary the number of features to scan, and when to switch from sequentially scanning one or more features to a location that has not yet been explored / scanned. The goal of automated operation is to generate enough useful data 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 over a predetermined 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 can operate on hardware with limited processing power that needs to react in real time / near real time. While Bayesian optimization is described, other statistical and / or machine learning approaches can be used for automated exploration or probing to generate molecular maps. In variations, such models perform 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 nets, 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 the like. Any one or more of the following algorithms may be implemented: random generalization, AdaBoost, stacked generalization, gradient boosting machine methods, random forests, etc., and any suitable form of algorithm.

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

[0411] In some aspects, devices and systems of the present disclosure include a non-transitory computer-readable medium containing instructions to cause a processor to: i) determine from the sensors 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, responsive 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, 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 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. In some embodiments, the controller is a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC).

[0413] Aspects of the present disclosure include devices for performing the functions of the methods described herein. The present disclosure includes a device for mapping one or more features of a polynucleotide sequence of a target polynucleotide passing through first and second pores, 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) the 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) a device for identifying, in a first cycle, 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, 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. (v) a processor; and (vi) a non-transitory computer-readable medium containing instructions that cause the processor to: a) determine from the one or more sensors the simultaneous presence of the 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 opposite second force acting on the target polynucleotide, wherein 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 the second direction; and d) repeat steps a) through c) and detect a third and fourth set of features in a second cycle.

[0414] In some cases, the instructions cause the processor to further repeat step 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 pore diameter is in the range of about 2 nm to about 50 nm. In some cases, the pore diameter is about 20 nm. In some cases, the first and / or second pore diameter is in the range of about 2 nm to about 50 nm. In some cases, the first and / or second pore diameter is in the range of about 2 nm to about 8 nm. In some cases, the first and / or second pore diameter is in the range of about 10 nm to about 20 nm. In some cases, the pore diameter is in the range of about 20 nm to about 30 nm. In some cases, the first and / or second pore diameter is in the range of about 30 nm to about 40 nm. In some cases, the first and / or second pore diameter 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 spaced about 500 nm apart. 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 includes 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) movement of the target polynucleotide, f) direction of the target polynucleotide, g) 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 for causing the processor to construct a local map of regions of the polynucleotide containing one or more features based on the scan. In some cases, the device further includes instructions for causing the processor to construct a consensus map of regions 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 one or more features based on training data and a probabilistic model. In some cases, the device further includes instructions for causing 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 includes instructions that cause the processor to calculate the distance between features from the velocities of the target polynucleotide features, i.e., from the time between features detected in the current signal from the first pore, the second pore, or both. In some cases, the device further includes instructions that cause the processor to calculate the velocities of the target polynucleotide features for every scan and to calculate statistics of the feature velocities using the velocity distribution. In some cases, the device further includes instructions that cause the processor to combine all of the feature velocities and calculate the time history of the polynucleotide velocity for a given scan and a given scanning direction.

[0422] In some cases, the device further includes instructions for causing 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 for causing the processor to perform an amplitude sweep of the polynucleotide in the first direction, a second direction, or both. In some cases, the device further includes instructions for causing 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 for causing 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 the accuracy of detection of one or more features. In some cases, the device further includes instructions for causing 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 for causing 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 for determining an optimal velocity range for 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 the 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 systems for practicing the methods described herein, including: a) a dual-pore, dual-amplification device for mapping one or more features of a polynucleotide sequence of a target polynucleotide through first and second pores, 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) the 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, in a first cycle; and (iv) identifying a first set of features from the target polynucleotide during translocation of the target polynucleotide through the first and second pores in a first direction, and a second set of features from the target polynucleotide during translocation of the target polynucleotide through the second and first pores in a second direction, in a first cycle. a device including one or more sensors that enable the simultaneous presence of a target polynucleotide in both pores; c) a processor; and d) a non-transitory computer-readable medium containing 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 opposite second force acting on the target polynucleotide, wherein 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 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 scan a varying number of features of the polynucleotide. 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 includes instructions that cause the processor to construct a local map of regions of the polynucleotide containing one or more features based on the scan. In some embodiments, the system further includes instructions that cause the processor to construct a consensus map of regions of the polynucleotide containing one or more features based on the scan. In some embodiments, the construction includes a machine learning algorithm trained to detect the one or more features based on training data and probabilistic models.

[0431] In some cases, the system further includes instructions for causing the processor to calculate a velocity of a 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. In some cases, the system further includes instructions for causing the processor to calculate a distance between features from the velocity of the feature of the target polynucleotide, i.e., from the time between features detected in the current signal from the first pore, the second pore, or both. In some cases, the system further includes instructions for causing the processor to calculate the velocity of the feature of the target polynucleotide for every scan and to calculate statistics of the feature velocities using the velocity distribution. In some cases, the system further includes instructions for causing 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 for causing 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 for causing 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 for causing the processor to perform multiple scans of the polynucleotide at multiple velocities. In some cases, the system further includes instructions for causing 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 for causing 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 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. In some cases, adjusting the voltage to perform multiple scans at different velocities improves the comprehensiveness of the data for which features are mapped. For example, at higher velocities (i.e., when the voltage difference is greater), molecules (e.g., polynucleotides, payload molecules, etc.) are more likely to be deterministic and are less affected by Brownian motion (e.g., Brownian motion will less likely "contaminate" 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 for causing 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 the 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 adjusting 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 passing 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 that enables a) determining the simultaneous presence of the target polynucleotide in both pores from the one or more sensors; b) scanning 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) when the first set of primers is detected in the first cycle in the first orientation, adjusting the first voltage, the second voltage, or both for the first and second pores 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) when the target polynucleotide is in both pores simultaneously in the second orientation, detecting the presence of the second set of primers in the first cycle; 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) through g) and detecting the third and fourth sets 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 passing 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) a device including 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. v) 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; 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. An adaptive controller can vary the number of features to find which molecular features and locations to rescan (e.g., bidirectional scanning) and / or when to move to different, previously unexplored locations in near real time. The adaptive controller can create real-time local maps of one or more features and generate datasets for creating a consensus map of the entire molecule. When implementing a machine learning algorithm, a 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 indicates 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 feature fluctuations. Using active logic based on field programmable gate arrays (FPGAs) or application specific integrated circuits (ASICs), the direction of molecular transition 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 specific types of "events" for experiments / reagents, generate characteristics for all types of molecular detections (e.g., relative to the value of a criterion), and generate characteristics for other distributions (e.g., distributions of reagent characteristics 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, predicted CpG distance / mapping, performance, predicted fraction of 5hmC vs. 5mC, predicted genomic distance, distance between molecular motifs, and density of molecular motifs. Models can be developed using a supervised learning approach, using algorithms to process tagging distributions or other aspects of the training data and place them into excursion types. 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 corrective actions, including one or more of: reversing the polarity of the voltage; applying an electrical stimulus at a specific voltage (and / or 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 / probing is performed; changing the number of molecular motifs for probing / probing; 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 their locations and spacing, and feed this into a deep Q-learning algorithm to develop adaptive logic and continuous refinement of the consensus mapping. This technique improves future decisions made by the system for automated molecular exploration and probing for map generation.

[0445] Additional 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 convenient at times 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 present 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 embodiment 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 methods for recapturing a polynucleotide in a nanopore, the method comprising providing a nanopore device for controlling the 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 fluid volume, the first fluid volume being a geometrically constrained enclosure having an opening opposite 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 translocate 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 in the first fluid volume. In some cases, the method includes applying a second voltage to reverse the direction of the polynucleotide located in the first fluid volume, wherein the second voltage translocates 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 so that the polynucleotide is maintained within the first pore. In some cases, the method includes detecting a second ionic current within 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 fluid 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 two or more pores, three or more pores, four or more pores, five or more pores, six or more pores, seven or more pores, eight or more pores, nine or more pores, or ten 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 constraining of the first fluid channel is increased while a first voltage applied at the first pore is maintained. In some embodiments, the geometric constraining of the first fluid channel is increased while a first voltage applied at the first pore is maintained with respect to the geometric constraining of the cavity. In some embodiments, the first voltage applied at the 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 to 1000 milliseconds, 1 to 20 milliseconds, 20 to 60 milliseconds, 60 to 120 milliseconds, 120 to 150 milliseconds, 150 to 300 milliseconds, 300 to 500 milliseconds, 500 to 1000 milliseconds, 1 to 20 seconds, 20 to 60 seconds, 60 to 120 seconds, 120 to 150 seconds, 150 to 300 seconds, 300 to 500 seconds, or 500 to 1000 seconds. In some embodiments, the first voltage applied at the first pore that captures and translocates a polynucleotide passing through the first pore (e.g., from a 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 translocation of a 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 openings 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, adjacent to each other, and 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 has a different shape. The fluid channels are not limited to the shapes and / or sizes described herein and can be any shape and / or size required by the specified conditions for their intended use. In some cases, the first fluid channel and / or the second fluid channel comprise 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 to 500 μm. In some cases, the first fluid channel and / or the second fluid channel have a width in the range of 50 to 100 μm, 100 to 150 μm, 150 to 200 μm, 200 to 250 μm, 250 to 300 μm, 300 to 350 μm, 350 to 400 μm, 400 to 450 μm, or 450 to 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 fluid channel and / or the second fluid channel have a depth ranging from 0.5 μm to about 2 μm. In some cases, the first fluid channel and / or the second fluid channel have a depth ranging from 0.5 to 1 μm, 1 to 1.5 μm, or 1.5 to 2 μm. In some cases, the first fluid channel and / or the second fluid channel have a depth ranging from 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 opposite sides 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 opposite sides of the second pore. In some cases, the first fluid volume and / or the second fluid volume includes two openings on opposite sides of the first pore and / or the second pore. In some cases, the two openings of the first fluid channel are coplanar and parallel to each other. In some cases, the two openings of the second fluid channel are coplanar 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 moves 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 moves 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 at a distance from at least the first pore in a range of 5 microns to 5 millimeters or more. In some cases, the second end of the polynucleotide is positioned at a distance from at least the first pore in a range of 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 period ranging from 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 polynucleotides from exiting the device for a period ranging from 5 ms to 5 minutes. In some cases, the method prevents polynucleotides from exiting a chamber of the device for a period ranging from 5 ms to 5 minutes. In some cases, the method prevents polynucleotides 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 with respect to the first voltage.

[0462] In some cases, the first pore has a diameter ranging from about 2 nm to about 50 nm. In some cases, the first pore has a diameter ranging from about 15 nm to about 30 nm. In some cases, the second pore has a diameter ranging from about 2 nm to about 50 nm. In some cases, the second pore has a diameter ranging from 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 within 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 to 1000 mV. In some cases, 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 includes adjusting a first voltage to the first pore, adjusting a third voltage to the second pore, or adjusting both, to change the direction of the polypeptide so that at least a portion of the polynucleotide translocates from the second pore through the first pore in a fourth direction (the fourth direction is from the second pore to the first pore). In some cases, adjusting the first voltage to the first pore, adjusting the third voltage to the second pore, or both, so that at least a portion of the polynucleotide translocates 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 includes 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 within 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 previously 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 the direction of the polynucleotide through the first and / or second pore by a controller, a processor, and a non-transitory computer-readable medium including instructions to the processor for changing 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 the detection of polynucleotides comprising polynucleotide sequences having lengths ranging from 5 base pairs to about 3,000,000 base pairs.

[0479] Method - Flossing and recapturing for one or more features The present disclosure provides methods for mapping one or more features of a polynucleotide sequence of a target polynucleotide bound to a plurality of probes through pores. The present disclosure further provides methods 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 automated methods for mapping one or more features of a target polynucleotide. The present disclosure also provides automated methods for sequencing a polynucleotide sequence. The present disclosure also provides methods for differential detection of methylated and unmethylated regions within 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 methods for 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; (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 either 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 migrated in a first direction (wherein the first direction is from the first pore to the second pore) through a first pore; setting a first initial voltage at the first pore and a second initial voltage at the second pore so that the target polynucleotide migrates through the first pore and the second pore; d) scanning for 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 direction; f) when the first set of features is detected in the first cycle in the first direction, adjusting the first voltage, the second voltage, or both for the first and second pores to change the orientation of the target polynucleotide so 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 direction; 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 a 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, the velocity of the target polynucleotide feature 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 a processor, a time history of the velocity of the polynucleotide using the velocities of all 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, using 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 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 a 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 a 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 attached 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.

[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 includes determining the position of one or more features of the target polynucleotide. In some cases, the method further includes determining the distance between each of the one or more features of the target polynucleotide. In some cases, the method further includes determining the distance between each feature in a first set of features of the target polynucleotide. In some cases, the method further includes determining the 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 in the polynucleotide sequence of the target polynucleotide. In some cases, the one or more characteristics include the position of one or more nucleosomes in 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 in the target polynucleotide, or the modification state and position of one or more histones contained within one or more nucleosomes in 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 histones contained within the nucleosome.

[0500] In some cases, the method further comprises identifying one or more characteristics of the target polynucleotide by measuring the 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, and 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 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 five or more scans, ten or more scans, fifteen or more scans, twenty or more scans, twenty-five or more scans, thirty or more scans, thirty-five or more scans, forty or more scans, forty-five or more scans, or fifty or more scans. In some cases, the second cycle includes one or more scans performed by the processor to detect a third set of features. In some cases, the second 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 second 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 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 a third, fourth, and fifth cycle, or as 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 characteristics include a DNA-binding protein.

[0506] In some cases, the method further includes 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 include 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 position of one or more methylcytosines (5mC) in the target polynucleotide. In some cases, the method further comprises determining the position of one or more 5-hydroxymethylcytosines (5hmC) in the target polynucleotide. In some cases, the method further comprises determining the position of one or more methylcytosines (5mC) and one or more 5-hydroxymethylcytosines (5hmC) in the target polypeptide. In some cases, the method further comprises determining one or more CpGs in 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 linked at the nick site of the target polynucleotide. In some cases, the polypeptide is monostreptavidin (MS) incorporated into the nick site of the polynucleotide sequence.

[0508] In some cases, the method further includes distinguishing between 5mC and 5hmC regions of the polynucleotide. In some cases, 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 cases, the one or more features include 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 derivatized 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 the 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 the 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 voltage with 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 scan or rescan cycles, 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, the features are 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, the features are 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, the features are 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 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 velocities. In some cases, performing multiple scans of the polynucleotide at multiple velocities improves the accuracy of detection of one or more features. In some cases, the method includes performing multiple scans of the polynucleotide at multiple velocities. 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, wherein the optimal velocity range for the polynucleotide reduces the 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 present 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 simultaneously through the first and second pores, 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, wherein the first and second pores are configured to detect when the target polynucleotide is 5mC or 5hmC in both pores. The method includes providing a device configured to simultaneously move one or more target polynucleotides 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 to cause at least a portion of the one or more target polynucleotides to move 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 labeled 5mC region of the polynucleotide sequence and each labeled 5hmC region 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; and 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, 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 characteristics 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 characteristics of at least a portion of the 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 (e.g., at least a portion of the target polynucleotide migrating from the first pore, through the chamber, to the second pore) in a third direction (the third direction is from the first pore 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 time period ranging from 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 experiences 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 in a time period ranging from 0.01 ms to 1000 ms. In some cases, the molecule exits the device in a time period ranging from 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 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 / s. 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 from 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 particle radius.

[0521] In some cases, a target polynucleotide is introduced into a 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 fluid channel) and / or a second direction (e.g., from the first fluid channel through the first pore to the chamber) selects 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, 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 velocity 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 the first and second pores provide independent sensors that synchronously measure different regions of the same polynucleotide, enabling sequential detection of one or more features. In some cases, the control logic is a controller. In some cases, the controller includes FPGA or ASIC circuitry and performs active feedback control. In some cases, the control logic is configured to promote 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 polynucleotides until the target polynucleotides exit 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 a 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 a 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 simultaneous movement of a target polynucleotide 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 within the first and second fluid channels, the first pore providing a first voltage at the first pore and the second voltage at the second pore. a) 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; (b) one or more electrodes configured to provide a second voltage at each pore, wherein each voltage is independently regulated; (c) one or more sensors configured to independently control the voltage at each pore and measure the 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; a) applying a first voltage at the first pore and a second voltage at the second pore so 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; b) scanning one or more features of the target polynucleotide; c) detecting a first set of features in a first cycle as the target polynucleotide passes through the first pore; and 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, thereby moving at least a portion of the target polynucleotide out of the first fluid 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 the first set of characteristics in the 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 set of features 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 translocates across the second pore and the first pore in a fourth direction (the fourth direction is 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 simultaneously within both pores 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 pore being a geometrically constrained housing having an opening on an opposite side of 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 transmitting the first voltage across 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 within 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 within the first pore; and g) detecting a second ionic current within 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 a respective direction 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; (iv) 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 fluid 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. 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, capturing the target polynucleotide in the first pore and allowing the target to pass through the first pore and into the first fluid channel; b) scan one or more features of the target polynucleotide; c) receive from the one or more sensors the first set of features; 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, responsive to the counting, adjusting one or both of the first and second voltages so that at least a portion of the target polynucleotide moves out of the first fluid 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 so 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 so 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 the 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 present disclosure include a method for determining the 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 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, wherein each voltage is 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 comprising dual amplification electronics configured such that a first and second pore can simultaneously translocate a target polynucleotide in a first direction or 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 translocates 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 the first set of primers in the first cycle when the target polynucleotide is in both pores in the 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 in the second cycle. h) changing the orientation of the target polynucleotide so that it moves from the second pore to the first pore in two directions; h) 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 the third and fourth sets of primers in a second cycle.

[0527] Finally, the terminology used in the specification has been chosen primarily for purposes of readability and direction, 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, 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 foregoing, certain non-limiting aspects of the disclosure, numbered 1 through 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 used or combined with any of the preceding or following individually numbered aspects. This provides support for all such combinations of aspects and is not intended to be limited to the combinations of aspects explicitly provided below. [Example]

[0529] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent all or the only examples performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation are expected. 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. Standard abbreviations may be used, such as bp for base pairs, kb for kilobases, pl for picoliters, s or sec for seconds, min for minutes, h or hr for hours, aa for amino acids, kb for kilobases, bp for base pairs, nt for nucleotides, im for intramuscular, ip for intraperitoneal, sc for subcutaneous, etc.

[0530] Example 1: Nanopore flossing DNA in a dual pore device Here, we present an active control technique, termed "flossing," 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 of lambda DNA are used as detectable features to actively trigger bidirectional control. Molecular noise is suppressed by averaging data from multiple scans, resulting in estimates of averaged inter-tag distances that can be compared with known values. Because nanopore feature mapping applications require linearization of DNA as it passes through the pore, a key advantage of flossing is that the second scan increases trans-pore linearization to >98%, compared to 35% for the same set of molecules passing through a single nanopore. In conjunction with barcoding methods, the dual-pore flossing technique enables applications in genome mapping and structural variation, or allows for 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 repeatedly scanning the trapped molecule back and forth in a nanopore device. Using a novel active control technique called "flossing," we 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 a 48.5 kbp double-stranded λ-DNA with a set of chemically incorporated sequence-specific protein tags. This experimental approach captures the benefits of existing carrier-strand DNA nanopore technology by enhancing the quality of information extracted from a single trapped molecule. By taking multiple statistically equivalent scans, stochastic fluctuations are reduced by scan averaging, providing an average tag-spatial estimate comparable to known inter-tag distances, even allowing for missed tags within a subset of scans.

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

[0533] Another distinctive feature of the presented approach is that an active controller periodically adjusted the voltage at one pore by feeding back the sensing current of the other pore in real time. Specifically, during control, repeated application of unbalanced, competing voltage forces was used to drive molecular movement in one direction, then in the reverse direction after real-time detection of a set number of tags, using a DNA "flossing" concept. Binding DNA to a predetermined state comes at the cost of complex instrumentation, high sensing noise, and low throughput, but it provided both rate control and minable data generation during molecular movement through a solid-state pore. In the presented flossing control method, the interrogated DNA molecule was expelled from the pore, and new DNA was 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 Figure 9 introduces the general flossing concept and shows repeated bidirectional scans 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 is defined as pore 1 and the right pore is defined 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 below.

[0536] The flushing control logic starts by first co-trapping the DNA tagged at both pores to reach the towing state. When co-trapping occurs, a voltage lower than that of pore 2 is applied across pore 1 (V1 < V2), inducing the movement of molecules towards pore 2 (from left to right or L to R). While monitoring I2, after the set number N of tags, V1 > V2 is detected, and the control logic readjusts the voltage at pore 1 so as to transfer through pore 2. The readjustment induces the return of the molecular movement towards pore 1 (R to L). After detecting that the same set number N of tags has transferred through pore 2, the logic resets V1 < V2 to start 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 cycle of multiple scans was started with molecules moving from L to R for the first 9 ms and continued until 172 ms when the 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 test are provided in Tables S1 - S2 (Figures 18 - 19). Although the general concept of flushing was described, the method will next be shown in more detail and with the results obtained using the method.

[0537] Initializing towing and specifying the scan voltage 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 co-capture and tug-of-war initialization of single molecules. 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 the 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 induce tug-of-war, slowing the DNA velocity during detection. The average duration of all co-capture events was calculated for each different set of V1 while holding V2 constant, resulting in a bell-shaped curve with a peak indicating the force-balancing voltage that maximizes the duration of co-capture. Figure 16c shows an example device with a peak average duration of 110 ms when both V1 and V2 were set to 500 mV. This data was generated with bare DNA without tags.

[0538] While molecules were captured with velocity and conformational control induced by the tug-of-war of the dual pore, bare double-stranded DNA did not present detectable features for monitoring molecular translocation. To enable in situ feature monitoring, MS-tagged DNA, tagged at up to seven sites on the DNA, was developed as a model reagent (SI Section 3, Figure 21). The tag features were used to generate interference for the speed and direction of each scan, which allowed for the identification of scanning voltages, as described next.

[0539] The scan voltage is found after the force-equilibrium 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 the force-equilibrium value while holding V2 at its force-equilibrium value to promote unperturbed DNA movement toward Pore 1 and Pore 2, respectively. The V1 scanning value is selected heuristically using the following guidance: The working scan rate should allow robust sensing of tag blockades over the recording bandwidth (i.e., not too fast) while ensuring a sufficiently high Peclet number so that the transition time distribution is well-defined (i.e., not too slow). That is, a wide transition time distribution increases the likelihood of variability, which can undermine the goal of mapping time versus distance between tags, as discussed in a later section. In practice, achieving a scan rate that is neither too fast nor too slow is achievable over a wide range of V1 scanning 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 ​​are identified, the complete flossing multiple scan logic can be applied. A detailed description of the logic implemented in the FPGA is provided in the supplemental material (SI Section 4, Figure 17). The logic output is informed here by representative flossing data

[10] . Figure 10a shows 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, as previously characterized. The detectability of the tag relative to the DNA baseline of I2 remained robust despite the baseline shift. 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] Figures 10b and 10c provide a close-up view of the signal for the first cycle, which includes the first and second scans and the 81st and 82nd scans. By convention, the multiple scan logic starts in the L-to-R direction, so odd-numbered scans correspond to L-to-R transitions and even-numbered scans correspond to R-to-L transitions. The event in Figure 10a includes 41 cycles and a total of 82 scans, all in under 0.66 seconds. As shown in Figure 1, the FPGA logic was designed to switch V1 when two tags were detected within I2. The FPGA detected tags when I2 dropped 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 transition in 9 to 19 ms. Following a 1.5 ms delay, after detecting tag B, the FPGA sets V1 = 850 mV, driving 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 transition that caused the FPGA to detect it) (Figure 10c).

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

[0543] From the probability plot in Figure 10d, 37% of 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 with n total scans indicates a system that successfully captures the first (n-1) scans but fails to capture the nth scan. Therefore, the probability P(n) is P(n)=p n-1 (1-p) (1) is.

[0544] Fitting the data to equation (1) yields 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 total number of scans per event can be found in SI Section 5. Naturally, varying the voltage settings will affect the event duration between tags, which in turn will affect the probability of tag detection.

[0545] The dependence of tag size on scan direction is observed in Figure 10a-c, where MS tags exhibit relatively shallow, fast spikes at the two higher voltages as they pass through the pore. The finding of fast, 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 tag events from hitting full depth (i.e., faster events are shallower).

[0546] Multiple scan experiments using a three-tag triggering configuration were also performed (Figure 19). Obtaining many scans using three tag triggers was more difficult than using two. In part, this was due to the fact that few molecules (<20%) displayed more than three tags. Also, even when the system co-captured a molecule with three tags in both pores, the probability of correctly detecting all three tags was low (Figure 19c). Failure to detect any one of the tags could result in the molecule moving to a new region, thereby reducing the scan count for the initially scanned three-tag region. Therefore, we focused our experiments on this initially proposed two-tag triggering to generate more data with higher scan counts.

[0547] Flossing increases the fraction of DNA that has mappable data. The application of nanopore feature mapping requires the linearization of DNA as it passes through the sensing pore. In this case, the fraction of DNA that can be linearized by flossing techniques is examined, where linearization refers to the removal of the DNA folds initially present within the pore when co-trapping is initiated. As an example, the molecule in Figures 10a-c partially folds in approximately 10 ms on the first cycle (Figure 10b), which is removed on the second cycle, demonstrating a tug-of-war controlled trend to induce and maintain DNA linearization. The probability of complete linearization over the scanning cycles through pore 2 was examined as a function of scan number to see whether the trends in Figures 10a-c were representative of the distribution. Indeed, Figure 11 shows that the linearization probability increased to 98% by the second scan. In the data, folding events were identified when current blockage was 1.5 times higher than the unfolded blockage and persisted for more than 180 μs. Figure 11a, b shows representative single-hole and multi-scan events with observable folding examples.

[0548] A quantitative mechanism for the progression of unfolding during flossing was proposed in Figure 11c. Proceeding from L to R aligns the migration and field forces at Pore 2, ultimately promoting refolding, which migrates through Pore 2 and into Channel 2. Subsequent migration from R to L pulls only the region of DNA under tension by the tug-of-war back through Pore 2, despite the opposing field force at Pore 2. Meanwhile, the folds not under the tension of the tug-of-war experience only the field force and are therefore retained in Channel 2 and move away from Pore 2. Figure 11d shows the percentage of unfolded events relative to the progression of the transition types that each of the 309 events passed through. Thus, the statistics for each pillar are indeed from the same group of molecules, the same data as in Figure 10. The 35% unfolding probability from the initial single-pore capture (Pore 1 of the device) is consistent with the other single-pore studies. Following co-trapping, 66% of the first scan was unfolded, consistent with the tug-of-war data without flossing.

[28] By the second scan, only 6 of 309 molecules (2%) remained folded, and only 5 remained folded by the final scan. Thus, the flossing process effectively linearizes 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 underlying 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 tag translocation through a single pore ("dwell time estimation"), which is the rate at which tag blockade occurs. The second approach, specific to dual-pore technology, 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 known as the pore-to-pore time. For pore-to-pore velocity assessment, the pore-to-pore time was correlated with the measured distance between the pores for each chip. By definition, the pore-to-pore approach utilizes the relationship between the two current signals I1 and I2, since time begins when the tag exits the entry pore and the tagged end enters the exit pore. With the applied voltage, the DNA in the common reservoir was expected to be fully stretched between the pores (0.34 nm / bp).

[0550] Figure 12 shows an example of adjacent pairs of scans in a multiple-scan event, illustrating how the separation distance between tags is estimated from each scan. For the scan from L to R (Figure 12a, c), tags A and B move through pore 1, and approximately 1 ms later, they move through pore 2. The signal pattern indicates that tags A and B were spatially closer than the inter-pore distance (0.64 μm). The FPGA monitored I2 for the two tags during the control logic. During the waiting period after detecting A and B in I1, a third tag, C, passes through pore 1. Visually, it was clear from the pore-to-pore passage time of A and B that tags B and C were roughly twice as far apart as the inter-pore distance. When V1 was changed to promote the reverse direction, from R to L (Figure 12b, d), the first observable tag in I1 was C returning through pore 1. Again, this logic detects A and B at I2 and this time both tags transit pore 1 before the logic activates V1, facilitating the transfer of L to R. Seeing three tags in I1 was a result of the separation of three physical tags close enough to apply to 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, tag blockade at I2 corresponds to a tag leaving the common reservoir and exiting pore 2 from L to R, whereas movement from R to L signifies that a tag enters the common reservoir at I2. While I2 shows two tags, the presence of a third tag at I1 for both scan directions provides an opportunity to quantify the separation distance between two tag pairs. The number of tags between pores was plotted against the signal shown, as were tag velocities based on dwell time and pore-to-pore velocity. These velocity values ​​versus time provide an indication of how molecules are moving along the diagram (Figures 12a and 12b) during the co-capture tug-of-war. The pore-to-pore velocity was moderately faster in the R to L direction (0.8 vs. 0.6 μm / ms), which corresponded to the larger voltage difference for R to L migration than for L to R migration (V = 250 mV, V = 400 mV).

[0552] The tag-to-tag separation distance (12c, d bottom) 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. Because the tag-to-tag time is calculated from the ascending edge of the detected tag blockade to the descending edge of the next detected tag blockade, the membrane thickness is added to calculate an additional spatial separation equal to any tag traversing the length of the pore. Tag-to-tag distance estimates are shown for both L-to-R and R-to-L scans. This result suggests that the prediction of each separation will vary across the two different scan directions. The prediction of tag-pair separation distances will also vary depending on 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 inter-tag velocity is constant and equal to the pore-to-pore velocity is not actually correct. This assumption appears to be better when the inter-tag 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 Figure 12). If the assumption that the DNA was moving at a constant pore-to-pore velocity in any case was correct on average, then an average of many rescans would be 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 tagged DNA from the model reagent.

[0553] Combining scans to improve tag-to-tag distance predictions The quality of error reduction achieved by averaging distances obtained from individual scans within a multiple-scan event was tested. Five different multiple-scan events containing at least 30 cycles are reported in Table 1. For each event, averaged distance estimates are shown for each scan direction, using the Pore 1 and Pore 2 estimates alone and combined with the Pore 2 estimate. The table reports the number of cycles equal to the number of scans in each direction and the number of tag pairs contributing to each separation distance estimate. 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 distance estimates. Friction explains why the probability of missing a tag within any one scan range increases with cycle count, as explained by Equation (1).

[0554] The correspondence between the averaged separation distances in Table 1 and the known tag-to-tag separations possible from the location map was evaluated. The known distances were calculated using the conventional 0.34 nm / bp, assuming DNA in a common reservoir fully stretched between pores. In event (i), the L to R data indicated sufficient variation in pore-to-pore velocity (as described in SI Section 11), so 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 absent from the molecule and tags 4, 5, and 7 are present, the adjacent tag-pair separation for event (i) is the most consistent of all possible adjacent tag-pair combinations possible according to the location map. In particular, the map showed neighboring 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 common for control logic using N=2 tag detection at I2 to trigger a change of direction. By comparing the averaged separation distance (separation distance) with the known inter-tag separation (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 possible 1.5 μm value if tag 6's position were assumed empty for the molecule in event (ii). 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 possible 0.3 μm value if tag 5's position were assumed empty for the molecule in event (iv).

[0556] The correspondence between distance estimates and mappable pairs was generally not as clear for individual scans (e.g., Figure 12) as it was for averaged scans and not possible for scans in which tags were missed (representative examples are shown in Figures 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 velocity data for events (i–v) in Table 1, as well as data for four additional multiple-scan events, are reported in Table S3 (Figure 21). The error in the estimate for each interval (separation) is obtained as the error about the average over the group of estimates, achieving a significant reduction in error through averaging.

[0557] The data in Table 1 (Figure 13) demonstrate 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 Figures 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 (Figure 21), which provides 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 aim of automating tag-pair time binning (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 variations 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 scans in a systematic way, the algorithm automatically groups tags and removes translational offsets across scans. Figures 14c and 14f show examples of aligned events, and the SI section titled "Tag Alignment Procedure" provides a detailed description of the approach. Essentially, the algorithm operates 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 relative to the other. Note that only a translational offset is applied; there is no overall expansion of the timescale. For each of these possible trial alignments, the algorithm calculates an alignment error metric 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 provides both a translational offset between scans and a correspondence of tags shared between scans. By iterating across all scans in a set, tags observed across scans can be grouped together and translational offsets can be 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 single-molecule scans.

[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 scan displayed a consistent number of tags for both pores. The barcodes were then corrected to units of distance by multiplying by the average pore-to-pore velocity. Finally, the reported error in the corrected tag position incorporates the error in velocity and the error in separation due to the standard population. Note that the sharpening of the plateau indicates the accuracy achieved through multiple scan averaging. In particular, when the extracted tag separations were sorted by size, the data were found to exhibit a clear plateau corresponding to the expected separations in the map. Spacings (separations) away from the expected spacing result from non-specific binding (e.g., tags non-specifically attached to random nicks present in λ-DNA) or from offsets caused by imperfections in the tag blockage analysis algorithm.

[0562] In single-pore operation, a sub-4 nm diameter membrane-based pore harboring a PNA-based motif (15 bp footprint) demonstrated resolution of 100 bp between tags, whereas a pipette-based pore achieved resolution as low as 200 bp. We observed that peaks exhibited good resolution for separations of 300 bp (e.g., the visible spacing between tags in the traces in Figure 11), and a lower limit of less than 300 bp is achievable as slower transit and larger bandwidths are the knobs to be turned in this setup. Essentially, this allows for resolving tags reasonably farther apart than the membrane thickness (~105 bp for the current chip).

[0563] in conclusion We developed an approach that provides multi-read coverage data by first trapping and linearizing individual long DNA molecules in a dual nanopore device and then using automated "flossing" control logic to move the molecules back and forth between dual nanopore current sensing. In light of the development of dual-pore technology, our rescanning approach solves a key challenge: although the longest trapping times can be achieved by balancing the competing forces at each pore, sub-diffusive dynamics persist as speeds are reduced, undermining mapping-based approaches that rely on the correspondence between the time at which a tag is detected and the tag's physical location along the DNA. Our approach avoids sub-diffusive dynamics during bidirectional scanning of molecules using speeds that are high enough to avoid wide transition time distributions while still allowing reliable tag detection. Genome scaling has the potential to move beyond experiments with heterogeneous samples contai...

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 binding molecules; 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 independently control the voltage and measure the current at each pore; the first and second pores are configured to allow target polynucleotides 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, wherein the first and second voltages are: a labeled 5mC region of one or more polynucleotide sequences; a tagged 5hmC region of one or more polynucleotide sequences; separately inducing translocation of the one or more target polynucleotides through 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. 10. 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 the resistance of the first channel is inversely proportional to the 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 characteristics of the polynucleotide and performs additional recapture of one or more characteristics 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 two times, at least three times, or at least five 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. 10. 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. 10. The method of claim 1, wherein the first voltage is maintained for a period of time after the target polynucleotide is captured and translocated through the first pore.

12. 2. The method of claim 1, further comprising determining the location of one or more 5mCs or 5hmCs 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 the 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 independently control the voltage and measure the current at each pore, wherein the first and second pores are 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 moves through the first pore and the second pore in the first direction, wherein the first direction is from the first pore to the second pore; d) scanning one or more primers for hybridization to said target polynucleotide; e) detecting the 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 steps c) through i) and detecting the third and fourth sets of primers in the second cycle; A method comprising:

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