Single-molecule and multi-molecule tracing methods and systems

Abstract

A method for sequencing by extension and related improvements to sequencing surrogate polymers in a nanopore are described. The surrogate polymer is formed from a template nucleic acid molecule. The surrogate polymer includes multiple units. Each unit includes a reporter code portion. The reporter codes correspond to different nucleotides. The surrogate polymer can be anchored to the nanopore. Embodiments described herein address these anchored surrogate polymers. To enable multiple reads on the surrogate polymer, a processive consensus technique can be applied. The surrogate polymer can be shifted forward a few units and then shifted backward a fewer units so that some of the same reporter codes are identified again. This method enables multiple reads of the same reporter code. The surrogate polymer ultimately passes through the nanopore in the forward direction. Periodically, a higher removal voltage can be applied to remove the surrogate polymer anchored to the nanopore.

Description

Related Applications

[0001] Cross-reference to related patent applications This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 412,774, filed October 3, 2022, which is incorporated by reference for all purposes. [Technical Field]

[0002] Embodiments described herein relate to nanopore-based sequencing methods and systems, including, in particular, methods and systems that generate consensus reads using Sequencing By eXpansion (SBX™). [Background technology]

[0003] Nanopore membrane devices with pore sizes on the order of one nanometer in inner diameter have shown promise for rapid nucleotide sequencing. When an electric potential is applied across a nanopore immersed in a conducting fluid, a small ionic current can exist due to the conduction of ions across the nanopore. The size of the current is sensitive to the pore size and the type of molecule within the nanopore. The molecule can be a specific reporter code corresponding to a specific nucleotide, thus allowing for the detection of the nucleotide at a specific position in the nucleic acid. As a way to measure the resistance of the molecule, it is possible to measure the voltage or other signal in a circuit with the nanopore (e.g., with an integrating capacitor) to detect which molecule is within the nanopore.

[0004] Nanopore-based sequencing chips can be used for DNA sequencing. Nanopore-based sequencing chips can incorporate a large number of sensor cells organized as an array. For example, an array of 1 million cells can include 1,000 rows and 1,000 columns of cells.

[0005] Due to manufacturing variations, the measured signal can vary from chip to chip and from cell to cell on the same chip. Therefore, it can be difficult to determine the correct molecule that may be or correspond to the correct nucleotide in a particular nucleic acid or other polymer within a cell. Furthermore, other time-dependent non-idealities in the measured signal can introduce inaccuracies. And because these circuits use biochemical circuit elements, such as lipid bilayers and nanopores, the variability of electrical properties can be much higher than with conventional semiconductor circuits. Furthermore, the sequencing process is inherently stochastic, and therefore variation can occur across a wide variety of systems, including sequencing devices that do not use nanopores.

[0006] Therefore, improved characterization techniques are desirable to improve the accuracy and stability of the sequencing process. Summary of the Invention

[0007] Embodiments described herein include improved methods and sequencing of surrogate polymers encoded with nucleic acid information within a nanopore. Surrogate polymers (also referred to herein as "Xpandomer" polymers) are formed by template-directed synthesis, which preserves the original genetic information of the target nucleic acid while increasing the linear separation of individual elements of sequence data. The surrogate polymer is formed from a template nucleic acid molecule. The surrogate polymer contains multiple units. Each unit contains one or more reporter code moieties. The reporter codes correspond to different nucleotides (e.g., A, T, C, G). The reporter codes generate different electrical signals in the nanopore, thus enabling identification of the nucleotide sequence. Each unit contains a translocation control element (TCE). To pass through the nanopore, the TCE requires a higher voltage to be applied compared to the baseline voltage to drive the rest of the unit through the nanopore. To enable multiple reads, the surrogate polymer can be shuttled through the nanopore several times. The surrogate polymer contains one leader segment, which can be anchored to the membrane on one side of the nanopore when retracting the surrogate polymer from the nanopore. The embodiments described herein address these anchored surrogate polymers.

[0008] To enable multiple reads on the surrogate polymer, a processive consensus technique can be applied. The surrogate polymer can be shifted forward a few units (e.g., 30) and then shifted back fewer units (e.g., 25) so that some of the same reporter codes are identified again. This method allows multiple reads of the same reporter code. The surrogate polymer finally passes through the nanopore in the forward direction. Periodically, a higher removal voltage can be applied to remove the surrogate polymer that has become stuck in the nanopore.

[0009] The removal voltage may be applied more frequently, but in a targeted manner. Cells (i.e., wells) in which the surrogate polymer is not determined to be anchored to the nanopore may be deactivated before the removal voltage is applied. The technique allows molecules of any length to be sequenced to a depth greater than one along their entire length.

[0010] In view of the above, one aspect of the present disclosure is a method for sequencing a target nucleic acid molecule, comprising: applying a first number of voltage pulses at a first level across a nanopore to displace a compound generated from the target nucleic acid molecule through the nanopore a first distance in a first direction, the compound comprising a plurality of units, each unit of the plurality of units comprising one type of reporter element from a plurality of types of reporter elements, each type of reporter element corresponding to the identity of a nucleotide in the target nucleic acid molecule, wherein application of the first number of voltage pulses causes a first subset of the plurality of units to pass through the nanopore; detecting at the nanopore the type of reporter element in the first subset; and applying a second number of voltage pulses at a second level across the nanopore to displace the compound through the nanopore a second distance in a second direction. applying a third number of voltage pulses at a third level across the nanopore to displace the compound a third distance in the first direction through the nanopore, wherein the first direction is opposite to the second direction, the voltage pulses of the first number of voltage pulses have an opposite polarity to the voltage pulses of the second number of voltage pulses, the second distance being less than the first distance, and the second number being less than the first number; applying a third number of voltage pulses at a third level across the nanopore to displace the compound a third distance in the first direction through the nanopore, wherein application of the third number of voltage pulses causes a second subset of the plurality of units to pass through the nanopore, the second subset and the first subset comprising some of the same units, the second subset comprising units that are not in the first subset, and the third distance being greater than the second distance; and detecting the type of reporter element in the second subset at the nanopore.

[0011] In some embodiments, the method further includes applying a fourth level of removal voltage across the nanopore to completely eject the compound from the nanopore, wherein the fourth level is greater than the first level, the second level, and the third level.

[0012] In some embodiments, the compound is a first compound of a plurality of compounds, the plurality of compounds being generated from a plurality of target nucleic acid molecules, the nanopore is a first nanopore of a plurality of nanopores, and each compound of the plurality of compounds is within one nanopore of the plurality of nanopores, and the method further comprises applying a first number of voltage pulses at a first level, a second number of voltage pulses at a second level, and a third number of voltage pulses at a third level to the plurality of nanopores.

[0013] In some embodiments, the method further comprises determining a plurality of sequences of a plurality of target nucleic acid molecules.

[0014] In some embodiments, the size distribution of the plurality of sequences has a mode greater than 300 nt.

[0015] In some embodiments, the method further includes applying a first number of voltage pulses at a first level, a second number of voltage pulses at a second level, and a third number of voltage pulses at a third level to the plurality of nanopores; determining that a first portion of the plurality of compounds has been displaced in the first portion of the plurality of nanopores by the first number of voltage pulses, the second number of voltage pulses, or the third number of voltage pulses; and applying a removal voltage at a fourth level across each nanopore in the second portion of the plurality of nanopores to completely eject the second portion of the plurality of compounds from each nanopore in the plurality of nanopores, wherein the fourth level is greater than the first level, the second level, and the third level, and the second portion of the plurality of nanopores does not include nanopores in the first portion of the plurality of nanopores.

[0016] In some embodiments, the method further comprises determining the sequence of the target nucleic acid molecule.

[0017] In some embodiments, determining the sequence of the target nucleic acid molecule comprises detecting the same type of reporter element for one or more units in both the first subset and the second subset.

[0018] In some embodiments, the method further comprises completely ejecting the compound from the nanopore.

[0019] In some embodiments, the step of completely ejecting the compound from the nanopore occurs during the application of the third number of voltage pulses.

[0020] In some embodiments, each unit of the plurality of units includes a translocation control element, and upon application of a first number of voltage pulses, the first number of translocation control elements pass through the nanopore, and the first number of voltage pulses is equal to the first number of translocation control elements.

[0021] In some embodiments, the method further includes applying a fourth level of voltage across the nanopore to displace the compound a fourth distance in a first direction through the nanopore during a voltage pulse of the first number of voltage pulses, wherein the fourth level of voltage has the same polarity as the voltage pulses of the first number of voltage pulses, the fourth level is less than the first level, and the compound after being displaced the fourth distance has a translocation control element within the nanopore.

[0022] In some embodiments, the second level is greater than the first level.

[0023] In some embodiments, the method further includes measuring a signal value for the nanopore having a voltage applied across the nanopore when reporter elements in a first subset of the plurality of units are within the nanopore; and using the signal value to determine the types of reporter elements in the first subset, thereby determining the identities of nucleotides in the target nucleic acid molecule.

[0024] In some embodiments, the first subset of the plurality of units comprises 30 or more units.

[0025] In some embodiments, the third level is equal to the first level.

[0026] In some embodiments, the target nucleic acid molecule is longer than 200 nt.

[0027] In some embodiments, the first number of voltage pulses is 30 or greater.

[0028] In some embodiments, the first number of voltage pulses may exceed the second number of voltage pulses by 5 or more.

[0029] Another aspect of the present disclosure is a computer product comprising a non-transitory computer-readable medium storing a plurality of instructions that, when executed, control a computer system to perform the aforementioned methods and embodiments thereof.

[0030] Another aspect of the present disclosure is a system comprising a computer product and one or more processors for executing instructions stored on a computer-readable medium.

[0031] Further aspects of the present disclosure are systems comprising means for performing any of the above methods, systems comprising one or more processors configured to perform any of the above methods, and systems comprising modules that respectively perform the steps of any of the above methods.

[0032] A better understanding of the nature and advantages of embodiments of the present invention may be obtained by reference to the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 illustrates components of a surrogate polymer according to an embodiment of the present invention. [Figure 2] 2 shows details of the XNTP structure according to an embodiment of the present invention. The molecular structure shown in FIG. 2 may not be chemically correct. FIG. 2 is provided for illustrative purposes. [Figure 3] FIG. 1 illustrates the attachment of extended oligomers to a solid substrate for surrogate polymer synthesis according to an embodiment of the present invention. [Figure 4] 4 shows the structure of an extension oligomer, a leader, and nearby components according to an embodiment of the present invention. The molecular structures shown in FIG. 4 may not be chemically correct. FIG. 4 is provided for illustrative purposes. [Figure 5A] FIG. 1 shows an example of a single-molecule to multi-molecule trace event using an exemplary extended sequencing waveform according to an embodiment of the present invention. [Figure 5B] FIG. 1 shows a molecule that does not eliminate the pore according to an embodiment of the present invention. [Figure 6] FIG. 10 illustrates one strategy for sequencing a surrogate polymer with the same forward and reverse voltages according to an embodiment of the present invention. [Figure 7] FIG. 1 illustrates a strategy for sequencing surrogate polymers with different forward and reverse voltages according to an embodiment of the present invention. [Figure 8] FIG. 1 illustrates a strategy for sequencing surrogate polymers with reduced light period duration according to an embodiment of the present invention. [Figure 9] FIG. 1 illustrates a light and dark period schedule according to a sequencing strategy in accordance with an embodiment of the present invention. [Figure 10] FIG. 1 illustrates a strategy for sequencing using a reverse nanopore according to an embodiment of the present invention. [Figure 11] FIG. 10 illustrates a voltage schedule for processive multi-pass read generation in accordance with an embodiment of the present invention. [Figure 12] FIG. 1 illustrates an assembled captured raw lead series according to an embodiment of the present invention. [Figure 13] FIG. 10 illustrates a meta period for removing a surrogate polymer from a nanopore according to an embodiment of the present invention. [Figure 14] FIG. 1 illustrates an assembled captured raw lead series according to an embodiment of the present invention. [Figure 15A] FIG. 1 shows the effect of different concentrations of redox couples in an open channel ADC according to an embodiment of the present invention. [Figure 15B] FIG. 1 shows the effect of different concentrations of redox couples in an open channel ADC according to an embodiment of the present invention. [Figure 16] FIG. 1 shows a flowchart of an exemplary process for sequencing a target nucleic acid molecule according to an embodiment of the present invention. [Figure 17] FIG. 1 illustrates a measurement system according to an embodiment of the present invention. [Figure 18] FIG. 1 is a block diagram of an exemplary computer system usable with systems and methods according to embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] The embodiments described herein include protocols for nanopore sequencing, including the Sequencing By eXpansion (SBX™) protocol. The protocol involves a molecular "read" portion that has the property of readily entering the pore in the forward direction but having a high barrier to passage through the pore in the reverse direction. Furthermore, embodiments include modified light and dark period voltage patterns designed to electrically trap surrogate polymer (e.g., Xpandomer) molecules within the nanopore and position the molecules in a controlled manner to allow multiple passes or reads with the same surrogate polymer molecule. Furthermore, embodiments include applying higher voltage pulses and / or longer cycle times in periodic light / dark cycles to ultimately remove molecules from the pore. Some embodiments include using selective application of higher voltage pulses to remove molecules only from specific pores during each global pulse application period. Benefits include enrichment of compound molecule trace events (i.e., subreads from the same molecule over a series of light periods). Other benefits may include increased pore occupancy and raw base call throughput. Furthermore, the protocol may result in more accurate multi-pass reads compared to single-pass reads. Embodiments may include performing reads of long surrogate polymer molecules with much shorter light periods without permanently cutting off. This may enable practical experimental conditions that result in short light period decay time constants.

[0035] Furthermore, embodiments may include the ability to dynamically select to spend more time on surrogate polymers from a particular UMI (molecular barcode) molecule family and less time on surrogate polymers from other UMI molecule families. UMIs can be added to sample nucleic acids during the sample preparation stage. All nucleic acid fragments from a particular sample have the same UMI. Different samples have different UMIs. UMIs allow different samples to be pooled together for sequencing while still identifying each nucleic acid molecule sample. During sequencing, it may be determined that sequences from a particular UMI family (i.e., sample) may require further sequencing. For example, the confidence of a particular base call may be low due to a relatively large number of different base calls at the same position. Fragments from these samples may be sequenced in excess using the methods described herein.

[0036] I. Sequencing by Extension Embodiments may be applied to nanopore-based sequencing by expansion (SBX), which is described in WO 2020 / 236526, filed May 14, 2020, entitled "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing," and U.S. Patent No. 7,939,259, filed June 19, 2008, entitled "High throughput nucleic acid sequencing by expansion," the entire contents of both of which are incorporated herein by reference for all purposes.

[0037] The sequencing-by-extension protocol is based on the polymerization of highly modified, non-natural nucleotide analogs called "XNTPs." Generally, SBX uses biochemical polymerization to transcribe the sequence of a DNA template onto measurable polymers called "Xpandomers." The transcribed sequences are encoded along the Xpandomer backbone in high-signal-to-noise reporters spaced approximately 10 nm apart, designed for high signal-to-noise and highly differentiated response. These differences result in significant performance improvements in the sequence read efficiency and accuracy of Xpandomers compared to natural DNA.

[0038] A. Surrogate Polymer Structure Figure 1 shows the components of a surrogate polymer (e.g., Xpandomer). Xpandomer contains an ordered sequence of XNTPs corresponding to the order of a target DNA sequence. Figure 1 is not to scale. XNTPs are expandable 5' triphosphate-modified unnatural nucleotide analogs compatible with template-dependent enzymatic polymerization. XNTPs have two distinct functional regions: a selectively cleavable phosphoramidate bond that connects the 5' α-phosphate to the nucleobase, and a symmetrically synthesized reporter tether (SSRT) that is attached within the nucleoside triphosphoramidate at a position that allows controlled extension by cleavage of the phosphoramidate bond. The SSRT contains linkers separated by selectively cleavable phosphoramidate bonds. Each linker is attached to one end of a reporter code.

[0039] Section 102 shows primer-directed Xpandomer synthesis. XNTP 104 is shown in a "constrained configuration" characteristic of XNTP substrates and the daughter strand product of template-dependent polymerization. The constrained configuration of polymerized XNTP is the precursor to the extended configuration (XNTP 108), as seen in the Xpandomer product. Section 106 shows cleavage to extend the Xpandomer. The transition from the constrained configuration to the extended configuration occurs upon cleavage of the P--N bond of the phosphoramidate within the primary backbone of the daughter strand.

[0040] During assembly, monomeric XNTP substrates (XATP, XCTP, XGTP, and XTTP) are polymerized onto the extendable end of a nascent daughter strand by a process of template-directed polymerization using a single-stranded template as a guide. This process typically begins with a primer and proceeds in a 5' to 3' direction. DNA polymerase 110 or other polymerases are typically used to form the daughter strand, and conditions are selected to yield a complementary copy of the template strand. After the daughter strand is synthesized, the coupled SSRTs form a constrained Xpandomer that further forms the daughter strand. The SSRT in the daughter strand has the "constrained configuration" of the XNTP substrate. The constrained configuration of the SSRT is a precursor to the extended configuration, as seen in the Xpandomer product.

[0041] In this example, once synthesis and extension are complete, each monomeric XNTP unit 112 in the Xpandomer contains two reporter codes 116a and 116b, with reporter code "levels" corresponding to the base types they encode, and a translocation control element (TCE) 120. The TCE controls the rate of translocation of the Xpandomer through the nanopore through a combination of steric hindrance, electrical repulsion, and / or preferential interactions with the nanopore. The resistance of the TCE to the driving force of the ionic current when positioned at the pore opening, and the resulting increase in applied voltage (i.e., voltage pulse) required to overcome stalling and resume translocation, can be customized by adjusting various properties of the TCE (and, in some embodiments, other elements of the reporter codes and SSRT), such as bulk, length, and / or charge density.

[0042] Branched chain 124 is a branched structure that terminates the TCE and links to reporter codes 116a and 116b. Enhancers 128a and 128b may assist in polymerase incorporation. Nucleotide 132 is attached to enhancer 128b and may include a cleavable linker 136. Cleavable linker 136 may be a photocleavable linker. Cleavable linker 136 may be cleaved to result in the extension shown in section 106.

[0043] Figure 2 shows the structural details of XNTP 200. In Figure 2, XNTP contains two reporter codes 204a and 204b and a translocation control element (TCE) 208. Different reporter codes are sized to block ion flow through the nanopore at different measurable levels. The reporter codes and other features can be designed by selecting specific phosphoramidite sequences.

[0044] In certain embodiments, the TCE is a polymer produced by solid-phase synthesis using phosphoramidite technology with appropriate monomer building blocks that terminate in a branched structure (i.e., a "branched chain"). Branched phosphoramidites include both symmetric and asymmetric branched chains. In one embodiment, the TCE branched chain 210 is a symmetrically branched CED phosphoramidite, with each arm of the branched chain linked to a reporter code. Exemplary symmetric chemical branched chains include 1,2,3-O-tris-(phosphodiester)propane, 1,3-bis-(5-O-phosphodiester-pentylamido)-2-O-phosphodiester-propane, and 1,4,7-O-tris-(phosphodiester)-heptane.

[0045] A UV chromophore 212 can be attached to the end of TCE 208. The UV chromophore 212 can enable visualization or quantification. Spacers 216a and 216b are attached to reporter codes 204a and 204b, respectively. Spacers 216a and 216b can be polyethylene glycol (PEG) units that can adjust the length they traverse within the pore. Enhancers 220a and 220b can be attached to spacers 216a and 216b. Enhancers 220a and 220b can be positively charged spermines that promote polymerase incorporation. Nucleotides 224 can be attached to enhancers 220a and 220b. Nucleotides 224 can include triphosphoramidate diesters. Structural elements of XNTP 200 can be adjusted to improve measurement within the nanopore.

[0046] 3 shows the attachment of an extension oligomer 304 to a solid substrate 308 in preparation for Xpandomer synthesis. The solid substrate 308 may be crosslinked to a maleimide. Step 312 shows the extension oligomer 304 not attached to the solid substrate 308. After click chemistry in step 316, the extension oligomer 304 is attached to the solid substrate 308. The extension oligomer 304 may be attached to a leader moiety 320. The leader moiety 320 may be attached to a photocleavable linker 324.

[0047] Figure 4 shows structure 400 of an extender oligo 404, a "leader" (L), and its nearby components. The extender oligo can be a 2'O-methyl extender oligo. Adjacent to the extender oligo 404 is a poly-C12 spacer (Z) 408, followed by a poly-C2 spacer (L) 412. Because photocleavage occurs immediately after Xpandomer synthesis and before loading any xpandomers onto a sequencer, the photocleavable spacer (PC) 416 and the segment above (i.e., azide (R) 420 and spacer (D) 424) are not part of the Xpandomer in the sequencing protocol described herein. Azide R 420 can be a 5'-azide. Spacer D 424 can be a polyPEG6 spacer. The truncated sequence of structure 400 is R(D) 10 (PC)(L) 25 The sequence at the end may be (Z)6(TCATAAGACGAACGGA), with the terminal sequence representing the extended oligo 404. During several Xpandomer sequencing operations following the flow of Xpandomer molecules into the fluidic channel above the membrane, the hydrophobic polyC12 spacer may partition into the membrane phase and initiate quasi-2D diffusion / migration along the membrane surface. 3D diffusion / migration and fluid flow may also play a role in the mass transport of Xpandomer toward the pore. Xpandomer capture is thought to occur when the highly negatively charged polyC2 spacer segment (L) is electrostatically drawn into the nanopore barrel during application of a "positive" bias across the membrane. Note that in the case of an inverted pore, the bottom of the pore barrel protrudes slightly from the bilayer membrane on the cis side.

[0048] B.Operation During the "light period," the Xpandomer molecule is captured by the nanopore and begins to move through it due to the combination of both the baseline and TCE-applied voltage pulses. The baseline voltage is sufficient to read the tag code at each XNTP position, and the short, high-voltage TCE pulses are designed to overcome the energy barrier associated with TCE. Ideally, each TCE pulse translocates over a single TCE barrier, thus further translocating the Xpandomer molecule into the pore in the forward direction by the amount of one "base" position.

[0049] During typical operation, the applied voltage pattern is designed so that there are a fixed number of TCE pulses during each light period, causing the Xpandomer to move in the "forward" direction by a number of bases corresponding to the number of TCE pulses, or until the Xpandomer has completely moved and is released into the fluidic "trans" chamber below the membrane.

[0050] During typical operation, an Xpandomer molecule may not fully translocate before the end of a single light period. This can occur due to a molecule being captured late in the light period and having an Xpandomer length with more base positions than there are TCE pulses remaining in the light period. A molecule can become stuck while attempting to translocate in the forward direction for a variety of reasons. A base position may exist with a defect (e.g., a failed cleavage event) that makes it impossible or very difficult for the molecule to pass that point. In such situations, and for other reasons, an Xpandomer may fail to fully translocate during the light period, regardless of the number of TCE pulses in the light period. In such situations, several base positions at the beginning of the read may be observed to sequence and generate the expected signal level until the defect position is reached. In that case, the final tag code level located just before the defect may be observed for the remainder of the light period. To prevent the pore from remaining permanently clogged, a large negative voltage may be applied for some time during the dark period to eject stuck molecules by strongly driving them in the reverse direction.

[0051] FIG. 5A shows an example of a single-molecule-multiple-molecule trace (SM3T) event using a typical extended sequencing waveform. The graph shows time in seconds on the x-axis. The graph shows voltage readings on the y-axis. Instead of voltage, other electrical measurements can be used, including voltage equivalents (e.g., ADC counts) or current. Dark periods 504 and 508 are normal dark periods when the pore is empty. Light period 512 shows signals 516a and 516b for molecule 1 and molecule 2, respectively.

[0052] Signal 520 shows molecule 3 during the light period. This event indicates that molecule 3 is anchored in the pore and does not disappear for several cycles (dark periods 524, 528, 532 and light periods, signals 536, 540, and 544). Eventually, the molecule disappears in the dark cycle, as indicated by the change from signal 548a to signal 548b (when molecule 3 disappears). This event may be due to the properties of the Xpandomer's leader segment, making it difficult for the leader to move in the reverse direction.

[0053] Figure 5B shows a possible mechanism behind the trace in Figure 5A. Diagram 552 shows the light period. The direction of migration is downwards. The uncut position 556 hits the pore after the next pulse. A normal tag code level is expected. Diagram 558 shows the dark period. The direction of migration is now upwards. The reader 560 has difficulty moving backward (upwards) through the pore. Eventually, the reader 560 passes through the pore.

[0054] Xpandomer molecules can be designed with properties in the leader portion of the Xpandomer that allow the leader to behave differently in the forward and reverse directions. During the light period (forward direction), the leader can have properties that allow it to be captured into the pore from the cis side with a relatively high capture rate under a moderately applied voltage. After capture, but still during the same light period, a TCE pulse steadily processes the molecule through the pore so that the leader can protrude from the underside of the pore (the trans side of the membrane).

[0055] During the dark period (reverse direction), if the molecule is still inside the pore when the dark period begins, it should begin to move in the reverse direction under a negative applied voltage. If the Xpandomer molecule reverses its position almost completely (i.e., is almost completely retracted), the leader can remain on the trans side of the barrel. In this regard, the desired property of the leader is that it has a high energy barrier to enter the barrel from the trans side and therefore a high resistance to moving through the barrel in the trans to cis direction (forward direction).

[0056] II. Sequencing Strategies Strategies are described herein to exploit asymmetric leader behavior (with respect to the direction through the pore) that can increase precision by reducing the number of Xpandomers that attach to the pore.

[0057] A. Full forward and full reverse at the same voltage Figure 6 shows one strategy for sequencing an Xpandomer with the same forward and reverse voltages. Figure 6 shows a nanopore with a membrane underneath the nanopore. The Xpandomer has a blocker 604 (e.g., streptavidin) at one end and a reader 608 at the other end. The reader threads through the pore (downward in the figure). The Xpandomer moves downward and is sequenced during the light period (e.g., figure 612) until the end of the molecule and / or the end of the light period. A voltage is applied to move the Xpandomer downward. The voltage includes a baseline voltage to move the Xpandomer downward so that the reporter element can be read. A higher voltage is applied to pass the Xpandomer through the TCE element. The blocker 604 prevents the Xpandomer from exiting the pore.

[0058] During the dark period (e.g., Fig. 616), the applied voltage is reversed, causing the Xpandomer to move upward. The same voltage is applied during the dark period as during the light period, but the voltage has the opposite polarity. The reader 608 may have difficulty passing through the membrane and / or pore in the opposite direction.

[0059] The distribution of SM3T durations can be exponential. This distribution may exhibit many shorter lengths, reflecting the anchoring of Xpandomer to the nanopore. The mode of duration should be equal to 1.

[0060] B. Full forward and full reverse at increased reverse voltage Figure 7 shows a strategy for sequencing an Xpandomer with different forward and reverse voltages. The nanopore and Xpandomer are configured similarly to Figure 6. The Xpandomer has a blocker 704 on one end and a reader 708 on the other end. The light period in Figure 712 can be the same as the light period in Figure 612. During the dark period, the reader may stick and be unable to pass through the nanopore. To address this situation, the reverse voltage is increased during several periodic dark cycles (e.g., the dark period in Figure 716). For example, once every 10 cycles or once every 20 cycles can include an increased reverse voltage to remove the stuck leader. This increased voltage can be referred to as the "removal voltage."

[0061] The shape of the distribution of SM3T durations may be altered from that of Figure 6. As fewer Xpandomers adhere to the nanopore, the distribution will exhibit longer lengths. The mode may not be equal to 1.

[0062] C. Limited photoperiod duration Figure 8 shows a strategy for sequencing Xpandomers with shortened light period duration. Figure 8 shows a nanopore with a membrane underneath it. The Xpandomer has a leader 804 at one end and no blocker at the other end. The leader 804 threads through the pore (downward in the figure). The Xpandomer moves downward and is sequenced during a light period (e.g., figure 808). The light period is shortened to prevent Xpandomers of a certain size from exiting the nanopore in the forward direction. Shorter Xpandomers may pass through and escape completely. Each light period may include a fixed, predetermined number of TCE voltage pulses. A fixed number of light periods may be applied. A dark period (e.g., figure 812) is then applied to reverse the direction of the Xpandomer. The dark period in figure 812 may be the same as the dark period in figure 616.

[0063] The shape of the distribution of SM3T durations can be altered from that of Figure 6. The mode does not have to be equal to 1. This distribution can collapse the read length distribution for most reads with a mode of + / - a few bases.

[0064] Figure 9 shows the light and dark period schedules for the sequencing strategy. The top graph shows the polarity of the voltage applied across the nanopore. During the light period, the polarity is indicated as 1. During the dark period, the polarity is indicated as -1. The top graph does not show the magnitude of the voltage, only the polarity. The bottom graph shows the position of the molecule within the pore. Position 1 corresponds to the unit of the Xpandomer closest to the reader. Higher position numbers (e.g., 200) are farther from the reader, and in this example, correspond to units of the Xpandomer closer to the opposite end of the Xpandomer.

[0065] As shown in Figure 9, light period 904 can include normal sequencing of Xpandomer 908. Sequencing can be limited by light period duration or complete migration and escape. Xpandomer 908, and all Xpandomers in Figure 9, do not have blockers at the ends of the molecules.

[0066] Xpandomer 912 may enter the nanopore later in the light period 916. After the light period ends, more bases (e.g., reporter elements) may remain. During the dark period, Xpandomer 912 exits the nanopore completely.

[0067] In Figure 9, the Xpandomer 912 has more units (e.g., TCE) than the number of pulses in the light period. During the dark period 920, a high voltage is applied to ensure that the reader reaches the pore. A lower voltage is then applied. Certain Xpandomers do not pass completely through the nanopore. The duration of the light period can be increased to allow more of the Xpandomers to pass through the nanopore. However, if the light period is too long, the Xpandomers may pass through the nanopore in only one direction. Furthermore, Xpandomers of different sizes may be present in the sample. A certain light period duration may result in shorter Xpandomers passing through the nanopore in only one direction, but not enough to sequence all the XNTPs in the longer Xpandomers.

[0068] Xpandomer 924 may reach the pore early in the light period 928. The molecule may have more bases than there are pulses remaining in the light period. Therefore, Xpandomer 924 does not pass all the way through. Almost exactly as many bases are read as there are pulses in the light period.

[0069] D. Reverse Nanopore Figure 10 shows a strategy for sequencing using a reverse nanopore. As has been done historically for SBT in HTP, the pore is inserted from the cis side. The Xpandomer contains a leader at one end and a blocker (e.g., blocker 1004) at the other end. The leader enters the membrane before the nanopore. The Xpandomer is sequenced as it is reversed from the nanopore.

[0070] In step 1008, the Xpandomer is captured. A high voltage (e.g., a TCE voltage) is applied for a longer period (e.g., 0.1-10 ms instead of 8 μs). The captured molecule 1012 with the blocker migrates to the end where further movement is stopped by the blocker. Also shown in step 1008 is a captured molecule 1016 without the blocker. The molecule 1016 may include a fragmented molecule. The molecule without the blocker may pass rapidly through the nanopore.

[0071] In step 1020, the Xpandomer 1012 is moved in the reverse direction, causing the Xpandomer 1012 to retract from the nanopore. The TCE voltage is applied in the reverse direction. Sequencing information is obtained as the molecule retracts. All captured molecules are expected to be located at the end (or non-cleaved position) of the molecule. The molecule is pulsed in the reverse direction, and data is acquired as the molecule retracts. The light period can be as long as the longest expected molecule in the sample. For example, for a ctDNA assay, the longest expected molecule may be 350 bp, corresponding to 350 pulses or 350 ms with a 1 ms inter-pulse duration.

[0072] In step 1030, an optional recharge may be performed before the next acquisition step. A dark period may be applied. If the accumulated voltages from steps 1 and 2 are not balanced, the dark voltage may serve to recharge the electrodes. During this period, no data is acquired. If phased array mode is being performed, this dark period is the duration of steps 1008 and 1020, allowing the other half of the chips (phases) to complete those two steps.

[0073] The leader can be modified to prevent easy migration when passing through the pore, starting from the entrance (i.e., membrane) side. The Xpandomer may need to be captured reasonably well when entering from the entrance side. High voltage can help capture the Xpandomer. The membrane may be more tolerant to high voltage for a longer period than other strategies (e.g., 8 μs pulse duration). A block can be added to the end of the Xpandomer.

[0074] Advantages of this strategy may include filtering out a portion of the fragmented molecules. Furthermore, read initiation may be synchronized. Furthermore, pore insertion may be from the cis side. For example, a well may be filled with a nanopore solution. The well may then be covered with a membrane, and the nanopore may be inserted from the cis side.

[0075] III. Processive Consensus Strategy Strategies may include reading the same XNTP multiple times within the nanopore and applying voltage to remove stuck Xpandomers. These strategies may increase accuracy by reducing the number of Xpandomers stuck in the pore and / or by repeatedly reading the XNTP through the nanopore.

[0076] A. Clear / recharge voltage applied to all cells Strategies for sequencing molecules do not necessarily require reading the molecule from beginning to end for each pass. Rather than the entire molecule passing through the nanopore for a complete read, a portion of the molecule may pass through the nanopore for a subread. Furthermore, the molecule may move forward and then backward to perform many short overlapping passes and a progressive manner. The duration of the light and dark periods may be shortened compared to other strategies. The number of high-voltage TCE pulses may be less than the number of TCE pulses in the Xpandomer. The number of TCE pulses in the light period will be greater than the number of reverse TCE pulses in the dark period.

[0077] As an example, the length distribution of Xpandomers may have a peak around 350 bp. The light period may include a duration of 30 TCE pulses. The dark period may have a total duration equal to the light period duration, but with an applied voltage pattern including 25 TCE pulses in the opposite direction. Equal durations of the light and dark periods allow for balancing of the charge on the electrodes, allowing them to regenerate and / or reset. The protocol may include an additional period containing a "removal voltage" to periodically remove occasionally stuck Xpandomers.

[0078] FIG. 11 shows a voltage schedule for processive multi-pass read generation. The graph shows voltage pulses for the upper and lower electrodes. The dashed lines indicate zero voltage for each respective electrode. Voltages above the dashed lines have positive polarity. Voltages below the dashed lines have negative polarity. During the light period, the upper electrode has positive pulses and the lower electrode has negative pulses. Light periods 1104, 1108, and 1112 are shown. During the dark period, the upper electrode has negative pulses and the lower electrode has positive pulses. Dark periods 1116 and 1120 are shown. The dark periods are shown as having five fewer pulses than the light periods.

[0079] During the light period, the Xpandomer moves in the downward direction as shown. The Xpandomer is sequenced during the light period. Each pulse should correspond to a lead. Capture of the Xpandomer can occur at any time during the light period. During the dark period, the Xpandomer moves in the reverse (upward) direction. No sequencing is performed during the reverse direction. If the reader returns to the front door, the Xpandomer may stick. A meta period, which includes a removal voltage, may remove the Xpandomer. The meta period is not shown in Figure 11.

[0080] After a sufficient number of cycles, the molecule (e.g., Xpandomer 1124) exits the pore in the forward direction.

[0081] Figure 12 is a diagram of an assembled captured raw read series obtained from a protocol similar to that used in Figure 11. The length of the Xpandomer corresponds to a target nucleic acid molecule that is 116 bp in length. The Xpandomer was run through a 30-pulse forward cycle and a 25-pulse reverse cycle. A total of 20 cycles were used to cover the entire length of the Xpandomer. The reads for each cycle are shown in section 1204. Because each cycle contains overlapping reads, individual nucleotides are sequenced several times. The captured consensus read is shown in read 1208. Below the captured consensus read, the number of times the nucleotide was sequenced is shown. For example, the initial subsequence of AAGCT is sequenced twice. The central portion beginning with TCTGGT is sequenced six times. The beginning of the Xpandomer can be sequenced multiple times if the initial forward and reverse cycles are set to have the same number of pulses before changing to a cycle in which the number of forward pulses in the light period is greater than the number of reverse pulses in the dark period. The end of the Xpandomer can be sequenced multiple times by continuing the forward and reverse pulses until the Xpandomer has completely exited the nanopore.

[0082] As an example, each half cycle can be 30 ms with a 1 ms pulse interval. The entire cycle can take 60 ms. The total time spent on the molecule can be 1200 ms. A total of 577 raw bases can be read in a total time of 1.2 seconds or a light time of 0.6 seconds. Excluding the 20 bases at either end, the 116 bp molecule is sequenced at a coverage depth of 6x.

[0083] Figure 13 shows the metaperiod for removing the Xpandomer from the nanopore. The metaperiod itself can include several AC periods and clearing cycles of light and dark. The AC periods can be similar to those typically used in sequencing, except that they are shorter than 1 or 2 seconds. The pulses indicated by segments 1304 and 1308 are clearing cycles of light and dark. The other pulses have normal voltages during the light and dark periods (i.e., the same as the TCE voltage pulses during non-metaperiods). The number of light pulses can be set to a number that would remove the Xpandomer if the reader were not stuck. For example, the number of pulses in the light period can be equal to the median number of TCE units in the length distribution of the Xpandomer. The dark period can be sufficient to return the Xpandomer to its reverse position.

[0084] The special light and dark periods may occur once, twice, or more times per meta period. The special light and dark periods may have higher applied voltages and / or may be of longer duration. In FIG. 13, the special periods are three times as long as the normal cycle of one light period and one dark period. The entire meta period is 10 AC periods long. A meta period may occur every 10, 15, or 20 AC periods. In FIG. 13, the special periods are three times as long, and the meta period is 10 AC periods long.

[0085] Regular light periods (e.g., light periods 1312a and 1312b) may have AC correction periods much shorter than 1 or 2 seconds. The number of pulses in the light period may be equal to the median of the input Xmer fragment length distribution. Regular dark periods (e.g., periods 1316a and 1316b) may have an initial high voltage period to rapidly reverse the molecules back to the reader position.

[0086] B. Clear / recharge voltage applied to a particular cell The processive consensus protocol described in Figures 11-13 can be modified. The meta period and clear / recharge voltages can be applied only to cells with stuck Xpandomers. The periods can be applied every 1, 2, 3, 4, or 5 AC periods instead of every 10 or 20 AC periods.

[0087] By the end of the dark period prior to each meta period (i.e., clear period), a determination is made for all active cells in the array as to whether a clear period should be experienced by each cell or whether the cell should be temporarily deactivated during the assertion of the global clear period. In preparation for the clear period, the deactivation mask may be updated.

[0088] During the light and dark clear periods, a deactivation mask is applied and high positive and negative voltages are applied to the global tip lines, and many or most cells may be temporarily deactivated, and thus electrically isolated, so that they do not experience the clearing voltage during either the light or dark clear periods, or both.

[0089] In one embodiment, approximately 1 ms may elapse to update the deactivation masks of all cells on the nanopore sequencer chip. The mask update time may be followed by a light and / or dark clearing period. The clearing period itself may last anywhere from 1 ms to tens of milliseconds. After the clearing period, another time of approximately 1 ms duration may be allotted to reactivate the cells with a second deactivation mask update.

[0090] C. Advantages of Processive Consensus Both the processive consensus applied to all cells and the processive consensus applied to a specific cell can involve individual light periods much shorter than those required to traverse the entire length of the target molecule. Furthermore, subreads generated in successive light periods can overlap at their ends. This results in a protocol that can sequence molecules of any length to a depth greater than one across their entire length. This method is an improvement over the strategy described in Figure 9, in which the length of the light period must be tailored to the specific input DNA fragment length distribution. Molecules above a certain length will undergo multiple passes because they are not eliminated after cycles of light and dark periods. Therefore, the maximum read length on a molecule may be equal to the number of TCE pulses in a light period. Therefore, the ends of molecules longer than that critical length will not be sequenced.

[0091] D. Reduction of duplication In some embodiments, it may be desirable to reduce the number of times a sequence is read. For example, a molecule may be read at a single depth of coverage or slightly greater (e.g., an average depth of between 1 and 2). To achieve this, the dark period can be configured so that there are few reverse TCE pulses compared to the forward light period TCE pulses.

[0092] FIG. 14 shows exemplary molecule 1404 and exemplary molecule 1408 of captured raw reads when the number of reverse TCE pulses is reduced. Read 1412 and read 1416 represent a series of captured raw reads. Read 1420 and read 1424 represent a captured consensus read at the "single-pass limit." In both examples shown in FIG. 14, the number of reverse TCE pulses is 5. In exemplary molecule 1404, the number of forward TCE pulses is 60. In exemplary molecule 1408, the number of forward TCE pulses is 65. Most nucleotides are read in a single pass, achieving the "single-pass limit." Nucleotides corresponding to reverse pulses are read in two passes. Increasing the number of forward TCE pulses and / or decreasing the number of reverse TCE pulses increases the proportion of nucleotides read at only a single depth.

[0093] By targeting depth coverage slightly greater than 1 based on a progressive consensus voltage pattern, shorter light period durations are obtained than simply sequencing the entire molecule in a single light period.

[0094] E. A shorter light period A short AC modulation period (i.e., light period) offers electrochemical and circuit-related advantages. For example, due to the way sequencing is performed using wet analog circuitry, the voltage across the membrane / pore decays during the light period. The dark period is used to "recharge" both the electrochemical cell in the well volume and the electrostatic working electrode capacitor at the bottom of the well. The decay rate of this light period is governed in part by the size of the working electrode capacitor and in part by the concentration and volume of the electrochemically active redox species in the well. While there are some practical advantages to lowering the concentration of electrochemically active redox species used during sequencing, doing so shortens the light period decay time constant. One way to address a shorter light period decay time constant is simply to shorten the duration of the light period itself. Processive consensus suggests that this allows for a substantial reduction in the light period duration without permanently cutting off leads of Xpandomer molecules with lengths longer than the number of TCE pulses in the light period.

[0095] Figures 15A and 15B show the effect of different concentrations of redox couple on the open channel ADC. The graphs show time in seconds on the x-axis and the open channel ADC at the beginning and end of the light period. Figure 15A has a high concentration of redox agent. Figure 15B has an 8-fold lower concentration of redox agent than Figure 15A. For Figure 15A, the difference between the open channel ADC at the beginning and end is 10.8%. For Figure 15B, the difference between the open channel ADC at the beginning and end is 60.2%. Figures 15A and 15B show that the lower the concentration, the shorter the light period decay time constant.

[0096] F. Exemplary Methods Figure 16 is a flowchart of an exemplary process 1600 for sequencing a target nucleic acid molecule. In some implementations, one or more process blocks of Figure 16 can be performed by a system 1700 including a detector 1702 and a logic system 1703. The target nucleic acid molecule can be 100-150 nt, 150-200 nt, 200-300 nt, 300-400 nt, or greater than 500 nt in length.

[0097] In block 1610, a first number of voltage pulses at a first level may be applied across the nanopore to displace the compound through the nanopore a first distance in a first direction. The compound may be generated from a target nucleic acid molecule. The compound may be a surrogate polymer or Xpandomer, as described herein and in WO 2020 / 236526 and U.S. Patent No. 7,939,259, the entire contents of both of which are incorporated herein by reference for all purposes. Prior to applying the first number of voltage pulses, the first compound may be captured by the nanopore. The compound may include multiple units. The units may be similar to the units shown in FIG. 1. Each unit of the multiple units may include one type of reporter element from multiple types of reporter elements (also referred to as reporter codes). Each type of reporter element may correspond to the identity of a nucleotide in the target nucleic acid molecule.

[0098] The first direction can be the same direction in which the compound passed through the nanopore when the compound was initially captured. For example, the compound can have a leader moiety. The leader moiety can be captured within the nanopore. The first direction can be a direction in which the leader moves further away from the nanopore.

[0099] Application of a first number of voltage pulses may cause a first subset of the plurality of units to pass through the nanopore. The first number of voltage pulses may be voltage pulses during a light period. The number of voltage pulses may correspond to the number of units in the plurality of units passing through the nanopore. Each unit of the plurality of units may include a translocation control element. Application of the first number of voltage pulses may cause the first number of translocation control elements to pass through the nanopore. The first number of voltage pulses may be equal to the first number of translocation control elements.

[0100] The first subset of the plurality of units may include 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or more than 100 units. The first number of voltage pulses may include 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or more than 100 pulses.

[0101] A baseline level voltage may be applied across the nanopore to translocate a unit through the nanopore for sequencing during the first number of voltage pulses. This lower level voltage may translocate the unit through the nanopore, but may not be sufficient to translocate the translocation control element through the nanopore. A baseline level voltage may be applied across the nanopore to displace the compound a distance in a first direction through the nanopore. The baseline level voltage may be of the same polarity as the voltage pulses of the first number of voltage pulses. The baseline level may be less than the first level. The compound after displacing the distance may have a translocation control element within the nanopore.

[0102] Thus, in one example, the following actions may be performed in block 1610: applying a first number of voltage pulses at a first level across the nanopore to displace a compound a first distance in a first direction through the nanopore, the compound comprising a plurality of units, each unit of the plurality of units comprising one type of reporter element from a plurality of types of reporter elements, and applying the first number of voltage pulses causes a first subset of the plurality of units to pass through the nanopore.

[0103] In block 1620, the types of reporter elements in the first subset may be detected. A signal value for the nanopore with a voltage applied across the nanopore may be measured when a reporter element in the first subset of units is within the nanopore. The types of reporter elements in the first subset are determined using the signal value. As a result of the correspondence between reporter element types and nucleotides, the identities of the nucleotides within the target nucleic acid molecule are also determined.

[0104] Thus, in one example, the following actions may be performed in block 1620: Detect the reporter element types in the first subset at the nanopore.

[0105] In block 1630, a second number of voltage pulses at a second level may be applied across the nanopore to displace the compound a second distance in a second direction through the nanopore. The second number of voltage pulses may be voltage pulses in a dark period. The first direction is opposite to the second direction. The voltage pulses of the first number of voltage pulses have an opposite polarity to the voltage pulses of the second number of voltage pulses. The second distance may be less than the first distance. The second number may be less than the first number.

[0106] The first number of voltage pulses can exceed the second number of voltage pulses by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, or more than 50. In some embodiments, the first number of voltage pulses can be equal to the second number of voltage pulses. Subsequent pulse cycles can be unequal, so that the pulses ultimately propel the compound through the nanopore.

[0107] Thus, in one example, the following actions may be performed in block 1630: applying a second number of voltage pulses at a second level across the nanopore to displace the compound a second distance in a second direction through the nanopore.

[0108] In block 1640, a third number of voltage pulses at a third level may be applied across the nanopore to displace the compound a third distance in the first direction through the nanopore. The third number of voltage pulses may be voltage pulses during another light period. Application of the third number of voltage pulses causes a second subset of the plurality of units to pass through the nanopore. The second subset and the first subset may include some of the same units. The second subset may include units not in the first subset. The third distance may be greater than the second distance. The third level may be equal to the first level. The third number of voltage pulses may be the same as or different from the first number of voltage pulses.

[0109] Thus, in one example, the following operations may be performed in block 1640: applying a third number of voltage pulses at a third level across the nanopore to displace the compound a third distance in a first direction through the nanopore, and applying the third number of voltage pulses causes a second subset of the plurality of units to pass through the nanopore.

[0110] In block 1650, the types of reporter elements in the second subset may be detected. The sequence of the target nucleic acid molecule may be determined. The sequence may be determined from the order of the types of reporter elements detected. The same type of reporter element may be detected for one or more units in both the first and second subsets. For example, the same reporter element may be detected as described in techniques involving processive consensus. In some embodiments, the reporter element in a particular unit may be detected two, three, four, five, six, seven, eight, nine, ten, or more times. The type of reporter element in the unit may be determined to be the type that is most frequently detected.

[0111] Thus, in one example, the following actions may be performed in block 1650: Detect the reporter element types in the second subset at the nanopore.

[0112] In embodiments, process 1600 may further include a step of completely ejecting the compound from the nanopore. The compound may exit the nanopore during several voltage pulses having the same polarity as the voltage pulses of the first number of voltage pulses. For example, the compound may exit the nanopore during the light period. The compound may completely exit the nanopore during application of the third number of voltage pulses.

[0113] Additional numbers of voltage pulses of alternating polarity may be applied. For example, a fourth number of voltage pulses may displace the compound in a second direction. Then, a fifth number of voltage pulses may displace the compound in a first direction. These cycles of voltage pulses may continue until the compound exits the nanopore.

[0114] In some embodiments, a fourth level of removal voltage may be applied across the nanopore to completely expel the compound from the nanopore. The fourth level may be greater than the first, second, and third levels. In some embodiments, the removal voltage may be applied for a duration longer than the durations of the first, second, and third number of pulses.

[0115] In some embodiments, several nanopores (e.g., in an array) may be used to sequence several nucleic acid molecules. The compound may be the first compound of a plurality of compounds. The plurality of compounds may be generated from a plurality of target nucleic acid molecules. The nanopore may be the first nanopore of a plurality of nanopores. Each compound of the plurality of compounds may be within one nanopore of the plurality of nanopores. A first number of voltage pulses at a first level, a second number of voltage pulses at a second level, and a third number of voltage pulses at a third level may be applied to the plurality of nanopores. The pulses may be applied in the same order as the first compound. A reporter element in each compound of the plurality of compounds may be detected. Multiple sequences of the plurality of nucleic acid molecules may be determined. The size distribution of the plurality of sequences may have a mode greater than 300 nt. The mode may be 200-300 nt, 300-400 nt, 400-500 nt, or greater than 500 nt.

[0116] In some embodiments, similar to the process described in Section III.B, a removal voltage may be applied to a specific nanopore but not to other nanopores. A first number of voltage pulses, a second number of voltage pulses at a second level, and a third number of voltage pulses at a third level may be applied to the multiple nanopores. It may be determined that a first portion of the multiple compounds is displaced in the first portion of the multiple nanopores by the first number of voltage pulses, the second number of voltage pulses, or the third number of voltage pulses. The first portion of the multiple compounds may not be anchored to the nanopore. A fourth level of removal voltage may be applied across each nanopore of the second portion of the multiple nanopores. The applied removal voltage may completely expel the second portion of the multiple compounds from each nanopore of the multiple nanopores. The fourth level may be greater than the first level, the second level, and the third level. In some embodiments, the removal voltage may be applied for a duration longer than the duration of any of the voltage pulses. The second portion of the plurality of nanopores may not include nanopores of the first portion of the plurality of nanopores.

[0117] In some embodiments, it can be determined that a first portion of the plurality of compounds is not displaced by the first number of voltage pulses, the second number of voltage pulses, or the third number of voltage pulses in a first portion of the plurality of nanopores. The first portion of the plurality of compounds can be anchored to the nanopores. A fourth level of removal voltage can be applied across each nanopore in the first portion of the plurality of nanopores.

[0118] A compound can be determined to be stuck if there is no characteristic change in the measured electrical signal when moving from one reporter element to the next. There is a characteristic change in the measured electrical signal when moving from one reporter element to the next. Sticking at a particular reporter element results in no expected change in the electrical signal after applying a moving voltage pulse. A sufficiently high sampling rate is used to distinguish between stuck compounds and compounds with moving but consecutive types of reporter elements.

[0119] Process 1600 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in conjunction with one or more other processes described elsewhere herein.

[0120] 16 illustrates example blocks of process 1600, in some implementations process 1600 may include additional, fewer, different, or differently arranged blocks than those illustrated in FIG 16. Additionally or alternatively, two or more of the blocks of process 1600 may be performed in parallel.

[0121] IV. Exemplary Systems FIG. 17 illustrates a measurement system 1700 according to an embodiment of the present invention. The illustrated system includes a sample 1705, such as an Xpandomer, in a sample holder 1701, which can be contacted with an assay 1708 to provide a signal of a physical property 1715. The assay 1708 can include nanopore-expanded sequencing. An example of a sample holder can be a well plate containing an Xpandomer. The physical property 1715 (e.g., voltage, current, or other electrical property) from the sample is detected by a detector 1702. The detector 1702 can take measurements at intervals (e.g., periodic intervals) to obtain data points that constitute a data signal. In one embodiment, an analog-to-digital converter converts the analog signal from the detector multiple times to a digital format. The detector 1702 can be a voltage or current measuring device. The sample holder 1701 and the detector 1702 can form an assay device. The data signal 1725 is transmitted from the detector 1702 to a logic system 1703. The data signal 1725 may be stored in local memory 1735 , external memory 1704 , or storage device 1745 .

[0122] Logic system 1703 may be or include a computer system, ASIC, microprocessor, etc. It may also include or be coupled to a display (e.g., a monitor, LED display, etc.) and user input devices (e.g., a mouse, keyboard, buttons, etc.). Logic system 1703 and other components may be part of a standalone or networked computer system, or may be attached or incorporated directly into an apparatus (e.g., a sequencing apparatus) including detector 1702 and / or sample holder 1701. Logic system 1703 may also include software implementing within processor 1720. Logic system 1703 may include a computer-readable medium storing instructions for controlling system 1700 to perform any of the methods described herein. For example, logic system 1703 may provide commands to a system including sample holder 1701 such that sequencing or other physical operations are performed. Such physical operations may be performed in a particular order, for example, with reagents being added and removed in a particular order. Such physical manipulation may be performed by a robotic system, including, for example, a robotic arm, so that the sample can be used to obtain and perform an assay.

[0123] Any of the computer systems referred to herein may utilize any suitable number of subsystems. An example of such a subsystem is shown in computer system 10 in FIG. 18. In some embodiments, a computer system includes a single computer device, and the subsystems may be components of the computer device. In other embodiments, a computer system may include multiple computer devices, each of which is a subsystem with internal components. Computer systems may include desktop and laptop computers, tablets, mobile phones, other mobile devices, and cloud-based systems.

[0124] The subsystems shown in FIG. 18 are interconnected via a system bus 75. Additional subsystems are shown, such as a printer 74, a keyboard 78, a storage device 79, and a monitor 76 (e.g., a display screen, such as an LED) coupled to a display adapter 82. Peripherals and input / output (I / O) devices coupled to the I / O controller 71 may be connected to the computer system by any number of means known in the art, such as input / output (I / O) ports 77 (e.g., USB, Thunderbolt, Lightning). For example, the I / O ports 77 or external interfaces 81 (e.g., Ethernet, Wi-Fi, etc.) may be used to connect the computer system 10 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via the system bus 75 not only enables information exchange between the subsystems but also allows the central processor 73 to communicate with each subsystem and control the execution of instructions from the system memory 72 or storage device 79 (e.g., a fixed disk such as a hard drive or an optical disk). The system memory 72 and / or storage device 79 may embody computer-readable media. Another subsystem is a data collection device 85 such as a camera, microphone, accelerometer, etc. Any data mentioned herein can be output from one component to another and to a user.

[0125] A computer system may include multiple identical components or subsystems connected together, for example, by an external interface 81, by an internal interface, or through a removable storage device that can be connected and disconnected from one component to another. In some embodiments, computer systems, subsystems, or devices may communicate over a network. In such cases, one computer may be considered a client and another computer may be considered a server, each of which may be part of the same computer system. The client and server may each include multiple systems, subsystems, or components.

[0126] Aspects of the embodiments may be implemented in the form of control logic using hardware circuitry (e.g., application-specific integrated circuits or field-programmable gate arrays) and / or using computer software with generally programmable processors in a modular or integrated fashion. As used herein, a processor may include a single-core processor, a multi-core processor on the same integrated chip, or multiple processing units located on a single circuit board or networked, as well as dedicated hardware. Based on the disclosure and teachings provided herein, those skilled in the art will know and understand other ways and / or methods for implementing embodiments of the present invention using hardware and combinations of hardware and software.

[0127] Any software components or functions described in this application may be implemented as software code executed by a processor using any suitable computer language, such as, for example, Java, C, C++, C#, Objective-C, Swift, or a scripting language, such as, for example, Perl or Python, using conventional or object-oriented techniques. The software code may be stored on a computer-readable medium for storage and / or transmission as a series of instructions or commands. Suitable non-transitory computer-readable media may include random access memory (RAM), read-only memory (ROM), magnetic media such as a hard drive or floppy disk, or optical media such as a compact disc (CD) or DVD (Digital Versatile Disc) or Blu-ray disc, flash memory, or the like. The computer-readable medium may be any combination of such storage or transmission devices.

[0128] Such programs may also be encoded and transmitted using carrier signals adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. Thus, computer-readable media may be created using data signals encoded with such programs. Computer-readable media encoded with program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer-readable medium may be located on or within an individual computer product (e.g., a hard drive, CD, or complete computer system), or may reside on or within different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing a user with any of the results described herein.

[0129] Any of the methods described herein may be implemented, in whole or in part, in a computer system including one or more processors that may be configured to perform the steps. Accordingly, embodiments may be directed to a computer system configured to perform the steps of any of the methods described herein, possibly including different components that perform each step or each group of steps. While the steps of the methods herein are presented as numbered steps, they may be performed simultaneously, at different times, or in different orders. Furthermore, some of these steps may be used with some of the other steps from other methods. Also, all or some of the steps may be optional. Furthermore, any of the steps of any of these methods may be performed using a module, unit, circuit, or other means of a system for performing these steps.

[0130] The specific details of particular embodiments may be combined in any suitable manner without departing from the spirit and scope of the embodiments of the invention, however, other embodiments of the invention may be directed to particular embodiments relating to each individual aspect or particular combinations of these individual aspects.

[0131] The foregoing descriptions of exemplary embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosure to the precise forms described, and many modifications and variations are possible in light of the above teachings.

[0132] The terms "a," "an," or "the" are intended to mean "one or more" unless specifically indicated otherwise. The use of "or" is intended to mean "inclusive or" rather than "exclusive or" unless specifically indicated otherwise. A reference to a "first" element does not necessarily require that a second element be provided. Furthermore, a reference to a "first" or "second" element does not limit the referenced element to a particular location unless explicitly stated. The term "based on" is intended to mean "based at least in part on."

[0133] All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for all purposes. None are admitted to be prior art.

Claims

1. 1. A method for sequencing a target nucleic acid molecule, comprising: applying a first number of voltage pulses at a first level across the nanopore to displace a compound generated from the target nucleic acid molecule through the nanopore in a first direction a first distance; The compound comprises a plurality of units, each unit of the plurality of units comprises one type of reporter element from a plurality of types of reporter elements; each type of reporter element corresponds to the identity of a nucleotide within said target nucleic acid molecule; applying the first number of voltage pulses causes a first subset of the plurality of units to pass through the nanopore; detecting the type of reporter element in the first subset at the nanopore; applying a second number of voltage pulses at a second level across the nanopore to displace the compound a second distance in a second direction through the nanopore; the first direction is opposite to the second direction; the voltage pulses of the first number of voltage pulses have an opposite polarity to the voltage pulses of the second number of voltage pulses; the second distance is less than the first distance; the second number being less than the first number; applying a third number of voltage pulses at a third level across the nanopore to displace the compound a third distance through the nanopore in the first direction; applying the third number of voltage pulses causes a second subset of the plurality of units to pass through the nanopore; the second subset and the first subset include portions of the same units; the second subset includes units that are not in the first subset; the third distance being greater than the second distance; and detecting the type of reporter element in the second subset at the nanopore; A method comprising:

2. 10. The method of claim 1, further comprising applying a fourth level of removal voltage across the nanopore to completely eject the compound from the nanopore, wherein the fourth level is greater than the first level, the second level, and the third level.

3. the compound is a first compound of a plurality of compounds; the plurality of compounds is generated from a plurality of target nucleic acid molecules; the nanopore is a first nanopore of a plurality of nanopores; 3. The method of claim 1 or 2, wherein each compound of the plurality of compounds is within one nanopore of the plurality of nanopores, applying the first number of voltage pulses at the first level, the second number of voltage pulses at the second level, and the third number of voltage pulses at the third level to the plurality of nanopores. The method further comprises:

4. 4. The method of claim 3, further comprising determining a plurality of sequences of the plurality of target nucleic acid molecules.

5. 5. The method of claim 4, wherein the size distribution of the plurality of sequences has a mode greater than 300 nt.

6. applying the first number of voltage pulses at the first level, the second number of voltage pulses at the second level, and the third number of voltage pulses at the third level to the plurality of nanopores; determining that a first portion of the plurality of compounds is displaced in a first portion of the plurality of nanopores by the first number of voltage pulses, the second number of voltage pulses, or the third number of voltage pulses; applying a fourth level of removal voltage across each nanopore of a second portion of the plurality of nanopores to completely eject a second portion of the plurality of compounds from each nanopore of the plurality of nanopores; the fourth level is greater than the first level, the second level, and the third level; the second portion of the plurality of nanopores does not include a nanopore of the first portion of the plurality of nanopores; 5. The method of claim 3 or 4, further comprising:

7. 7. The method of claim 1, further comprising determining the sequence of the target nucleic acid molecule.

8. 8. The method of claim 7, wherein determining the sequence of the target nucleic acid molecule comprises detecting the same type of reporter element for one or more units in both the first subset and the second subset.

9. The method of claim 1 , further comprising completely pumping the compound out of the nanopore.

10. 10. The method of claim 9, wherein completely ejecting the compound from the nanopore occurs during the application of the third number of voltage pulses.

11. each unit of the plurality of units includes a movement control element; application of the first number of voltage pulses causes a first number of translocation control elements to pass through the nanopore; the first number of voltage pulses equals the first number of movement control elements; 11. The method according to any one of claims 1 to 10.

12. applying a fourth level voltage across the nanopore to displace the compound a fourth distance through the nanopore in the first direction during the first number of voltage pulses; the voltage at the fourth level is of the same polarity as the voltage pulses in the first number of voltage pulses; the fourth level is less than the first level; the compound after being displaced the fourth distance has a translocation control element within the nanopore. The method of claim 11 further comprising:

13. The method of claim 1 , wherein the second level is greater than the first level.

14. measuring a signal value for the nanopore having a voltage applied across the nanopore when a reporter element in the first subset of the plurality of units is within the nanopore; using the signal values ​​to determine the types of reporter elements in the first subset, thereby determining the identities of nucleotides in the target nucleic acid molecule; 14. The method of any one of claims 1 to 13, further comprising:

15. 15. The method of claim 1, wherein the first subset of the plurality of units comprises 30 or more units.

16. 16. The method of claim 1, wherein the third level is equal to the first level.

17. 17. The method of any one of claims 1 to 16, wherein the target nucleic acid molecule is longer than 200 nt.

18. 18. The method of claim 1, wherein the first number of voltage pulses is 30 or greater.

19. 19. The method of any one of claims 1 to 18, wherein the first number of voltage pulses may exceed the second number of voltage pulses by 5 or more.

20. 20. A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that, when executed, control a computer system to perform the method of any one of claims 1 to 19.

21. A computer product according to claim 20; one or more processors for executing instructions stored on the computer-readable medium; A system comprising:

22. A system comprising means for carrying out the method according to any one of claims 1 to 19.

23. 20. A system comprising one or more processors configured to perform the method of any one of claims 1 to 19.

24. A system comprising modules for respectively implementing the steps of the method according to any one of claims 1 to 19.

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