Single-molecule and multi-molecule tracing method and system

Abstract

Methods of sequencing by extension and related improvements in sequencing of replacement polymers in nanopores are described herein. The substitute polymer is formed from a template nucleic acid molecule. The replacement polymer includes a plurality of units. Each unit includes a report code portion. The report code corresponds to a different nucleotide. Alternative polymers may be caught in the nanopores. The embodiments described herein address these stuck alternative polymers. In order to allow for a plurality of reads of the replacement polymer, a sequential consensus technique may be applied, in particular, to allow for a plurality of times of reading of the replacement polymer. The replacement polymer may be moved forward by several units, and then moved backward by fewer units, in order to again identify some of the same reporting codes. This method allows multiple reads of the same report code. The replacement polymer ultimately passes through the nanopore in a forward direction. A higher purge voltage may be periodically applied to purge any stuck replacement polymer in the nanopore.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 412,774, filed on October 3, 2022, which is incorporated by reference for all purposes. Technical Field

[0003] Embodiments described herein relate to nanopore-based sequencing methods and systems. Specifically, the methods and systems include methods for using sequencing by extension (SBX) TM ) to generate consistent reads. Background Art

[0004] Nanopore membrane devices with pores with an internal diameter on the order of one nanometer have shown promise in rapid nucleotide sequencing. When a voltage potential is applied to a nanopore immersed in a conductive fluid, there may be a small ionic current generated by the conduction of ions across the nanopore. The magnitude of the current is sensitive to the pore size and which molecule is in the nanopore. The molecule can be a specific reporter code corresponding to a specific nucleotide, allowing the detection of nucleotides at specific positions of the nucleic acid. The voltage or other signal in the circuit containing the nanopore (for example, on an integrated capacitor) can be measured as a way to measure the resistance of the molecule, so that which molecules are in the nanopore can be detected.

[0005] Nanopore-based sequencing chips can be used for DNA sequencing. Nanopore-based sequencing chips can include a large number of sensor units configured as an array. For example, an array of one million units can include 1000 rows x 1000 columns of units.

[0006] Due to manufacturing variability, the measured signal may vary from chip to chip and may also vary from unit to unit of the same chip. Therefore, it may be difficult to determine the correct molecule, which may be or correspond to the correct nucleotide in a specific nucleic acid or other polymer in the unit. In addition, other time-related non-idealities in the measured signal may also lead to inaccuracies. Moreover, because these circuits use biochemical circuit elements, such as lipid bilayers, nanopores, etc., the variability of electrical properties may be much higher than that of traditional semiconductor circuits. In addition, the sequencing process is essentially random, and therefore variability may occur in various systems (including sequencing devices that do not use nanopores).

[0007] Therefore, improved characterization techniques are desired to improve the accuracy and robustness of the sequencing process. Summary of the invention

[0008] Embodiments described herein relate to sequencing methods and improvements to sequencing of alternative polymers encoded with nucleic acid information in nanopores.Alternative polymers (also referred to herein as "Xpandomer" polymers) are formed by template-guided synthesis, which retains the original genetic information of the target nucleic acid while also increasing the linear separation of the individual elements of the sequence data.The alternative polymer is formed from a template nucleic acid molecule.Alternative polymers include multiple units.Each unit includes one or more report code portions.The report code may correspond to different nucleotides (e.g., A, T, C, G).The report code may generate different electrical signals in the nanopore and therefore allow the identification of nucleotide sequences.Each unit includes a translocation control element (TCE).In order to pass through the nanopore, TCE needs to apply a higher voltage compared to the baseline voltage used to drive the rest of the unit through the nanopore.Alternative polymers may be passed forward and backward through the nanopore multiple times to allow multiple sequence readings.Alternative polymers include a leader sequence fragment, which may get stuck in the membrane on one side of the nanopore when the alternative polymer is pushed out of the nanopore.Embodiments described herein address these stuck alternative polymers.

[0009] In order to allow multiple sequence reads of alternative polymers, a sequential consistent technique can be applied. The alternative polymer can be moved forward by a few units (e.g., 30) and then moved backward by fewer units (e.g., 25) to identify some of the same report codes again. This method allows multiple reads of the same report code. The alternative polymer eventually passes through the nanopore in the forward direction. A higher clearing voltage can be applied periodically to clear any stuck alternative polymer in the nanopore.

[0010] The clearing voltage can be applied more frequently but in a targeted manner. The units (ie, pores) of the surrogate polymer that are not determined to be stuck in the nanopore can be deactivated before applying the clearing voltage. The technique allows molecules of any length to be sequenced over their entire length at a depth greater than one.

[0011] In view of the foregoing, one aspect of the present disclosure is a method for sequencing a target nucleic acid molecule, the method comprising: applying a first number of voltage pulses at a first level across a nanopore to cause a compound to be displaced a first distance in a first direction through the nanopore, the compound being produced from the target nucleic acid molecule, wherein the compound comprises a plurality of units, each of the plurality of units comprising a type of reporter element among a plurality of types of reporter elements, each type of reporter element corresponding to an identifier of a nucleotide in the target nucleic acid, and applying the first number of voltage pulses to cause a first subset of the plurality of units to pass through the nanopore; detecting the type of reporter element in the first subset in the nanopore; applying a second number of voltage pulses at a second level across the nanopore to cause the compound to pass through the nanopore shifting a second distance in a second direction, wherein the first direction is opposite to the second direction, voltage pulses in the first number of voltage pulses have opposite polarity to voltage pulses in the second number of voltage pulses, the second distance is less than the first distance, and the second number is less than the first number; applying a third number of voltage pulses at a third level across the nanopore to shift the compound a third distance in the first direction through the nanopore, wherein applying the third number of voltage pulses causes a second subset of the plurality of cells to pass through the nanopore, the second subset and the first subset include some of the same cells, the second subset includes cells that are not in the first subset, and the third distance is greater than the second distance; and detecting in the nanopore a type of reporter element in the second subset.

[0012] In some embodiments, the method further comprises applying a clearing voltage at a fourth level across the nanopore to cause the compound to completely pass out of the nanopore, wherein the fourth level is greater than the first level, the second level, and the third level.

[0013] In some embodiments, the compound is a first compound among a plurality of compounds, the plurality of compounds are produced from a plurality of target nucleic acid molecules, the nanopore is a first nanopore among a plurality of nanopores, and each compound of the plurality of compounds is in a nanopore among the plurality of nanopores, the method further comprising 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.

[0014] In some embodiments, the method further comprises determining a plurality of sequences of the plurality of target nucleic acid molecules.

[0015] In some embodiments, the size distribution of the plurality of sequences has a mode greater than 300 nt.

[0016] In some embodiments, the method further includes: 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 being 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, applying a clearing voltage at a fourth level across each nanopore in a second portion of the plurality of nanopores to cause the second portion of the plurality of compounds to completely pass through a corresponding 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 a nanopore in the first portion of the plurality of nanopores.

[0017] In some embodiments, the method further comprises determining the sequence of the target nucleic acid molecule.

[0018] 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.

[0019] In some embodiments, the method further comprises allowing the compound to completely pass through the nanopore.

[0020] In some embodiments, completely passing the compound through the nanopore occurs during application of the third number of voltage pulses.

[0021] In some embodiments, each cell in the plurality of cells comprises a translocation control element, applying the first number of voltage pulses causes the first number of translocation control elements to pass through the nanopore, and the first number of voltage pulses is equal to the first number of translocation control elements.

[0022] In some embodiments, the method further includes applying a voltage at a fourth level across the nanopore between voltage pulses in the first number of voltage pulses to cause the compound to be translocated a fourth distance in the first direction through the nanopore, wherein the voltage at the fourth level is the same polarity as the voltage pulses in the first number of voltage pulses, the fourth level is less than the first level, and the compound after being translocated the fourth distance has a translocation control element in the nanopore.

[0023] In some embodiments, the second level is greater than the first level.

[0024] In some embodiments, the method further includes measuring a signal value for the nanopore having a voltage applied across the nanopore when the reporter element in the first subset of the plurality of units is in the nanopore; and using the signal value to determine the type of the reporter element in the first subset, thereby determining the identity of the nucleotide in the target nucleic acid molecule.

[0025] In some embodiments, the first subset of the plurality of cells includes 30 or more cells.

[0026] In some embodiments, the third level is equal to the first level.

[0027] In some embodiments, the target nucleic acid molecule is longer than 200 nt.

[0028] In some embodiments, the first number of voltage pulses is 30 or more.

[0029] In some embodiments, the first number of voltage pulses may exceed the second number of voltage pulses by 5 or more.

[0030] 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 method and embodiments thereof.

[0031] Another aspect of the present disclosure is a system that includes a computer product and one or more processors for executing instructions stored on the computer readable medium.

[0032] Another aspect of the present disclosure is a system comprising an apparatus for performing any of the above methods, a system comprising one or more processors configured to perform any of the above methods, and a system comprising modules that respectively perform the steps of any of the above methods.

[0033] The nature and advantages of embodiments of the present invention may be better understood with reference to the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Alternative polymer compositions according to embodiments of the present invention are shown.

[0035] Figure 2 The XNTP structural details according to an embodiment of the present invention are shown. Figure 2 The molecular structures shown may not be chemically correct. Figure 2 It is provided for illustrative purposes.

[0036] Figure 3Attachment of extended oligomers to solid substrates for alternative polymer synthesis according to embodiments of the present invention is illustrated.

[0037] Figure 4 The structures of an extended oligomer, a leader sequence, and nearby components according to an embodiment of the present invention are illustrated. Figure 4 The molecular structures shown may not be chemically correct. Figure 4 It is provided for illustrative purposes.

[0038] Figure 5A An example of a single-molecule-multi-molecule tracing event with a typical sequencing-by-extension waveform according to an embodiment of the present invention is shown.

[0039] Figure 5B Molecules that are not pore cleared according to an embodiment of the present invention are illustrated.

[0040] Figure 6 One strategy for sequencing alternative polymers involving identical forward and reverse voltages according to an embodiment of the present invention is shown.

[0041] Figure 7 A strategy for sequencing alternative polymers involving different forward and reverse voltages according to an embodiment of the present invention is shown.

[0042] Figure 8 A strategy for sequencing surrogate polymers involving shortened bright period durations according to an embodiment of the invention is shown.

[0043] Fig. 9 A timing schedule of light periods and dark periods with a sequencing strategy according to an embodiment of the present invention is shown.

[0044] Fig.10 A strategy for sequencing using an inverted nanopore according to an embodiment of the present invention is shown.

[0045] Fig.11 A voltage schedule for sequential multi-pass read segment generation according to an embodiment of the present invention is illustrated.

[0046] Fig.12 An assembled captured raw read series according to an embodiment of the present invention is illustrated.

[0047] Fig.13 Meta-periods of clearing a surrogate polymer from a nanopore are shown, in accordance with an embodiment of the present invention.

[0048] Fig.14 Shown are captured raw read series assembled according to an embodiment of the present invention.

[0049] Fig.15A and Fig. 15B The effect of different concentrations of redox couple in an open channel ADC according to an embodiment of the present invention is shown.

[0050] Fig.16 is a flow chart of an example process for sequencing a target nucleic acid molecule according to an embodiment of the present invention.

[0051] Fig.17 A measuring system according to an embodiment of the invention is shown.

[0052] Fig.18 A block diagram of an exemplary computer system that may be used with systems and methods according to embodiments of the present invention is shown. DETAILED DESCRIPTION

[0053] Embodiments described herein include protocols for nanopore sequencing, including sequencing by extension (SBX TM ) scheme. The scheme includes a "leader" portion of the molecule that has the property of easily entering the hole in the forward direction but having a high barrier to translocation through the hole in the reverse direction. In addition, the embodiment includes a voltage pattern applied during the modified light and dark periods, which is designed to electrically capture the surrogate polymer (e.g., Xpandomer) molecule in the nanopore and position the molecule in a controlled manner so as to allow multiple ring tests or sequence readings of the same surrogate polymer molecule. In addition, the embodiment includes applying higher voltage pulses and / or longer cycle times on periodic light / dark cycles to eventually remove molecules from the hole. Some embodiments include selectively applying higher voltage pulses to remove molecules only from specific holes in each global pulse application period. Advantages include enrichment of compound molecule tracing events (i.e., subsequence readings (subreads) from the same molecule in a series of light periods). Other advantages may include an increase in hole occupancy and original base recognition (base call) flux. In addition, compared with single-ring sequence reading, the scheme can produce more accurate multi-ring sequence readings. Embodiments may include running with much shorter bright periods without permanently cutting off the reads of the long surrogate polymer molecules. This may allow for realistic experimental conditions that produce short bright period decay time constants.

[0054] In addition, embodiments may include the ability to dynamically choose to spend more time on alternative polymers from a particular UMI (Unique Molecular Identifier) ​​family of molecules and less time on alternative polymers from other UMI families of molecules. UMIs may be added to sample nucleic acids during the sample preparation phase. Each nucleic acid segment from a particular sample will have the same UMI. Different samples will have different UMIs. UMIs allow different samples to be pooled together for sequencing while identifying the sample for each nucleic acid molecule. During sequencing, one may determine that sequences (i.e., samples) from a particular UMI family may require additional sequencing. For example, due to a relatively high number of different base calls for the same position, the confidence of certain base calls may be low. Segments from these samples may be sequenced additional times using the methods described herein.

[0055] I. Sequencing by Extension

[0056] Embodiments may be applied to sequencing by extension (SBX) using nanopores. Sequencing by extension is described in WO 2020 / 236526 A1, filed May 14, 2020, "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing," and US 7,939,259 B2, filed June 19, 2008, "High throughput nucleic acid sequencing by expansion," both of which are incorporated herein by reference in their entirety for all purposes.

[0057] The sequencing by extension scheme is based on the polymerization of highly modified non-natural nucleotide analogs (called "XNTPs"). In general, SBX uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer". The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are separated by about 10nm and designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to natural DNA.

[0058] A. Alternative Polymer Structures

[0059] Figure 1Components of a surrogate polymer (eg, Xpandomer) are shown. An Xpandomer includes a sequence of ordered XNTPs corresponding to the order of a target DNA sequence. Figure 1 Not drawn to scale. XNTPs are expandable, 5' triphosphate-modified non-natural nucleotide analogs compatible with template-dependent enzymatic polymerization. XNTPs have two distinct functional regions; namely: a selectively cleavable phosphoramidite bond that connects the 5' α-phosphate to the nucleobase; and a symmetrically synthesized reporter tether (SSRT) attached to certain positions within the nucleoside triphosphoramidite that allow controlled extension by cleavage of the phosphoramidite bond. SSRTs include linkers, and these linkers are separated by selectively cleavable phosphoramidite bonds. Each linker is attached to one end of the reporter code.

[0060] Segment 102 shows primer-directed Xpandomer synthesis. XNTP 104 is illustrated in a "constrained configuration," a characteristic of the daughter strand product of XNTP substrate and template-dependent polymerization. The constrained configuration of the polymerized XNTP is a precursor to the extended configuration (XNTP 108), as seen in the Xpandomer product. Segment 106 illustrates cleavage to extend the Xpandomer. The transition from the constrained configuration to the extended configuration occurs when the PN bond of the phosphoramidate within the primary backbone of the daughter strand is broken.

[0061] During assembly, monomeric XNTP substrates (XATP, XCTP, XGTP and XTTP) are polymerized on the extendable end of the nascent daughter chain by a template-directed polymerization process using a single-stranded template as a guide. Typically, the process starts with a primer and proceeds in a 5' to 3' direction. Typically, DNA polymerase 110 or other polymerases are used to form the daughter chain, and conditions are selected to obtain a complementary copy of the template strand. After the daughter chain is synthesized, the coupled SSRT forms a constrained Xpandomer that further forms the daughter chain. The SSRT in the daughter chain has a "restricted configuration" of the XNTP substrate. The constrained configuration of the SSRT is a precursor to the unfolded configuration, as seen in the Xpandomer product.

[0062] In this example, once synthesis and expansion are complete, each monomeric XNTP unit 112 in the Xpandomer contains two reporter codes 116a and 116b, where the reporter code "level" corresponds to the base type it encodes, and a translocation control element (TCE) 120. The TCE controls the rate at which the Xpandomer translocates through the nanopore by a combination of stereoisomers, electrical repulsion, and / or preferential interactions with the nanopore. When positioned at the pore orifice, the resistance of the TCE to the driving force of ionic current and the subsequent increase in applied voltage (i.e., voltage pulse) necessary to overcome the stall and resume translocation can be customized by modulating various properties of the TCE (and, in some embodiments, the reporter codes and other elements of the SSRT), such as volume, length, and / or charge density.

[0063] Branch 124 is a branched structure that terminates the TCE and is linked to reporting codes 116a and 116b. Enhancers 128a and 128b can facilitate polymerase incorporation. Nucleotide 132 is attached to enhancer 128b and can include a cleavable linker 136. Cleavable linker 136 can be a photocleavable linker. Cleavable linker 136 can be cleaved to cause the extension shown in segment 106.

[0064] Figure 2 The structural details of XNTP 200 are shown. Figure 2 In the embodiment of the invention, the XNTP includes two reporter codes 204a and 204b, and a translocation control element (TCE) 208. Different sizes of reporter codes can prevent ions from flowing through the nanopore at different measurable levels. Reporter codes and other features can be designed by selecting the sequence of specific phosphoramidites.

[0065] In certain embodiments, TCE is a polymer produced by solid phase synthesis using a phosphoramidite method, having a suitable monomer structural unit terminated with a branched structure (i.e., "branch"). Branched phosphoramidites include both symmetrical branches and asymmetrical branches. In one embodiment, TCE branch 210 is a symmetrical branched CED phosphoramidite, wherein each arm of the branch is connected to a reporting code. Exemplary symmetrical chemical branches include 1,2,3-O-tri-(phosphodiester)-propane, 1,3-bis-(5-O-phosphodiester-pentylamide)-2-O-phosphodiester-propane and 1,4,7-O-tri-(phosphodiester)-heptane.

[0066] UV chromophore 212 may be attached to the end of TCE 208. UV chromophore 212 may allow visualization or quantification. Spacers 216a and 216b are attached to reporting codes 204a and 204b, respectively. Spacers 216a and 216b may be polyethylene glycol (PEG) units that may adjust the length passed through the hole. Enhancers 220a and 220b may be attached to spacers 216a and 216b. Enhancers 220a and 220b may be positively charged spermine that facilitates polymerase incorporation. Nucleotides 224 may be attached to enhancers 220a and 220b. Nucleotides 224 may include triphosphate diesters. The structural elements of XNTP 200 may be adjusted for improved measurements within the nanopore.

[0067] Figure 3 Attachment of an extended oligomer 304 to a solid substrate 308 is illustrated in preparation for Xpandomer synthesis. The solid substrate 308 may be cross-linked with maleimide. Stage 312 shows that the extended oligomer 304 is not attached to the solid substrate 308. After click chemistry at stage 316, the extended oligomer 304 is attached to the solid substrate 308. The extended oligomer 304 may be attached to a leader sequence portion 320. The leader sequence portion 320 may be attached to a photocleavable linker 324.

[0068] Figure 4 The structure 400 of an extension oligonucleotide 404, a "leader sequence" (L), and nearby components is illustrated. The extension oligonucleotide can be a 2'O-methyl extension oligonucleotide. Adjacent to the extension oligonucleotide 404 is a poly-C12 spacer (Z) 408, followed by a poly-C2 spacer (L) 412. The photocleavable spacer (PC) 416 and the excess fragments (i.e., azide (R) 420 and spacer (D) 424) are not part of the Xpandomer in the sequencing scheme described herein, because photocleavage occurs immediately after Xpandomer synthesis and before any xpandomer is loaded on the sequencer. Azide R 420 can be 5'-azide. Spacer D 424 can be a poly-PEG6 spacer. The abbreviated sequence for structure 400 can be R(D) 10 (PC)(L) 25(Z)6(TCATAAGACGAACGGA), where the sequence at the end represents the extended oligonucleotide 404. During some Xpandomer sequencing operations after the xpandomer molecules flow into the fluid channel above the membrane, the hydrophobic poly-C12 spacer may partition to the membrane phase, initiating quasi-2D diffusion / migration along the surface of the membrane. 3D diffusion / migration and fluid flow may also play a role in the mass transport of the Xpandomer toward the pore. It is believed that Xpandomer capture occurs when the highly negatively charged poly-C2 spacer fragment (L) is electrostatically attracted into the barrel of the nanopore during a "positive" applied bias across the membrane. Note that for the inverted pore, the bottom of the barrel of the pore protrudes slightly from the bilayer membrane on the cis side.

[0069] B. Operation

[0070] During the "light period", the Xpandomer molecules are captured and begin to translocate through the nanopore due to a combination of both baseline and TCE applied voltage pulses. The baseline voltage is sufficient to read the tag code at each XNTP position, and the short, higher voltage TCE pulses are designed to overcome the energy barrier associated with the TCE. Ideally, each TCE pulse will result in translocation across a single TCE barrier, moving the Xpandomer further in the forward direction into the pore by the amount of one "base" position.

[0071] During typical operation, the applied voltage pattern is designed so that there are a fixed number of TCE pulses during each bright period, which causes the Xpandomer to translate in the “forward” direction for a number of bases corresponding to the number of TCE pulses, or until the Xpandomer is fully translocated and released into the fluid “trans” chamber beneath the membrane.

[0072] During typical operation, Xpandomer molecules may not completely translocate before the end of a single bright period. This may occur due to the capture of molecules in the late bright period and having an Xpandomer length with more base positions than the remaining TCE pulses in the bright period. For a variety of reasons, molecules may be stuck when attempting to translocate in the forward direction. There may be base positions with defects (such as failed cutting events), which makes it impossible or very difficult for molecules to translocate beyond this point. In such cases, and for other reasons, Xpandomer may not be able to completely translocate during the bright period, regardless of the number of TCE pulses in the bright period. In such cases, it can be observed that multiple base positions at the beginning of the sequence reading are sequenced and generate expected signal levels until the defect position is reached. The last tag code level just before the defect can then be observed in the remainder of the bright period. In order to prevent the hole from remaining permanently blocked, a large negative voltage can be applied over a period of time in the dark period to remove any stuck molecules by driving them forcefully in the reverse direction.

[0073] Figure 5A An example of a single molecule-multimolecule tracer (SM3T) event with a typical sequencing waveform by extension is shown. The figure shows time (in seconds) on the x-axis. The figure shows voltage readings on the y-axis. Other electrical measurements can be used instead of voltage, including voltage equivalents (e.g., ADC counts) or current. Dark periods 504 and 508 are normal dark periods, where the hole is clean. Bright period 512 shows signals 516a and 516b of molecule 1 and molecule 2, respectively.

[0074] Signal 520 shows molecule 3 during the bright period. This event shows that molecule 3 was stuck in the hole and did not clear over several cycles (dark periods 524, 528, 532 and signals 536, 540, and 544 in the bright period). Eventually, the molecule cleared in the dark cycle, as indicated by the change from signal 548a to signal 548b (when molecule 3 cleared). This event may be due to the nature of the Xpandomer leader sequence fragment, which makes it difficult for the leader sequence to translocate in the reverse direction.

[0075] Figure 5B Illustrated Figure 5A Figure 552 shows a bright period. The translocation direction is downward. Uncut position 556 will hit the hole after the next pulse. Normal label code level is expected. Figure 558 shows a dark period. The translocation direction is now upward. It is difficult for the leader sequence 560 to translocate in the reverse direction (upward) through the hole. Eventually, the leader sequence 560 passes through the hole.

[0076] Xpandomer molecules can be designed to have properties in the leader sequence portion of the Xpandomer that cause the leader sequence to behave differently in the forward and reverse directions. During the bright period (forward direction), the leader sequence may have properties that allow the leader sequence to be captured into the pore from the cis side with a relatively high capture rate under reasonably applied voltages. After capture, but still during the same bright period, the leader sequence may protrude from the bottom side of the pore (the reverse side of the membrane) because the TCE pulse causes the molecule to steadily advance through the pore.

[0077] During the dark period (reverse direction), if the molecule was still in the pore at the beginning of the dark period, the molecule should begin to translocate in the reverse direction under the negative applied voltage. Once the Xpandomer molecule has almost completely reversed its position (i.e., it has almost completely moved backward), the leader sequence may remain on the trans side of the barrel. At this point, the desired property of the leader sequence is that the leader sequence has a high energy barrier to entering the barrel from the trans side and is therefore highly resistant to translocation through the barrel in the trans to cis direction (forward direction).

[0078] II. Strategies for Sequencing

[0079] This article describes strategies that exploit asymmetric leader sequence behavior relative to the direction of passage through the pore. These strategies can improve accuracy by reducing the number of Xpandomers that get stuck in the pore.

[0080] A. Full forward and full reverse with the same voltage

[0081] Figure 6 One strategy for sequencing Xpandomers involving identical forward and reverse voltages is shown. Figure 6 A nanopore is shown with a membrane below the nanopore. The Xpandomer has a blocker 604 (e.g., streptavidin) on one end and a leader sequence 608 on the other end. The leader sequence line is punctured (downward in the figure). The Xpandomer moves downward and is sequenced during the bright period (e.g., Figure 612) until the end of the molecule and / or the end of the bright period. A voltage is applied to move the Xpandomer downward. The voltage includes a baseline voltage that moves the Xpandomer downward so that a sequence reading can be performed on the reporter element. In order to move the Xpandomer through the TCE element, a higher voltage is applied. The blocker 604 prevents the Xpandomer from leaving the hole.

[0082] During the dark period (e.g., Figure 616), the applied voltage is reversed and the Xpandomer translocates in the upward direction. The same voltage is applied during the dark period as during the light period, but the voltage has the opposite polarity. It may be difficult for the leader sequence 608 to pass through the membrane and / or pore in the opposite direction.

[0083] The distribution of SM3T durations may be exponential. The distribution may show many shorter lengths, reflecting that the Xpandomer is stuck in the nanopore. The mode of the durations should be equal to 1.

[0084] B. Full forward and full reverse with increased reverse voltage

[0085] Figure 7 A strategy for sequencing an Xpandomer involving different forward and reverse voltages is shown. The nanopore and Xpandomer are similar to Figure 6 is configured. The Xpandomer has a blocker 704 on one end and a leader sequence 708 on the other end. The bright period in Figure 712 can be the same as the bright period in Figure 612. During the dark period, the leader sequence may be stuck and may not be able to pass through the nanopore. To address this situation, the reverse voltage is increased for some periodic dark cycles (e.g., the dark period in Figure 716). For example, one dark period in every 10 dark periods or one dark period in every 20 dark periods may include an increased reverse voltage to remove any stuck leader sequences. This increased voltage may be referred to as a "clearing voltage."

[0086] The shape of the distribution of SM3T duration can be obtained from Figure 6 The distribution of Xpandomers changes. Fewer Xpandomers are stuck in the nanopores, so the distribution shows longer lengths. The mode may not be equal to 1.

[0087] C. Limited light period duration

[0088] Figure 8 A strategy for sequencing Xpandomers involving shortened bright period durations is shown. Figure 8 A nanopore is shown with a membrane underneath. The Xpandomer has a leader sequence 804 on the other end and no blocker on the other end. The leader sequence 804 line is punctured (in the figure in the downward direction). The Xpandomer moves downward and is sequenced during the bright period (e.g., Figure 808). The bright period is shortened to prevent Xpandomers of certain sizes from traveling through and leaving the nanopore in the forward direction. Shorter Xpandomers may completely translocate through and escape. Each bright period may include a fixed, predetermined number of TCE voltage pulses. A fixed number of bright periods may be applied. Then a dark period (e.g., Figure 812) is 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.

[0089] The shape of the distribution of SM3T duration can be obtained from Figure 6The distribution of the sequence read lengths changes. The mode may not be equal to 1. This distribution may shrink the sequence read length distribution to most reads within the mode + / - a few bases.

[0090] Fig. 9 A light period and dark period timing schedule with a sequencing strategy is shown. The upper graph shows the polarity of the voltage applied across the nanopore. During the light period, the polarity is shown as 1. During the dark period, the polarity is shown 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 in the pore. Position 1 corresponds to the cell of the Xpandomer closest to the leader sequence. In this example, a high position number (e.g., 200) corresponds to a cell of the Xpandomer that is away from the leader sequence and closer to the opposite end of the Xpandomer.

[0091] like Fig. 9 As shown, bright period 904 may include normal sequencing by Xpandomer 908. Sequencing may be limited by the duration of the bright period or by complete translocation and escape. Fig. 9 Xpandomer 908 and all Xpandomers in have no blocking agent on the tail end of the molecule.

[0092] Xpandomer 912 may enter the nanopore late in the light period 916. After the light period ends, more bases (eg, reporter elements) may remain. During the dark period, Xpandomer 912 is completely removed from the nanopore.

[0093] exist Fig. 9 In the example of FIG. 1 , Xpandomer 912 has more cells (e.g., TCE) than the number of pulses in the bright period. During the dark period 920, a high voltage is applied to ensure that the leader sequence reaches the hole. A lower voltage is then applied. A particular Xpandomer will never completely translocate through the nanopore. The duration of the bright period can be increased so that more Xpandomers pass through the nanopore. However, too long a bright period may cause the Xpandomer to translocate through the nanopore in only one direction. In addition, there may be Xpandomers of different sizes in the sample. A specific bright period duration may cause a shorter Xpandomer to translocate through the nanopore in only one direction, while also being insufficient to sequence all XNTPs in a longer Xpandomer.

[0094] Xpandomer 924 may reach the pore early in bright period 928. This molecule has more bases than the pulses remaining in the bright period. Therefore, Xpandomer 924 does not fully translocate across. Almost as many bases are sequenced as there are pulses in the bright period.

[0095] D. Inverted Nanopore

[0096] Fig.10 A strategy for sequencing using an inverted nanopore is shown. The pore is inserted from the cis side, as has been done historically for SBT on HTP. The Xpandomer includes a leader sequence at one end and a blocker (e.g., blocker 1004) at the other end. The leader sequence enters the membrane before the nanopore. The Xpandomer is sequenced as it is reversed out of the nanopore.

[0097] In step 1008, the Xpandomer is captured. A high voltage (e.g., a TCE voltage) is applied for a longer period of time (e.g., 0.1 to 10 milliseconds instead of 8 μs). The captured molecule 1012 with a blocker moves to the end so that further translocation is prevented by the blocker. Also shown in step 1008 are captured molecules 1016 without a blocker. The molecules 1016 may include segmented molecules. Molecules without a blocker can quickly pass through the nanopore.

[0098] In step 1020, Xpandomer 1012 is moved in the reverse direction to withdraw Xpandomer 1012 from the nanopore. TCE voltage is applied in the reverse direction. Sequencing information is obtained when the molecule exits. All captured molecules are expected to be located at the ends of the molecule (or at uncut positions). The molecules are pulsed in the reverse direction, and data is acquired as the molecules are being withdrawn. The bright period may be as long as the longest expected molecule in the sample. For example, for ctDNA determination, the longest expected molecule may be 350bp, corresponding to 350 pulses or 350ms with a 1ms inter-pulse duration.

[0099] In step 1030, an optional recharge may be performed before the next capture step. A dark period may be applied. If the accumulated voltages from steps 1 and 2 are not balanced, the dark voltage may help recharge the electrodes. During this period, no data is acquired. If a phased array mode is being run, this dark period may be the duration of steps 1008 and 1020, allowing the other half (phase) of the chip to complete these two steps.

[0100] The leader sequence can be modified so that it is not easily translocated when moving through the pore starting from the vestibule (i.e., membrane) side. The Xpandomer may need fairly good capture when entering from the vestibule side. High voltage may help capture the Xpandomer. The membrane may tolerate high voltage for a longer time than other strategies (e.g., with 8 μs pulse duration). Blocks can be added to the ends of the Xpandomer.

[0101] The benefits of this strategy may include filtering out a small fraction of segmented molecules. In addition, the start of sequence reads may be synchronized. Furthermore, pore insertion may be from the cis side. For example, the 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.

[0102] III. Sequential Consistency Strategy

[0103] Strategies can include taking sequence reads of the same XNTP multiple times in the nanopore and applying a voltage to clear stuck Xpandomers. These strategies can improve accuracy by reducing the number of Xpandomers stuck in the pore and / or by repeating sequence reads of XNTPs that pass through the nanopore.

[0104] A. Clear / recharge voltage applied to all cells

[0105] The strategy for sequencing molecules may not involve each loop test and the molecules are read from the beginning to the end. A part of the molecule can be moved through the nanopore to read the subsequence, rather than the whole molecule moving through the nanopore to read the complete sequence. In addition, the molecule can move forward and then move backward to perform many short overlapping loop tests and sequential methods. Compared with other strategies, the bright period and dark period duration may be shortened. The number of high voltage TCE pulses can be less than the number of TCE in the Xpandomer. The number of TCE pulses in the bright period will be greater than the number of reverse TCE pulses in the dark period.

[0106] As an example, the Xpandomer length distribution may have a peak around 350 bp. The light period may include a duration of 30 TCE pulses. The dark period duration may be equal to the light period duration in total time, but with an applied voltage pattern that includes 25 TCE pulses in the reverse direction. Having equal durations of light and dark periods allows for balancing the charge on the electrodes, regenerating and / or resetting the electrodes. The scheme may include additional periods that include a "clearing voltage" to periodically remove Xpandomers that occasionally get stuck.

[0107] Fig.11The voltage schedule for sequential multi-loop measurement sequence readout generation is illustrated. The figure shows voltage pulses for the top electrode and for the bottom electrode. The dotted line indicates a voltage of 0 polarity for each corresponding electrode. The voltage above the dotted line has a positive polarity. The voltage below the dotted line has a negative polarity. During the bright period, the top electrode has a pulse of positive polarity, while the bottom electrode has a pulse of negative polarity. Bright periods 1104, 1108, and 1112 are shown. During the dark period, the top electrode has a pulse of negative polarity, while the bottom electrode has a pulse of positive polarity. Dark periods 1116 and 1120 are shown. The dark period is illustrated as having five fewer pulses than the bright period.

[0108] During the bright period, the Xpandomer moves in the downward direction as shown. The Xpandomer is sequenced during the bright period. Each pulse should correspond to a sequence read. Capture of the Xpandomer may occur at any time during the bright period. During the dark period, the Xpandomer moves in the reverse (upward) direction. No sequencing is performed during the reverse direction. If the leader sequence retreats to the vestibule, the Xpandomer may get stuck. The meta-period with the clearing voltage may move out of the Xpandomer. Fig.11 The meta-period is not shown.

[0109] After a sufficient number of cycles, the molecule (eg, Xpandomer 1124) exits the pore in the forward direction.

[0110] Fig.12 Is caused by Fig.11 12. The Xpandomer length corresponds to a target nucleic acid molecule of 116 bp in length. The Xpandomer moves with a forward cycle of 30 pulses and a reverse cycle of 25 pulses. A total of 20 cycles are used to cover the entire length of the Xpandomer. The reads for each cycle are shown in section 1204. Since each cycle contains overlapping reads, a single nucleotide will be sequenced several times. The common reads captured are shown at reads 1208. The number of times the nucleotides have been sequenced is shown below the common reads captured. For example, the initial subsequence of AAGCT is sequenced twice. The middle part starting with TCTGGT is sequenced six 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 bright period has more forward pulses than the dark period with reverse pulses, the start of the Xpandomer can be sequenced multiple times. By continuing the forward and reverse pulses, the ends of the Xpandomer can be sequenced multiple times until the Xpandomer has completely left the nanopore.

[0111] As an example, each half cycle may be 30ms with 1ms interpulse interval. A complete cycle may take 60ms. The total time spent on one molecule may be 1200ms. A total of 577 original bases can be sequenced in 1.2s total time or 0.6s bright time. In addition to the 20 bases at either end, the 116bp molecule is sequenced at 6 times coverage depth.

[0112] Fig.13 A meta-period for clearing the Xpandomer from the nanopore is shown. The meta-period itself may contain multiple AC periods and a light and dark clearing cycle. The AC period may be similar to those typically used in sequencing, except that it is shorter than 1 or 2 seconds. The pulses indicated by segments 1304 and 1308 are light and dark clearing cycles. For the light period and dark period, the other pulses have normal voltage (i.e., the same TCE voltage pulses as during non-meta periods). If the leader sequence is not stuck, the number of pulses in the light period can be set to the number of cleared Xpandomers. For example, the number of pulses in the light period can be equal to the median number of TCE units in the Xpandomer length distribution. The dark period may be sufficient to drive the Xpandomer back to the reverse position.

[0113] Each elementary period may perform one, two or more special light periods and dark periods. The special light periods and dark periods may have a higher applied voltage and / or a longer duration. Fig.13 In , the special period is three times the duration of the normal cycle of one light period and one dark period. The entire meta period is 10 AC periods long. The meta period can be applied every 10, 15 or 20 AC periods. Fig.13 The special period is 3 times longer and the meta period is 10 AC periods long.

[0114] Normally bright periods (e.g., bright periods 1312a and 1312b) may have AC-modified periods much shorter than 1 or 2 seconds. The number of pulses in a bright period may be equal to the median of the input xmer segment length distribution. Normally dark periods (e.g., periods 1316a and 1316b) may initially have a high voltage in the period to quickly drive the molecule back to the leading sequence position in the reverse direction.

[0115] B. Clear / recharge voltage applied to a specific cell

[0116] Can modify about Figures 11 to 13 The sequentially consistent scheme described. The meta-periods and clear / recharge voltages can be applied only to cells with stuck Xpandomers. These periods can be applied every first, second, third, fourth, or fifth AC period instead of every 10th or 20th AC period.

[0117] At the end of the dark period preceding each meta-period (i.e., purge period), a decision is made for each active cell in the array whether each cell should undergo a purge period or whether the cell should be temporarily deactivated during assertion of the global purge period. The deactivation mask may be updated in preparation for the purge period.

[0118] During the bright and dark clear periods, a deactivation mask is applied and high positive and negative voltages are applied to the global chip lines. Many or most cells may be temporarily deactivated and thus electrically isolated so that they do not experience clear voltages during the bright or dark clear periods or both.

[0119] In one embodiment, it may take about 1 ms to update the deactivation mask for all cells on the nanopore sequencer chip. The mask update time may be followed by a light and / or dark purge period. The purge period itself may last anywhere from one millisecond to tens of milliseconds. After the purge period, another time period of about 1 ms may be allocated to reactivate the cell with a second deactivation mask update.

[0120] C. Advantages of sequential consistency

[0121] Sequential consensus, applied to all cells and to specific cells, may include individual bright periods that are much shorter than the time required to traverse the full length of the target molecule. In addition, subsequence reads generated in consecutive bright periods may overlap at the ends. This creates a scheme in which molecules of any length can be sequenced at a depth greater than one over their entire length. The method is about Fig. 9 Improvements of the described strategy, wherein the bright period length will need to be adjusted for a specific input DNA segment length distribution. Molecules equal to or greater than a certain length will undergo multiple loop tests, because the molecules will not clear after the cycle of bright and dark periods. Therefore, the maximum sequence read length on a molecule can be equal to the number of TCE pulses in the bright period. Therefore, the ends of molecules longer than this critical length will not be sequenced.

[0122] D. Reduce overlap

[0123] 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 coverage or at a slightly greater depth (e.g., an average depth between 1 and 2). To achieve this, the dark period may be set so that there are fewer TCE pulses in the reverse direction than in the forward direction during the bright period.

[0124] Fig.14Example molecules 1404 and 1408 of raw reads captured when the number of reverse TCE pulses is reduced are shown. Reads 1412 and 1416 show a series of raw reads captured. Reads 1420 and 1424 show common reads captured with "single ring limit". Fig.14 In the two examples shown, the number of reverse TCE pulses is 5. In example molecule 1404, the number of forward TCE pulses is 60. In example molecule 1408, the number of forward TCE pulses is 65. Most nucleotides are sequenced using a single loop measurement, achieving "single loop measurement limit". The nucleotides corresponding to the reverse pulses are nucleotides that are sequenced in two loop measurements. Increasing the number of forward TCE pulses and / or reducing the number of reverse TCE pulses increases the proportion of nucleotides that are sequenced at only a single depth.

[0125] Based on a sequential uniform voltage pattern, targeting a depth coverage slightly greater than 1 results in shorter bright-phase durations than simply sequencing the entire molecule in one single bright-phase.

[0126] E. Shorter light period

[0127] Short AC modulation periods (i.e., bright periods) bring electrochemical and circuit-related advantages. For example, due to the way in which sequencing is performed using wet analog circuits, the voltage across the membrane / pore decays during the bright period. The dark period is used to "recharge" the electrochemical cell in the volume of the well and the electrostatic working electrode capacitor at the bottom of the well. The rate at which this bright period decays depends in part on the size of the working electrode capacitor and in part on the concentration and volume of the electrochemically active redox species in the well. There are some practical advantages in reducing the concentration of the electrochemically active redox species used during sequencing, but doing so produces a shorter bright period decay time constant. One way to handle a shorter bright period decay time constant is to simply shorten the duration of the bright period itself. Sequential consistency allows for a significant reduction in the duration of the bright period without permanently cutting off the reads for Xpandomer molecules having a length longer than the number of TCE pulses in the bright period.

[0128] Fig.15A and Fig. 15B The effect of different concentrations of redox couple in the open channel ADC is shown.The graph shows time in seconds on the x-axis, and the open channel ADC at the beginning and end of the bright period. Fig.15A Has a high concentration of redox agents. Fig. 15B With ratio Fig.15A Eight times lower redox concentration. Fig.15A , the difference between the start and end of the open channel ADC is 10.8%. Fig. 15B, the difference between the open channel ADCs for the beginning and the end is 60.2%. Fig.15A and Fig. 15B It is shown that lower concentrations produce shorter bright period decay time constants.

[0129] F. Exemplary Methods

[0130] Fig.16 is a flow chart of an example process 1600 for sequencing a target nucleic acid molecule. In some embodiments, Fig.16 One or more process blocks of can be performed by system 1700, including detector 1702 and logic system 1703. The target nucleic acid molecule can be 100 nt to 150 nt, 150 nt to 200 nt, 200 nt to 300 nt, 300 nt to 400 nt, or more than 500 nt long.

[0131] At block 1610, a first number of voltage pulses at a first level may be applied across the nanopore to displace a compound a first distance in a first direction through the nanopore. The compound may be produced by a target nucleic acid molecule. The compound may be a surrogate polymer or Xpandomer, as described herein and in WO 2020 / 236526A1 and US 7,939,259 B2, 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 a plurality of units. The units may be similar to Figure 1 Each unit in the plurality of units may include one type of reporter element among a plurality of types of reporter elements (also referred to as reporter codes). Each type of reporter element may correspond to the identity of a nucleotide in a target nucleic acid molecule.

[0132] The first direction may be in the same direction that the compound moved through the nanopore when the compound was initially captured. For example, the compound may have a leader sequence portion. The leader sequence portion may be captured in the nanopore. The first direction may be in the direction that the leader sequence moved away from the nanopore.

[0133] Applying a first number of voltage pulses may cause a first subset of the plurality of cells to pass through the nanopore. The first number of voltage pulses may be voltage pulses in a bright period. The number of voltage pulses may correspond to the number of cells in the plurality of cells that pass through the nanopore. Each cell in the plurality of cells may include a translocation control element. Applying a first number of voltage pulses may cause a 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.

[0134] The first subset of the plurality of cells may include 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, or more than 100 cells. The first number of voltage pulses may include 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, or more than 100 pulses.

[0135] A voltage at a baseline level across the nanopore may be applied to move the cell through the nanopore for sequencing between pulses in the first number of voltage pulses. This lower level of voltage may move the cell through the nanopore, but may not be sufficient to move the translocation control element through the nanopore. A voltage at a baseline level may be applied across the nanopore to displace the compound a distance in a first direction through the nanopore. The voltage at the baseline level may have the same polarity as the voltage pulses in the first number of voltage pulses. The baseline level may be less than the first level. The compound after being displaced this distance may have a translocation control element in the nanopore.

[0136] Thus, in one example, at box 1610, the following actions may be taken: applying a first number of voltage pulses at a first level across the nanopore to cause a compound to shift a first distance in a first direction through the nanopore, wherein the compound includes a plurality of units, each unit of the plurality of units includes a 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.

[0137] At block 1620, the type of reporter element in the first subset can be detected. When the reporter element in the first subset of the plurality of units is in the nanopore, a signal value can be measured for the nanopore with a voltage applied across the nanopore. The type of reporter element in the first subset is determined using the signal value. Due to the correspondence between the type of reporter element and the nucleotide, the identity of the nucleotide in the target nucleic acid molecule is also determined.

[0138] Thus, in one example, at block 1620, the following actions may be taken: detecting in the nanopore the types of reporter elements in the first subset.

[0139] At 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 in the first number of voltage pulses have opposite polarity to the voltage pulses in 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.

[0140] The first number of voltage pulses may exceed the second number of voltage pulses by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, or more than 50. In some embodiments, the first number of voltage pulses may be equal to the second number of voltage pulses. Subsequent cycles of pulses may then be unequal such that the pulses ultimately propel the compound through the nanopore.

[0141] Thus, in one example, at block 1630, the following actions may be taken: applying a second number of voltage pulses at a second level across the nanopore to translocate the compound a second distance in a second direction through the nanopore.

[0142] At 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 in another bright period. Applying the third number of voltage pulses causes a second subset of the plurality of cells to pass through the nanopore. The second subset and the first subset may include some of the same cells. The second subset may include cells that are 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.

[0143] Thus, in one example, at box 1640, the following actions may be taken: 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 applying the third number of voltage pulses causes a second subset of the plurality of cells to pass through the nanopore.

[0144] At block 1650, the type of reporter element 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 cells in both the first subset and the second subset. For example, the same reporter element may be detected as described in the technique involving sequential consistency. In some embodiments, the reporter element in certain cells may be detected 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. The type of reporter element at the cell may be determined as the most frequently detected type.

[0145] Thus, in one example, at block 1650, the following actions may be taken: detecting in the nanopore the type of reporter element in the second subset.

[0146] In an embodiment, process 1600 may further include causing the compound to completely pass through the nanopore. The compound may pass through the nanopore during a plurality of voltage pulses having the same polarity as the voltage pulses in the first number of voltage pulses. For example, the compound may pass through the nanopore during the bright period. During application of the third number of voltage pulses, the compound may completely pass through the nanopore.

[0147] Additional numbers of voltage pulses of alternating polarity may be applied. For example, a fourth number of voltage pulses may cause the compound to shift in the second direction. Then a fifth number of voltage pulses may cause the compound to shift in the first direction. These cycles of voltage pulses may continue until the compound passes through the nanopore.

[0148] In some embodiments, a clearing voltage at a fourth level may be applied across the nanopore to allow the compound to completely pass out of the nanopore. The fourth level may be greater than the first level, the second level, and the third level. In some embodiments, the clearing voltage may be applied for a duration longer than the duration of the first number, the second number, and the third number of pulses.

[0149] In some embodiments, several nanopores (e.g., in an array) can be used to sequence several nucleic acid molecules. The compound can be the first compound among a plurality of compounds. A plurality of compounds can be produced from a plurality of target nucleic acid molecules. The nanopore can be the first nanopore among a plurality of nanopores. Each compound among a plurality of compounds can be located in a nanopore among a 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 can be applied to a plurality of nanopores. Pulses can be applied in the same order as for the first compound. Report elements in each compound among a plurality of compounds can be detected. A plurality of sequences of a plurality of nucleic acid molecules can be determined. The size distribution of a plurality of sequences can have a mode greater than 300nt. The mode can be 200 to 300nt, 300 to 400nt, 400 to 500nt, or greater than 500nt.

[0150] In some embodiments, a clearing voltage may be applied to certain nanopores but not other nanopores, similar to the process described in Section III.B. A first number of voltage pulses to a plurality of nanopores, 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 a plurality of nanopores. A first portion of a plurality of compounds may be determined to be displaced in a first portion of a plurality of nanopores by a first number of voltage pulses, a second number of voltage pulses, or a third number of voltage pulses. The first portion of a plurality of compounds may not be stuck in the nanopores. A clearing voltage at a fourth level may be applied across each nanopore in a second portion of a plurality of nanopores. The applied clearing voltage may cause the second portion of a plurality of compounds to completely pass through the corresponding nanopores in a plurality of nanopores. The fourth level may be greater than the first level, the second level, and the third level. In some embodiments, a clearing voltage may be applied for a duration longer than the duration of any pulse voltage in the voltage pulse. The second portion of a plurality of nanopores may not include a nanopore in a first portion of a plurality of nanopores.

[0151] In some embodiments, a first portion of the plurality of compounds may be determined to not be displaced in a first portion of the plurality of nanopores by a first number of voltage pulses, a second number of voltage pulses, or a third number of voltage pulses. The first portion of the plurality of compounds may be stuck in the nanopores. A clearing voltage at a fourth level may be applied across each nanopore in the first portion of the plurality of nanopores.

[0152] A compound can be determined to be stuck when there is no characteristic change in the measured electrical signal as it moves 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. When stuck at a particular reporter element, there is no change in the expected electrical signal after applying a translocation voltage pulse. A sufficiently high sampling rate will be used to distinguish a stuck compound from a compound that is moving but has a continuous type of reporter element.

[0153] Process 1600 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in combination with one or more other processes described elsewhere herein.

[0154] although Fig.16 An example block diagram of process 1600 is shown, but in some implementations, process 1600 may include Fig.16 Additional blocks, fewer blocks, different blocks, or blocks arranged in a different manner than those depicted in the process 1600. Additionally or alternatively, two or more blocks of the blocks of process 1600 may be executed in parallel.

[0155] IV. Example System

[0156] Fig.17A measurement system 1700 according to an embodiment of the present invention is shown. The system as shown includes a sample 1705, such as an Xpandomer in a sample holder 1701, wherein the sample 1705 can be contacted with an assay 1708 to provide a signal of a physical property 1715. The assay 1708 can include sequencing by extension using a nanopore. An example of a sample holder can be a well plate including 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 measure 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 into a digital form multiple times. 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 sent to a logic system 1703 from the detector 1702. The data signal 1725 may be stored in the local memory 1735 , the external memory 1704 , or the storage device 1745 .

[0157] The logic system 1703 may be or may include a computer system, an ASIC, a microprocessor, etc. It may also include or be coupled to a display (e.g., a monitor, an LED display, etc.) and a user input device (e.g., a mouse, a keyboard, a button, etc.). The logic system 1703 and other components may be part of a stand-alone or network-connected computer system, or they may be directly attached to or incorporated into a device (e.g., a sequencing device) including a detector 1702 and / or a sample holder 1701. The logic system 1703 may also include software executed in the processor 1720. The logic system 1703 may include a computer-readable medium that stores instructions for controlling the system 1700 to perform any of the methods described herein. For example, the logic system 1703 may provide a command to a system including a sample holder 1701 so as to perform sequencing or other physical operations. Such physical operations may be performed in a specific order, for example, where reagents are added and removed in a specific order. Such physical operations may be performed by a robotic system (e.g., including a robotic arm), such as may be used to obtain a sample and perform a determination.

[0158] Any computer system mentioned herein may utilize any suitable number of subsystems. Examples of such subsystems are Fig.18 1 is shown in computer system 10. In some embodiments, the computer system includes a single computer device, wherein the subsystem can be a component of the computer device. In other embodiments, the computer system can include multiple computer devices, each of which is a subsystem with internal components. The computer system can include desktop and laptop computers, tablet computers, mobile phones, other mobile devices, and cloud-based systems.

[0159] Fig.18 The subsystems shown are interconnected via a system bus 75. Additional subsystems are shown, such as a printer 74, a keyboard 78, a storage device 79, a monitor 76 (e.g., a display screen, such as an LED, which is coupled to a display adapter 82), etc. The peripheral devices and input / output (I / O) devices coupled to the I / O controller 71 can be connected to the computer system through any number of devices known in the art, such as an input / output (I / O) port 77 (e.g., USB, Thunderbolt, Lightning). For example, the I / O port 77 or an external interface 81 (e.g., Ethernet, Wi-Fi, etc.) can 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 through the system bus 75 allows the central processor 73 to communicate with each subsystem and control the execution of multiple instructions from the system memory 72 or the storage device 79 (e.g., a fixed disk, such as a hard drive, or an optical disk), as well as the exchange of information between the subsystems. The system memory 72 and / or the storage device 79 may contain computer-readable media. Another subsystem is a data collection device 85, such as a camera, microphone, accelerometer, etc. Any data mentioned herein may be output from one component to another and may be output to a user.

[0160] The computer system may include multiple identical components or subsystems, for example, connected together via an external interface 81, via an internal interface, or via a removable storage device that can be connected or moved from one component to another. In some embodiments, the computer system, subsystem, or device may communicate via a network. In this case, one computer may be considered a client and another computer may be considered a server, wherein each computer may be considered part of the same computer system. The client and server may each include multiple systems, subsystems, or components.

[0161] Aspects of the embodiments may be implemented in a modular or integrated manner using hardware circuits (e.g., application specific integrated circuits or field programmable gate arrays) and / or using computer software with a general programmable processor in the form of control logic. As used herein, a processor may include a single-core processor, a multi-core processor on the same integrated chip, or multiple processing units on a single circuit board or networked, and dedicated hardware. Based on the disclosure and inspiration provided herein, those of ordinary skill in the art will know and understand other ways and / or methods of implementing embodiments of the present invention using hardware and combinations of hardware and software.

[0162] Any suitable computer language, such as, for example, Java, C, C++, C#, Objective-C, Swift, or a scripting language, such as Perl or Python, can be used to implement any software component or function described in this application as a software code executed by a processor using, for example, conventional techniques or object-oriented techniques. The software code can be stored on a computer-readable medium as a series of instructions or commands for storage and / or transmission. 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 disk (CD) or DVD (digital versatile disc) or Blu-ray disc, flash memory, etc. The computer-readable medium may be any combination of such storage or transmission devices.

[0163] Such programs may also be encoded and transmitted using carrier signals, which are adjusted to be suitable for transmission via wired networks, optical networks and / or wireless networks that meet various protocols including the Internet. In this way, computer-readable media may be created using data signals encoded through such programs. Computer-readable media encoded with program code may be packaged with compatible devices, or provided separately from other devices (e.g., downloaded via the Internet). Any such computer-readable media may reside on or inside a single computer product (e.g., a hard drive, a CD, or an entire computer system), and may be present on or inside different computer products within a system or network. A computer system may include a monitor, a printer, or other suitable displays for providing any results mentioned herein to a user.

[0164] Any method described herein can be performed in whole or in part by a computer system including one or more processors, which can be configured to perform steps. Therefore, embodiments can be directed to a computer system configured to perform the steps of any method described herein, and may have different components for performing corresponding steps or corresponding step groups. Although presented in numbered steps, the steps of the methods described herein can be performed simultaneously or at different times or in different orders. In addition, some steps can be used together with some steps in other methods. In addition, all or part of the steps can be optional. In addition, any step of any method can be performed with other devices of a module, unit, circuit or system for performing these steps.

[0165] Without departing from the spirit and scope of the embodiments of the present invention, the specific details of the specific embodiments may be combined in any suitable manner. However, other embodiments of the present invention may be directed to specific embodiments related to each individual aspect, or specific combinations of these individual aspects.

[0166] The above description of exemplary embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form described, and many modifications and variations are possible in light of the above teachings.

[0167] Unless specifically stated to the contrary, statements of "a," "an," or "the" are intended to mean "one or more." Unless specifically stated to the contrary, the use of "or" is intended to mean "inclusive or," not "exclusive or." Reference to a "first" component does not necessarily require that a second component be provided. Furthermore, unless explicitly stated, reference to a "first" or "second" component does not limit the referenced components to specific locations. The term "based on" is intended to mean "based at least in part on."

[0168] All patents, patent applications, publications, and specifications mentioned herein are incorporated by reference in their entirety for all purposes. None is admitted to be prior art.

Claims

1. A method for sequencing a target nucleic acid molecule, the method comprising: applying a first number of voltage pulses at a first level across a nanopore to translocate a compound through the nanopore a first distance in a first direction, the compound being generated from the target nucleic acid molecule, wherein: The compound comprises a plurality of units, Each unit of the plurality of units comprises a type of reporting element of a plurality of types of reporting elements, Each type of reporter element corresponds to the identity of a nucleotide in the target nucleic acid molecule, and applying the first number of voltage pulses to cause a first subset of the plurality of cells to pass through the nanopore; detecting in the nanopore a type of reporter element in the first subset; applying a second number of voltage pulses at a second level across the nanopore to translocate the compound a second distance in a second direction through the nanopore, wherein: The first direction is opposite to the second direction, the voltage pulses in the first number of voltage pulses have opposite polarity to the voltage pulses in the second number of voltage pulses, The second distance is smaller than the first distance, and the second amount is less than the first amount; applying a third number of voltage pulses at a third level across the nanopore to translocate the compound a third distance in the first direction through the nanopore, wherein: applying the third number of voltage pulses to cause a second subset of the plurality of cells to pass through the nanopore, the second subset and the first subset include some of the same cells, The second subset includes elements that are not in the first subset, and The third distance is greater than the second distance; and The type of reporter elements in the second subset is detected in the nanopore.

2. The method according to claim 1, further comprising: A clearing voltage at a fourth level is applied across the nanopore to cause the compound to completely pass out of the nanopore, wherein the fourth level is greater than the first level, the second level, and the third level.

3. The method according to claim 1 or 2, wherein: The compound is a first compound among a plurality of compounds, The plurality of compounds are generated from a plurality of target nucleic acid molecules, The nanopore is a first nanopore of a plurality of nanopores, and Each compound in the plurality of compounds is located in a nanopore in the plurality of nanopores, The method further comprises: 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 are applied to the plurality of nanopores.

4. The method according to claim 3, further comprising: A plurality of sequences of the plurality of target nucleic acid molecules are determined.

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

6. The method according to claim 3 or 4, further comprising: 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 being 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 clearing voltage at a fourth level across each nanopore in a second portion of the plurality of nanopores to cause a second portion of the plurality of compounds to completely pass out of a corresponding 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.

7. The method according to any one of claims 1 to 6, further comprising determining the sequence of the target nucleic acid molecule.

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

9. The method of claim 1, further comprising allowing the compound to completely pass through the nanopore.

10. The method of claim 9, wherein causing the compound to completely pass through the nanopore occurs during application of the third number of voltage pulses.

11. The method according to any one of claims 1 to 10, wherein: Each unit of the plurality of units comprises a translocation control element, applying the first number of voltage pulses to cause a first number of translocation control elements to pass through the nanopore, and The first number of voltage pulses is equal to the first number of translocation control elements.

12. The method according to claim 11, further comprising: applying a voltage at a fourth level across the nanopore between voltage pulses in the first number of voltage pulses to translocate the compound a fourth distance in the first direction through the nanopore, wherein: the voltage at the fourth level being 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 translocated the fourth distance has a translocation control element in the nanopore.

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

14. The method according to any one of claims 1 to 13, further comprising: 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 cells is in the nanopore; The signal value is used to determine the type of reporter element in the first subset and thereby determine the identity of the nucleotide in the target nucleic acid molecule.

15. The method of any one of claims 1 to 14, wherein the first subset of the plurality of cells comprises 30 or more cells.

16. The method according to any one of claims 1 to 15, wherein the third level is equal to the first level.

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

18. The method of any one of claims 1 to 17, wherein the first number of voltage pulses is 30 or more.

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

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

21. A system comprising: The computer product according to claim 20, and One or more processors for executing instructions stored on a computer-readable medium.

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

23. 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 performing the steps of the method according to any one of claims 1 to 19.

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