Systems and methods for tuning sequencing parameters

Abstract

In one embodiment, a system comprises an array (140) of nanopore sequencing cells. A sensor is configured to detect a change in an average pore voltage of the array (140) of sequencing cells that corresponds to capacitance changes in membranes of the sequencing cells during sequencing read operations through nanopores (216; 316; 347) of the sequencing cells. The system further includes a controller communicatively coupled to the sensor and to switches (401) of the sequencing cells. The controller is configured to determine that the change in average pore voltage is outside a predetermined range, and to control the switches (401) of the sequencing cells to change an integration time of sampling by A / D converters (435) of the sequencing cells by changing a time in which voltage sources (405, 420) of the sequencing cells charge capacitors (408, 426) of the sequencing cells in order to compensate for the capacitance changes of the membranes of the sequencing cells.

Description

Attorney Docket No. P39049-WOSYSTEMS AND METHODS FOR TUNING SEQUENCING PARAMETERSTECHNICAL FIELD

[0001] The present disclosure relates generally to systems and methods for detecting capacitance changes in nanopore membranes, and more particularly to systems and methods for tuning integration times when sampling nanopore sequencing cells to compensate for the capacitance changes.BACKGROUND

[0002] A nanopore based sequencing chip is an analytical tool that can be used for DNA sequencing. These devices can incorporate a large number of sensor cells configured as an array. For example, a sequencing chip can include an array of one million cells, with, for example, 1000 rows by 1000 columns of cells. Each cell of the array can include a membrane and a protein pore having a pore size on the order of one nanometer in internal diameter. Such nanopores have been shown to be effective in rapid nucleotide sequencing.

[0003] When a voltage potential is applied across a nanopore immersed in a conducting fluid, a small ion current attributed to the conduction of ions across the nanopore can exist. The size of the current is sensitive to the pore size and the type of molecule positioned within the nanopore. The molecule can be a particular tag attached to a particular nucleotide, thereby allowing detection of a nucleotide at a particular position of a nucleic acid. A voltage or other signal in a circuit including the nanopore can be measured (e.g., at an integrating capacitor) as a way of measuring the resistance of the molecule, thereby allowing detection of which molecule is in the nanopore.

[0004] However, capacitance change of the membrane during a sequencing run can lead to changes in measurements of the molecule, which can lead to sequencing errors. Accordingly, improved techniques for compensating membrane capacitance changes are desired.BRIEF SUMMARY

[0005] Various embodiments provide techniques and systems related to compensating for capacitance changes.

[0006] In some embodiments, a system includes an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; anAttorney Docket No. P39049-WO analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides, the voltage levels measured over a period of time called an integration time; and a controller configured to: measure an average RC time constant for the array of sequencing cells; and adjust the integration time to achieve a target integration time to RC time constant ratio.

[0007] In some embodiments, the RC time constant is measured when the nanopore is in an open channel state.

[0008] In some embodiments, the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

[0009] In some embodiments, the target integration time to RC time constant ratio is determined by measuring the RC time constant before the sequencing read operations are inititiated.

[0010] In some embodiments, a method is provided. The method includes measuring an average RC time constant for an array of sequencing cells, where each sequencing cell is configured to support a membrane that includes a nanopore, where each sequencing cell includes a measuring circuit; and adjusting an integration time to achieve a target integration time to RC time constant ratio, where the integration time is a period of time that is used to measure a voltage level by the measuring circuit that corresponds to a base of a nucleotide.

[0011] In some embodiments, the RC time constant is measured when the nanopore is in an open channel state.

[0012] In some embodiments, the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

[0013] In some embodiments, the target integration time to RC time constant ratio is determined by measuring the RC time constant before the array of sequencing cells are used to sequence a molecule.

[0014] In some embodiments, a system includes an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform comprising a brightAttorney Docket No. P39049-WO period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, where the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and adjust the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

[0015] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0016] In some embodiments, the controller is configured to decrease the recharge voltage when the measured average voltage is greater than the target average voltage.

[0017] In some embodiments, the controller is configured to increase the recharge voltage when the measured average voltage is less than the target average voltage.

[0018] In some embodiments, a method is provided. The method includes applying a sequencing waveform across an array of sequencing cells, where the sequencing waveform includes a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, where the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; and adjusting the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

[0019] In some embodiments, the average voltage is measured when the sequencing cells are in an open channel state.

[0020] In some embodiments, the method further includes decreasing the recharge voltage when the measured average voltage is greater than the target average voltage.

[0021] In some embodiments, the method further includes increasing the recharge voltage when the measured average voltage is less than the target average voltage.

[0022] In some embodiments, a system includes an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides, the voltage levels measured over a period of time called an integration time; and a controller configured to: measure an average RC time constant for the array of sequencing cells; adjust the integration time to achieve a targetAttorney Docket No. P39049-WO integration time to RC time constant ratio; apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, where the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and adjust the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

[0023] In some embodiments, both the average voltage and the average RC time constant are measured when the nanopore is in an open channel state.

[0024] In some embodiments, the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

[0025] In some embodiments, the target integration time to RC time constant ratio is determined by measuring the RC time constant before the sequencing read operations are inititiated.

[0026] In some embodiments, the controller is configured to decrease the recharge voltage when the measured average voltage is greater than the target average voltage.

[0027] In some embodiments, the controller is configured to increase the recharge voltage when the measured average voltage is less than the target average voltage.

[0028] In some embodiments, a method is provided. The method includes measuring an average RC time constant for an array of sequencing cells, where each sequencing cell is configured to support a membrane that includes a nanopore, where each sequencing cell includes a measuring circuit; adjusting an integration time to achieve a target integration time to RC time constant ratio, where the integration time is a period of time that is used to measure a voltage level by the measuring circuit that corresponds to a base of a nucleotide; applying a sequencing waveform across the array of sequencing cells, where the sequencing waveform includes a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, where the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; and adjusting the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

[0029] In some embodiments, the RC time constant is measured when the nanopore is in an open channel state.Attorney Docket No. P39049-WO

[0030] In some embodiments, the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

[0031] In some embodiments, the target integration time to RC time constant ratio is determined by measuring the RC time constant before the array of sequencing cells are used to sequence a molecule.

[0032] In some embodiments, the average voltage is measured when the sequencing cells are in an open channel state.

[0033] In some embodiments, the method further includes decreasing the recharge voltage when the measured average voltage is greater than the target average voltage.

[0034] In some embodiments, the method further includes increasing the recharge voltage when the measured average voltage is less than the target average voltage.

[0035] In some embodiments, a system is provided. The system includes an array of sequencing cells, each sequencing cell configured to support a membrane that includes a nanopore, each sequencing cell further including a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform including a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, where the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; determine a voltage error, where the voltage error includes a difference between the average voltage and a target voltage; determine a recharge voltage adjustment based on a proportional-integral-derivative control function; and adjust a recharge voltage by the recharge voltage adjustment.

[0036] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0037] In some embodiments, a method is provided. The method includes applying a sequencing waveform across an array of sequencing cells, where the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, where the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, where the voltage error includes a difference between the average voltage and a target voltage; determining aAttorney Docket No. P39049-WO recharge voltage adjustment based on a proportional-integral-derivative control function; and adjusting a recharge voltage by the recharge voltage adjustment.

[0038] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0039] In some embodiments, a system is provided. The system includes an array of sequencing cells, each sequencing cell configured to support a membrane that includes a nanopore, each sequencing cell further including a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform including a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, where the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and determine a recharge voltage using a look-up table based on the measured average voltage and a target voltage.

[0040] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0041] In some embodiments, a method is provided. The method includes applying a sequencing waveform across an array of sequencing cells, where the sequencing waveform includes a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, where the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, where the voltage error includes a difference between the average voltage and a target voltage; and determining a recharge voltage using a look-up table based on the measured average voltage and a target voltage.

[0042] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0043] In some embodiments, a system is provided. The system includes an array of sequencing cells, each sequencing cell configured to support a membrane including a nanopore, each sequencing cell further including a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitorAttorney Docket No. P39049-WO and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform including a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, where the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and determine a recharge voltage using an algebraic equation based on the average voltage and the target voltage.

[0044] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0045] In some embodiments, a method is provided. The method includes applying a sequencing waveform across an array of sequencing cells, where the sequencing waveform includes a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, where the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, wherein the voltage error includes a difference between the average voltage and a target voltage; and determining a recharge voltage using an algebraic equation based on the average voltage and the target voltage.

[0046] In some embodiments, the average voltage is measured when the nanopore is in an open channel state.

[0047] A better understanding of the nature and advantages of embodiments of the present invention can be gained with reference to the following detailed description and the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. l is a top view of an embodiment of a nanopore sensor chip having an array of nanopore cells.

[0049] FIG. 2 illustrates an embodiment of a nanopore cell in a nanopore sensor chip that can be used to characterize a polynucleotide or a polypeptide.

[0050] FIG. 3 A illustrates an embodiment of a nanopore cell performing nucleotide sequencing using a nanopore based sequencing -by-synthesis (Nano-SBS) technique. FIG. 3B illustrates an embodiment of a nanopore cell performing sequencing using different sequencing chemistry involving passing an Xpandomer through a nanopore.Attorney Docket No. P39049-WO

[0051] FIG. 4 illustrates an embodiment of an electric circuit in a nanopore cell.

[0052] FIG. 5 A and FIG. 5B show example data points captured from a cell during a single AC modulation period, and its respective bright period and dark period.

[0053] FIG. 6 is a flow chart that illustrates an embodiment of a high level controller logic that has been programmed and / or configured to perform the adjustment of integration time based on an integration time constant measurement as described herein.

[0054] FIG. 7 is a flow chart that illustrates an embodiment of a high level controller logic that has been programmed and / or configured to perform the adjustment of the recharge voltage based on a voltage measurement that corresponds to the voltage across the nanopore as described herein.

[0055] FIG. 8A illustrates the effect of adjusting the recharge voltage on base call levels, while FIG. 8B illustrates the effect of adjusting the integration time.

[0056] FIG. 9. is a flow chart that illustrates another embodiment of a high level controller logic that has been programmed and / or configured to adjust the recharge voltage using a PID control function

[0057] FIG. 10 is a flow chart that illustrates another embodiment of a high level controller logic that has been programmed and / or configured to adjust the recharge voltage using a look-up table.

[0058] FIG. 11 is a flow chart that illustrates another embodiment of a high level controller logic that has been programmed and / or configured to adjust the recharge voltage using an algebraic equation.

[0059] FIG. 12 is a computer system, according to certain aspects of the present disclosureTERMS

[0060] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Methods, devices, and materials similar or equivalent to those described herein can be used in the practice of disclosed techniques. The following terms are provided to facilitate understanding of certain terms used frequently and are not meant to limit the scope of the present disclosure. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0061] A “nanopore” refers to a pore, channel or passage formed or otherwise provided in a membrane. A membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed of a polymeric material (i.e., anAttorney Docket No. P39049-WO amphiphilic triblock copolymer), or a combination of the two. The nanopore can be disposed adjacent or in proximity to a sensing circuit or an electrode coupled to a sensing circuit, such as, for example, a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. In some examples, a nanopore has a characteristic width or diameter on the order of 0.1 nanometers (nm) to about 1000 nm. In some implementations, a nanopore may be a protein.

[0062] A “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphorami dites, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0063] The term “nucleotide,” in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, can be understood to refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.

[0064] The term “tag” refers to a detectable moiety that can be atoms or molecules, or a collection of atoms or molecules. A tag can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature, which signature can be detected with the aid of a nanopore. Typically, when a nucleotide is attached to the tag it is called a “Tagged Nucleotide.” The tag can be attached to the nucleotide via the phosphate moiety.Attorney Docket No. P39049-WO

[0065] “Xpandomer” or “XP” refers to a polymer synthesized by transcription of the sequence of a nucleic acid template. The transcribed sequence is encoded along the XP backbone in high signal -to-noise reporters that are separated by ~10 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of XPs relative to natural DNA. XPs are used in to carry out sequencing- by-expansi on (“SBX”) and the building blocks of XPs are XNTPs, which are non-natural nucleotide analogs used in XP synthesis to transcribe the sequence of a nucleic acid template. XNTPs are expandable, 5' triphosphate modified non-natural nucleotide analogs compatible with template dependent enzymatic polymerization.

[0066] The term “template” refers to a single stranded nucleic acid molecule that is copied into a complementary strand of DNA nucleotides for DNA synthesis. In some cases, a template can refer to the sequence of DNA that is copied during the synthesis of mRNA.

[0067] The term “primer” refers to a short nucleic acid sequence that provides a starting point for DNA synthesis. Enzymes that catalyze the DNA synthesis, such as DNA polymerases, can add new nucleotides to a primer for DNA replication.

[0068] A “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides. The term encompasses both a full length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art and include but are not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. They include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no sequence similarity among the various families. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3' to 5' exonuclease activity and 5' to 3' exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3' to 5' exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerizing and 3' to 5' exonuclease activity. In E. coli, three types of DNA polymerases have been found — DNA polymerases I (family A), II (family B), and III (family C). In eukaryotic cells, three different family B polymerases — DNA polymerases a, P, and a — are implicated in nuclear replication, and a family A polymerase — polymerase y — is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerasesAttorney Docket No. P39049-WO typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA- dependent.

[0069] The term “bright period” generally refers to the time period when a tag of a tagged nucleotide is forced into a nanopore by an electric field applied through an AC signal. The term “dark period” generally refers to the time period when a tag of a tagged nucleotide is pushed out of the nanopore by the electric field applied through the AC signal. An AC cycle can include the bright period and the dark period. In different embodiments, the polarity of the voltage signal applied to a nanopore cell to put the nanopore cell into the bright period (or the dark period) can be different.

[0070] The term “signal value” refers to a value of the sequencing signal output from a sequencing cell. According to certain embodiments, the sequencing signal is an electrical signal that is measured and / or output from a point in a circuit of one or more sequencing cells, e.g., the signal value is (or represents) a voltage or a current. The signal value can represent the results of a direct measurement of voltage and / or current and / or may represent an indirect measurement, e.g., the signal value can be a measured duration of time for which it takes a voltage or current to reach a specified value. A signal value can represent any measurable quantity that correlates with the resistivity of a nanopore and from which the resistivity and / or conductance of the nanopore (threaded and / or unthreaded) can be derived. As another example, the signal value can correspond to a light intensity, e.g., from a fluorophore attached to a nucleotide being added to a nucleic acid with a polymerase.

[0071] The term “osmolarity”, also known as osmotic concentration, refers to a measure of solute concentration. Osmolarity measures the number of osmoles of solute particles per unit volume of solution. An osmole is a measure of the number of moles of solute that contribute to the osmotic pressure of a solution. Osmolarity allows the measurement of the osmotic pressure of a solution and the determination of how the solvent will diffuse across a semipermeable membrane (osmosis) separating two solutions of different osmotic concentration.

[0072] The term “osmolyte” refers to any soluble compound that when dissolved into a solution increases the osmolarity of that solution.Attorney Docket No. P39049-WODETAILED DESCRIPTION

[0073] According to certain embodiments, techniques and systems disclosed herein relate to compensating for capacitance changes in membranes, such as lipid bilayer membranes.

[0074] Example nanopore systems, circuitry, and sequencing operations are initially described, followed by example techniques to compensate for membrane capacitance changes in DNA sequencing cells. Embodiments of the invention can be implemented in numerous ways, including as a process, a system, and a computer program product embodied on a computer readable storage medium and / or a processor, such as a processor configured to execute instructions stored on and / or provided by a memory coupled to the processor.

[0075] I. Nanopore Based Sequencing Chip

[0076] FIG. 1 is a top view of an embodiment of a nanopore sensor chip 100 having an array 140 of nanopore cells 150. Each nanopore cell 150 includes a control circuit integrated on a silicon substrate of nanopore sensor chip 100. In some embodiments, side walls 136 are included in array 140 to separate groups of nanopore cells 150 so that each group can receive a different sample for characterization. Each nanopore cell can be used to sequence a nucleic acid. In some embodiments, nanopore sensor chip 100 includes a cover plate 130. In some embodiments, nanopore sensor chip 100 also includes a plurality of pins 110 for interfacing with other circuits, such as a computer processor.

[0077] In some embodiments, nanopore sensor chip 100 includes multiple chips in a same package, such as, for example, a Multi -Chip Module (MCM) or System-in-Package (SiP). The chips can include, for example, a memory, a processor, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), data converters, a high-speed VO interface, etc.

[0078] In some embodiments, nanopore sensor chip 100 is coupled to (e.g., docked to) a nanochip workstation 120, which can include various components for carrying out (e.g., automatically carrying out) various embodiments of the processes disclosed herein. These processes can include, for example, analyte delivery mechanisms, such as pipettes for delivering lipid suspension or other membrane structure suspension, analyte solution, and / or other liquids, suspension or solids. The nanochip workstation components can further include robotic arms, one or more computer processors, and / or memory. A plurality of polynucleotides can be detected on array 140 of nanopore cells 150. In some embodiments, each nanopore cell 150 is individually addressable.

[0079] II. Nanopore Sequencing CellAttorney Docket No. P39049-WO

[0080] Nanopore cells 150 in nanopore sensor chip 100 can be implemented in many different ways. For example, in some embodiments, tags of different sizes and / or chemical structures are attached to different nucleotides in a nucleic acid molecule to be sequenced. In some embodiments, a complementary strand to a template of the nucleic acid molecule to be sequenced may be synthesized by hybridizing differently polymer-tagged nucleotides with the template. In some implementations, the nucleic acid molecule and the attached tags both move through the nanopore, and an ion current passing through the nanopore can indicate the nucleotide that is in the nanopore because of the particular size and / or structure of the tag attached to the nucleotide. In some implementations, only the tags are moved into the nanopore. There can also be many different ways to detect the different tags in the nanopores.

[0081] A. Nanopore Sequencing Cell Structure

[0082] FIG. 2 illustrates an embodiment of an example nanopore cell 200 in a nanopore sensor chip, such as nanopore cell 150 in nanopore sensor chip 100 of FIG. 1, that can be used to characterize a polynucleotide or a polypeptide. Nanopore cell 200 can include a well 205 formed of dielectric layers 201 and 204; a membrane, such as a lipid bilayer 214 formed over well 205; and a sample chamber 215 on lipid bilayer 214 and separated from well 205 by lipid bilayer 214. Well 205 can contain a volume of electrolyte 206, and sample chamber 215 can hold bulk electrolyte 208 containing a nanopore, e.g., a soluble protein nanopore transmembrane molecular complexes (PNTMC), and the analyte of interest (e.g., a nucleic acid molecule to be sequenced).

[0083] Nanopore cell 200 can include a working electrode 202 at the bottom of well 205 and a counter electrode 210 disposed in sample chamber 215. A signal source 228 can apply a voltage signal between working electrode 202 and counter electrode 210. A single nanopore (e.g., a PNTMC) can be inserted into lipid bilayer 214 by an electroporation process caused by the voltage signal, thereby forming a nanopore 216 in lipid bilayer 214. The individual membranes (e.g., lipid bilayers 214 or other membrane structures) in the array can be neither chemically nor electrically connected to each other. Thus, each nanopore cell in the array can be an independent sequencing machine, producing data unique to the single polymer molecule associated with the nanopore that operates on the analyte of interest and modulates the ionic current through the otherwise impermeable lipid bilayer 214.

[0084] As shown in FIG. 2, nanopore cell 200 can be formed on a substrate 230, such as a silicon substrate. Dielectric layer 201 can be formed on substrate 230. Dielectric material used to form dielectric layer 201 can include, for example, glass, oxides, nitrides, and the like. An electric circuit 222 for controlling electrical stimulation and for processingAttorney Docket No. P39049-WO the signal detected from nanopore cell 200 can be formed on substrate 230 and / or within dielectric layer 201. For example, a plurality of patterned metal layers (e.g., metal 1 to metal 6) can be formed in dielectric layer 201, and a plurality of active devices (e.g., transistors) can be fabricated on substrate 230. In some embodiments, signal source 228 is included as a part of electric circuit 222. Electric circuit 222 can include, for example, amplifiers, integrators, analog-to-digital converters, noise filters, feedback control logic, and / or various other components. Electric circuit 222 can be further coupled to a processor 224 that is coupled to a memory 226, where processor 224 can analyze the sequencing data to determine sequences of the polymer molecules that have been sequenced in the array.

[0085] Working electrode 202 can be formed on dielectric layer 201 and can form at least a part of the bottom of well 205. In some embodiments, working electrode 202 is a metal electrode. For non-faradaic conduction, working electrode 202 can be made of metals or other materials that are resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, working electrode 202 can be a platinum electrode with electroplated platinum. In another example, working electrode 202 can be a titanium nitride (TiN) working electrode. Working electrode 202 can be porous, thereby increasing its surface area and a resulting capacitance associated with working electrode 202. Because the working electrode of a nanopore cell can be independent from the working electrode of another nanopore cell, the working electrode can be referred to as a cell electrode in this disclosure.

[0086] Dielectric layer 204 can be formed above dielectric layer 201. Dielectric layer 204 forms the walls surrounding well 205. Dielectric material used to form dielectric layer 204 can include, for example, glass, oxide, silicon mononitride (SiN), polyimide, or other suitable hydrophobic insulating material. The top surface of dielectric layer 204 can be silanized. The silanization can form a hydrophobic layer 220 above the top surface of dielectric layer 204. In some embodiments, hydrophobic layer 220 has a thickness of about 1.5 nanometer (nm).

[0087] Well 205 formed by the dielectric layer walls 204 includes volume of electrolyte 206 above working electrode 202. Volume of electrolyte 206 can be buffered and can include one or more of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KC1), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCh), strontium chloride (SrCh), manganese chloride (MnCh), and magnesium chloride (MgC12). In some embodiments, volume of electrolyte 206 has a thickness of about three microns (pm).Attorney Docket No. P39049-WO

[0088] As also shown in FIG. 2, a membrane can be formed on top of dielectric layer 204 and spanning across well 205. In some embodiments, the membrane includes a lipid monolayer 218 formed on top of hydrophobic layer 220. As the membrane reaches the opening of well 205, lipid monolayer 208 can transition to lipid bilayer 214 that spans across the opening of well 205. The lipid bilayer can comprise or consist of lipids, such as a phospholipid, for example, selected from diphytanoyl -phosphatidylcholine (DPhPC), 1,2- diphytanoyl-sn-glycero-3 -phosphocholine, l,2-di-O-phytanyl-sn-glycero-3 -phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methylester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol, 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 1000], 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000], 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-lactosyl, GM1 Ganglioside, Lysophosphatidylcholine (LPC), or any combination thereof. Other phospholipid derivatives may also be used, such as phosphatidic acid derivatives (e.g., DMPA, DDPA, DSP A), phosphatidylcholine derivatives (e.g., DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol derivatives (e.g., DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine derivatives (e.g., DMPE, DPPE, DSPE DOPE), phosphatidylserine derivatives (e.g., DOPS), PEG phospholipid derivatives (e.g., mPEG-phospholipid, polyglycerin-phospholipid, functionalized-phospholipid, terminal activated-phospholipid), diphytanoyl phospholipids (e.g., DPhPC, DOPhPC, DPhPE, and DOPhPE), for example. In some embodiments, the bilayer can be formed using non-lipid based materials, such as amphiphilic block copolymers (e.g., poly(butadiene)-block-poly(ethylene oxide), PEG diblock copolymers, PEG triblock copolymers, PPG triblock copolymers, and poloxamers) and other amphiphilic copolymers, which may be nonionic or ionic. In some embodiments, the bilayer can be formed from a combination of lipid based materials and non-lipid based materials. In some embodiments, the bilayer materials can be delivered in a solvent phase including one or more organic solvents such as alkanes (e.g., decane, tridecane, hexadecane, etc.), and / or one or more silicone oils (e.g., AR-20).Attorney Docket No. P39049-WO

[0089] As shown, lipid bilayer 214 is embedded with a single nanopore 216, e.g., formed by a single PNTMC. As described above, nanopore 216 can be formed by inserting a single PNTMC into lipid bilayer 214 by electroporation. Nanopore 216 can be large enough for passing at least a portion of the analyte of interest and / or small ions (e.g., Na+, K+, Ca2+, CI ) between the two sides of lipid bilayer 214.

[0090] Sample chamber 215 is over lipid bilayer 214 and can hold a solution of the analyte of interest for characterization. The solution can be an aqueous solution containing bulk electrolyte 208 and buffered to an optimum ion concentration and maintained at an optimum pH to keep the nanopore 216 open. Nanopore 216 crosses lipid bilayer 214 and provides the only path for ionic flow from bulk electrolyte 208 to working electrode 202. In addition to nanopores (e.g., PNTMCs) and the analyte of interest, bulk electrolyte 208 can further include one or more of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KC1), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCh), strontium chloride (SrCh), manganese chloride (MnCh), and magnesium chloride (MgCh).

[0091] Counter electrode (CE) 210 can be an electrochemical potential sensor. In some embodiments, counter electrode 210 is shared between a plurality of nanopore cells and can therefore be referred to as a common electrode. In some cases, the common potential and the common electrode can be common to all nanopore cells, or at least all nanopore cells within a particular grouping. The common electrode can be configured to apply a common potential to the bulk electrolyte 208 in contact with the nanopore 216. Counter electrode 210 and working electrode 202 can be coupled to signal source 228 for providing electrical stimulus (e.g., voltage bias) across lipid bilayer 214, and can be used for sensing electrical characteristics of lipid bilayer 214 (e.g., resistance, capacitance, and ionic current flow). In some embodiments, nanopore cell 200 can also include a reference electrode 212.

[0092] In some embodiments, various checks are made during creation of the nanopore cell as part of calibration. Once a nanopore cell is created, further calibration steps can be performed, e.g., to identify nanopore cells that are performing as desired (e.g., one nanopore in the cell). Such calibration checks can include physical checks, voltage calibration, open channel calibration, and identification of cells with a single nanopore.

[0093] B. Detection Signals of Nanopore Sequencing Cell

[0094] Nanopore cells in nanopore sensor chip, such as nanopore cells 150 in nanopore sensor chip 100, can enable parallel sequencing using a single molecule nanopore based sequencing by synthesis (Nano-SBS) technique.Attorney Docket No. P39049-WO

[0095] FIG. 3A illustrates an embodiment of a nanopore cell 300 performing nucleotide sequencing using the Nano-SBS technique. In the Nano-SBS technique, a template 332 to be sequenced (e.g., a nucleotide acid molecule or another analyte of interest) and a primer can be introduced into bulk electrolyte 308 in the sample chamber of nanopore cell 300. As examples, template 332 can be circular or linear. A nucleic acid primer can be hybridized to a portion of template 332 to which four differently polymer-tagged nucleotides 338 can be added.

[0096] In some embodiments, an enzyme (e.g., a polymerase 334, such as a DNA polymerase) is associated with nanopore 316 for use in the synthesizing a complementary strand to template 332. For example, polymerase 334 can be covalently attached to nanopore 316. Polymerase 334 can catalyze the incorporation of nucleotides 338 onto the primer using a single stranded nucleic acid molecule as the template. Nucleotides 338 can comprise tag species (“tags”) with the nucleotide being one of four different types: A, T, G, or C. When a tagged nucleotide is correctly complexed with polymerase 334, the tag can be pulled (e.g., loaded) into the nanopore by an electrical force, such as a force generated in the presence of an electric field generated by a voltage applied across lipid bilayer 314 and / or nanopore 316. The tail of the tag can be positioned in the barrel of nanopore 316. The tag held in the barrel of nanopore 316 can generate a unique ionic blockade signal 340 due to the tag's distinct chemical structure and / or size, thereby electronically identifying the added base to which the tag attaches.

[0097] Alternatively, the nanopore devices contemplated by the disclosure are used for Sequencing by Expansion ("SBX") as shown in FIG. 3B, a nanopore-based nucleic acid sequencing method that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer molecule referred to as an xpandomer (“XP molecule” or “XP”). See e.g., U.S. Pat. No. 7,939,259, entitled, “High Throughput Nucleic Acid Sequencing by Expansion;” and PCT publication WO2020236526A1, entitled “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing.” In the SBX process, a target nucleic acid sequence is encoded along the backbone XP sequence with reporter constructs that are separated by ~10 nm that are designed to provide high signal-to-noise, well-differentiated response signals during nanopore translocation. The enhanced signal-to-noise provided by the different response signals provides significantly increased sequence read efficiency and accuracy of XPs relative to native nucleic acid molecules.Attorney Docket No. P39049-WO

[0098] SBX chemistry sequences nucleic acids by creating an XP from a nucleic acid template. This is achieved by encoding the nucleic acid information on a surrogate polymer of extended length which is easier to detect. The surrogate polymer, i.e., XP, is formed by template directed synthesis which preserves the original genetic information of the target nucleic acid, while also increasing linear separation of the individual elements of the sequence data.

[0099] In the embodiment shown in Fig. 3B, the XP molecule, 345, includes a translocational control element (TCE, 350) which serves to arrest XP translocation through a nanopore (347). The TCE (350) is surrounded by reporter codes 351 and 352. In this embodiment, TCE (350) has a larger physical bulk relative to that of the reporter codes 351 and 352. XP translocation through the nanopore 347 is arrested when TCE 350 encounters the pore aperture 353. In certain embodiments, both the bulk of the TCE and the charge densities of the reporter codes (i.e., the local electric field at the arrest site) contribute to translocation arrest. During the pause, reporter code 354 is held in the barrel of the nanopore and blocks the flow of current through the pore in a characteristic and detectable manner. To overcome the arrest, a voltage pulse is applied to the system, which forces the TCE to enter and pass through the pore. Translocation then resumes until the next TCE encounters the pore aperture. In one embodiment, SBX chemistry is facilitated by the inclusion of additives that, e.g., enhance the translocation rate of XP molecules through a pore, including but not limited to, stabilizers such as EDTA and redox reagents. In a specific embodiment, concentrations from about lOmM to about 300mM of one or more organic and inorganic redox-capable species are included, e.g., ferri / ferro- cyanide, metal bipyridine compounds such as iron tris- bipyridine or cobalt tris-bipyridine, and modified ferrocenes.

[0100] In the SBS and SBX processes, an electrical signal, e.g., resistance or conductance, of the nanopore including the loaded (threaded) tag or XP can be measured via a signal value (e.g., voltage or a current passing through the nanopore), thereby providing an identification of the species and thus the nucleotide at the position of the template nucleic acid. In some embodiments, a direct current (DC) signal is applied to the nanopore cell (e.g., so that the direction in which the species moves through the nanopore is not reversed). However, operating a nanopore sensor for long periods of time using a direct current can change the composition of the electrode, unbalance the ion concentrations across the nanopore, and have other undesirable effects that can affect the lifetime of the nanopore cell. Applying an alternating current (AC) waveform can reduce the electro-migration to avoid these undesirable effects and have certain advantages as described below. The nucleic acidAttorney Docket No. P39049-WO sequencing methods described herein are fully compatible with applied AC voltages, and therefore an AC waveform can be used to achieve these advantages.

[0101] As used herein, a “loaded” or “threaded” tag is one that is positioned in and / or remains in or near the nanopore for an appreciable amount of time, e.g., 0.1 millisecond (ms) to 10,000 ms. In some cases, a tag is loaded in the nanopore prior to being released from the nucleotide. In some instances, the probability of a loaded tag passing through (and / or being detected by) the nanopore after being released upon a nucleotide incorporation event is suitably high, e.g., 90% to 99%.

[0102] In some embodiments when using tag chemistry, before polymerase 334 is connected to nanopore 316, the conductance of nanopore 316 is high, such as, for example, about 300 picosiemens (300 pS). As the tag is loaded in the nanopore, a unique conductance signal (e.g., signal 340) is generated due to the tag's distinct chemical structure and / or size. For example, the conductance of the nanopore can be about 60 pS, 80 pS, 100 pS, or 120 pS, each corresponding to one of the four types of tagged nucleotides. The polymerase can then undergo an isomerization and a transphosphorylation reaction to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule. Note that when using Xpandomer chemistry, a polymerase is not needed to be attached to the nanopore since the Xpandomer is synthesized separately before being introduced into the nanopore cell, and therefore only needs to be passed through the nanopore. In addition, the conductance measurements of the reporter codes within a pore can differ from the tag conductances. For example, the reporter code conductances within a pore can be 2, 3, 4, 5, 6 times the conductances of the corresponding tag conductances.

[0103] In some cases, some of the tagged nucleotides may not match (complementary bases) with a current position of the nucleic acid molecule (template). The tagged nucleotides that are not base-paired with the nucleic acid molecule can also pass through the nanopore. These non-paired nucleotides can be rejected by the polymerase within a time scale that is shorter than the time scale for which correctly paired nucleotides remain associated with the polymerase. Tags bound to non-paired nucleotides can pass through the nanopore quickly and be detected for a short period of time (e.g., less than 10 ms), while tags bounded to paired nucleotides can be loaded into the nanopore and detected for a long period of time (e.g., at least 10 ms). Therefore, non-paired nucleotides can be identified by a downstream processor based at least in part on the time for which the nucleotide is detected in the nanopore.Attorney Docket No. P39049-WO

[0104] A conductance (or equivalently the resistance) of the nanopore including the loaded (threaded) tag or reporter code of the Xpandomer can be measured via a signal value (e.g., voltage or a current passing through the nanopore), thereby providing an identification of the tag or reporter species and thus the nucleotide at the current position. In some embodiments, a direct current (DC) signal is applied to the nanopore cell (e.g., so that the direction in which the tag moves through the nanopore is not reversed). However, operating a nanopore sensor for long periods of time using a direct current can change the composition of the electrode, unbalance the ion concentrations across the nanopore, and have other undesirable effects that can affect the lifetime of the nanopore cell. Applying an alternating current (AC) waveform can reduce the electro-migration to avoid these undesirable effects and have certain advantages as described below. The nucleic acid sequencing methods described herein that utilize tagged nucleotides are fully compatible with applied AC voltages, and therefore an AC waveform can be used to achieve these advantages.

[0105] The ability to re-charge the electrode during the AC detection cycle can be advantageous when sacrificial electrodes, electrodes that change molecular character in the current-carrying reactions (e.g., electrodes comprising silver), or electrodes that change molecular character in current-carrying reactions are used. An electrode can deplete during a detection cycle when a direct current signal is used. The recharging can prevent the electrode from reaching a depletion limit, such as becoming fully depleted, which can be a problem when the electrodes are small (e.g., when the electrodes are small enough to provide an array of electrodes having at least 500 electrodes per square millimeter). Electrode lifetime in some cases scales with, and is at least partly dependent on, the width of the electrode.

[0106] Suitable conditions for measuring ionic currents passing through the nanopores are known in the art and examples are provided herein. The measurement can be carried out with a voltage applied across the membrane and pore. In some embodiments, the voltage used ranges from -400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0 mV, and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. The voltage used can be more preferably in the range from 100 mV to 240 mV and most preferably in the range from 160 mV to 240 mV. It is possible to increase discrimination between different nucleotides by a nanopore using an increased applied potential. Sequencing nucleic acids using AC waveforms and tagged nucleotides is described in US Patent Publication No. US 2014 / 0134616 entitled “Nucleic Acid Sequencing Using Tags,” filed on Nov. 6, 2013, whichAttorney Docket No. P39049-WO is herein incorporated by reference in its entirety. In addition to the tagged nucleotides described in US 2014 / 0134616, sequencing can be performed using nucleotide analogs that lack a sugar or acyclic moiety, e.g., (S)-glycerol nucleoside triphosphates (gNTPs) of the five common nucleobases: adenine, cytosine, guanine, uracil, and thymine (Horhota et al., Organic Letters, 8:5345-5347

[2006] ).

[0107] C. Electric Circuit of Nanopore Sequencing Cell

[0108] FIG. 4 illustrates an embodiment of an electric circuit 400 (which may include portions of electric circuit 222 in FIG. 2) in a nanopore cell, such as nanopore cell 400. As described above, in some embodiments, electric circuit 400 includes a counter electrode 410 that can be shared between a plurality of nanopore cells or all nanopore cells in a nanopore sensor chip and can therefore also be referred to as a common electrode. The common electrode can be configured to apply a common potential to the bulk electrolyte (e.g., bulk electrolyte 208) in contact with the lipid bilayer (e.g., lipid bilayer 214) in the nanopore cells by connecting to a voltage source VLIQ 420. In some embodiments, an AC non-Faradaic mode is utilized to modulate voltage VLIQ with an AC signal (e.g., a square wave) and apply it to the bulk electrolyte in contact with the lipid bilayer in the nanopore cell. In some embodiments, VLIQ is a square wave with a magnitude of ±200-250 mV and a frequency between, for example, 25 and 400 Hz. The bulk electrolyte between counter electrode 410 and the lipid bilayer (e.g., lipid bilayer 214) can be modeled by a large capacitor (not shown), such as, for example, 100 pF or larger.

[0109] FIG. 4 also shows an electrical model 422 representing the electrical properties of a working electrode 402 (e.g., working electrode 202) and the lipid bilayer (e.g., lipid bilayer 214). Electrical model 422 includes a capacitor 426 (C Bilayer) that models a capacitance associated with the lipid bilayer and a resistor 428 (RPORE) that models a variable resistance associated with the nanopore, which can change based on the presence of a particular tag or reporter code in the nanopore. Electrical model 422 also includes a capacitor 424 having a double layer capacitance (Cooubie Layer) and representing the electrical properties of working electrode 402 and well 205. Working electrode 402 can be configured to apply a distinct potential independent from the working electrodes in other nanopore cells.

[0110] Pass device 406 is a switch that can be used to connect or disconnect the lipid bilayer and the working electrode from electric circuit 400. Pass device 406 can be controlled by control line 407 to enable or disable a voltage stimulus to be applied across the lipid bilayer in the nanopore cell. Before lipids are deposited to form the lipid bilayer, the impedance between the two electrodes may be very low because the well of the nanopore cellAttorney Docket No. P39049-WO is not sealed, and therefore pass device 406 can be kept open to avoid a short-circuit condition. Pass device 406 can be closed after lipid solvent has been deposited to the nanopore cell to seal the well of the nanopore cell.

[0111] Circuitry 400 can further include an on-chip integrating capacitor 408 (ncap). Integrating capacitor 408 can be pre-charged by using a reset signal 403 to close switch 401, such that integrating capacitor 408 is connected to a voltage source VPRE 405. In some embodiments, voltage source VPRE 405 provides a constant reference voltage with a magnitude of, for example, 900 mV. In other embodiments, VLIQ can be held constant while VPRE is varied. When switch 401 is closed, integrating capacitor 408 can be pre-charged to the reference voltage level of voltage source VPRE 405.

[0112] After integrating capacitor 408 is pre-charged, reset signal 403 can be used to open switch 401 such that integrating capacitor 408 is disconnected from voltage source VPRE 405. At this point, depending on the level of voltage source VLIQ, the potential of counter electrode 410 can be at a higher level than that of the potential of working electrode 402 (and integrating capacitor 408), or vice versa. For example, during a positive phase of a square or rectangular wave from voltage source VPRE (e.g., the bright or dark period of the AC voltage source signal cycle), the potential of working electrode 414 is at a level higher than the potential of counter electrode 416. During a negative phase of the square or rectangular wave from voltage source VPRE (e.g., the dark or bright period of the AC voltage source signal cycle), the potential of working electrode 414 is at a level lower than the potential of counter electrode 416. Thus, in some embodiments, integrating capacitor 408 can be further charged during the bright period from the pre-charged voltage level of voltage source VPRE 405 to a higher level, and discharged during the dark period to a lower level, due to the potential difference between counter electrode 410 and working electrode 402. In other embodiments, the charging and discharging occur in dark periods and bright periods, respectively.

[0113] Integrating capacitor 408 can be charged or discharged for a fixed period of time, depending on the sampling rate of an analog-to-digital converter (ADC) 435, which can be higher than 1 kHz, 5 kHz, 10 kHz, 100 kHz, or more. For example, with a sampling rate of 1 kHz, integrating capacitor 408 can be charged / discharged for a period of about 1 ms, and then the voltage level can be sampled and converted by ADC 435 at the end of the integration period. A particular voltage level would correspond to a particular tag species or reporter code in the nanopore, and thus correspond to the nucleotide at a current position on the template.Attorney Docket No. P39049-WO

[0114] After being sampled by ADC 435, integrating capacitor 408 can be precharged again by using reset signal 403 to close switch 401, such that integrating capacitor 408 is connected to voltage source VPRE 405 again. The steps of pre-charging integrating capacitor 408, waiting for a fixed period of time for integrating capacitor 408 to charge or discharge, and sampling and converting the voltage level of integrating capacitor by ADC 435 can be repeated in cycles throughout the sequencing process.

[0115] A digital processor 430 can process the ADC output data, e.g., for normalization, data buffering, data filtering, data compression, data reduction, event extraction, or assembling ADC output data from the array of nanopore cells into various data frames. In some embodiments, digital processor 430 performs further downstream processing, such as base determination. Digital processor 430 can be implemented as hardware (e.g., in a graphics processing unit (GPU), FPGA, ASIC, etc.) or as a combination of hardware and software.

[0116] Accordingly, the voltage signal applied across the nanopore can be used to detect particular states of the nanopore. One of the possible states of the nanopore is an openchannel state when a tag-attached polyphosphate or an Xpandomer molecule is absent from the barrel of the nanopore, also referred to herein as the unthreaded state of the nanopore. Another four possible states of the nanopore each correspond to a state when one of the four different types of tag-attached polyphosphate nucleotides (A, T, G, or C) or reporter codes that correspond to the different nucleotides is held in the barrel of the nanopore. Yet another possible state of the nanopore is when the lipid bilayer is ruptured.

[0117] When the voltage level on integrating capacitor 408 is measured after a fixed period of time, the different states of a nanopore can result in measurements of different voltage levels. This is because the rate of the voltage decay (decrease by discharging or increase by charging) on integrating capacitor 408 (i.e., the steepness of the slope of a voltage on integrating capacitor 408 versus time plot) depends on the nanopore resistance (e.g., the resistance of resistor RPORE 428). More particularly, as the resistance associated with the nanopore in different states is different due to the molecules' (tags' or reporter codes) distinct chemical structures (i.e. size differences), different corresponding rates of voltage decay can be observed and can be used to identify the different states of the nanopore. The voltage decay curve can be an exponential curve with an RC time constant T=RC, where R is the resistance associated with the nanopore (i.e., RPORE resistor 428) and C is the capacitance associated with the membrane (i.e., CBiiayer capacitor 426) in parallel with R. A time constant of the nanopore cell can be, for example, about 200-500 ms. The decay curve may not fitAttorney Docket No. P39049-WO exactly to an exponential curve due to a variety of reasons, but the decay curve can be similar to an exponential curve and be monotonic, thus allowing detection of tags or reporter codes.

[0118] In some embodiments, the resistance associated with the nanopore in an open-channel state is in the range of 100 MOhm to 20 GOhm. In some embodiments, the resistance associated with the nanopore in a state where a tag or reporter code is inside the barrel of the nanopore can be within the range of 200 MOhm to 40 GOhm. In other embodiments, integrating capacitor 408 is omitted, as the voltage leading to ADC 435 will still vary due to the voltage decay in electrical model 422.

[0119] The rate of the decay of the voltage on integrating capacitor 408 can be determined in different ways. As explained above, the rate of the voltage decay can be determined by measuring a voltage decay during a fixed time interval. For example, the voltage on integrating capacitor 408 can be first measured by ADC 435 at time ti, and then the voltage is measured again by ADC 435 at time t2. The voltage difference is greater when the slope of the voltage on integrating capacitor 408 versus time curve is steeper, and the voltage difference is smaller when the slope of the voltage curve is less steep. Thus, the voltage difference can be used as a metric for determining the rate of the decay of the voltage on integrating capacitor 408, and thus the state of the nanopore cell.

[0120] In other embodiments, the rate of the voltage decay is determined by measuring a time duration that is required for a selected amount of voltage decay. For example, the time required for the voltage to drop or increase from a first voltage level VI to a second voltage level V2 can be measured. The time required is less when the slope of the voltage vs. time curve is steeper, and the time required is greater when the slope of the voltage vs. time curve is less steep. Thus, the measured time required can be used as a metric for determining the rate of the decay of the voltage on integrating capacitor ncap408, and thus the state of the nanopore cell. One skilled in the art will appreciate the various circuits that can be used to measure the resistance of the nanopore, e.g., including signal value measurement techniques, such as voltage or current measurements.

[0121] In some embodiments, electric circuit 400 does not include a pass device (e.g., pass device 406) and an extra capacitor (e.g., integrating capacitor 408 (ncap)) that are fabricated on-chip, thereby facilitating the reduction in size of the nanopore based sequencing chip. Due to the thin nature of the membrane (lipid bilayer), the capacitance associated with the membrane (e.g., capacitor 426 (Cniiayer)) alone can suffice to create the required RC time constant without the need for additional on-chip capacitance. Therefore, capacitor 426 can be used as the integrating capacitor, and can be pre-charged by the voltage signal VPRE andAttorney Docket No. P39049-WO subsequently be discharged or charged by the voltage signal VLIQ. The elimination of the extra capacitor and the pass device that are otherwise fabricated on-chip in the electric circuit can significantly reduce the footprint of a single nanopore cell in the nanopore sequencing chip, thereby facilitating the scaling of the nanopore sequencing chip to include more and more cells (e.g., having millions of cells in a nanopore sequencing chip).

[0122] D. Data Sampling in Nanopore Cell

[0123] To perform sequencing of a nucleic acid, the voltage level of integrating capacitor (e.g., integrating capacitor 408 (ncap) or capacitor 426 (Cuiiayer)) can be sampled and converted by the ADC (e.g., ADC 435) while a tagged nucleotide is being added to the nucleic acid. The tag of the nucleotide can be pushed into the barrel of the nanopore by the electric field across the nanopore that is applied through the counter electrode and the working electrode, for example, when the applied voltage is such that VLIQ is lower than VpRE.

[0124] Alternatively, when using Xpandomer chemistry, the voltage level can be sampled when the reported code is positioned within the barrel of the nanopore.

[0125] 1. Threading

[0126] A threading event is when a tagged nucleotide is attached to the template (e.g., nucleic acid fragment), and the tag moves in and out of the barrel of the nanopore. This movement can happen multiple times during a threading event. When the tag is in the barrel of the nanopore, the resistance of the nanopore can be higher, and a lower current can flow through the nanopore.

[0127] During sequencing, a tag may not be in the nanopore in some AC cycles (referred to as an open-channel state), where the current is the highest because of the lower resistance of the nanopore. When a tag is attracted into the barrel of the nanopore, the nanopore is in a bright mode. When the tag is pushed out of the barrel of the nanopore, the nanopore is in a dark mode.

[0128] When using Xpandomers, a threading event is when an Xpandomer molecules is inserted into the barrel of the nanopore.

[0129] 2. Bright and Dark Period

[0130] During an AC cycle, the voltage on integrating capacitor can be sampled multiple times by the ADC. For example, in one embodiment, an AC voltage signal is applied across the system at, e.g., about 100 Hz when using tag chemistry, or between about 0.1 Hz to 10 Hz and typically at less than 1 Hz when using Xpandomer chemistry, and an acquisition rate of the ADC can be about 2000 Hz per cell. Thus, there can be about 20 data pointsAttorney Docket No. P39049-WO(voltage measurements) captured per AC cycle (cycle of an AC waveform). Data points corresponding to one cycle of the AC waveform can be referred to as a set. In a set of data points for an AC cycle, there can be a subset captured when, for example, VLIQ is lower than VPRE, which can correspond to a bright mode (period) when the tag is forced into the barrel of the nanopore. Another subset can correspond to a dark mode (period) when the tag is pushed out of the barrel of the nanopore by the applied electric field when, for example, VLIQ is higher than VPRE.

[0131] When using Xpandomers, the Xpandomer molecule generally remains threaded during both the bright and dark periods of the AC cycle.

[0132] 3. Measured Voltages

[0133] For each data point, when the switch 401 is opened, the voltage at the integrating capacitor (e.g., integrating capacitor 408 (ncap) or capacitor 426 (Cuiiayer)) will change in a decaying manner as a result of the charging / discharging by VLIQ, e.g., as an increase from VPRE to VLIQ when VLIQ is higher than VPRE or a decrease from VPRE to VLIQ when VLIQ is lower than VPRE. The final voltage values can deviate from VLIQ as the working electrode charges. The rate of change of the voltage level on the integrating capacitor ncap408 can be governed by the value of the resistance of the bilayer, which can include the nanopore, which can in turn include a molecule (e.g., a tag of a tagged nucleotides or a reporter code) in the nanopore. The voltage level can be measured at a predetermined time after switch 401 opens.

[0134] Switch 401 can operate at the rate of data acquisition. Switch 401 can be closed for a relatively short time period between two acquisitions of data, typically right after a measurement by the ADC. The switch allows multiple data points to be collected during each sub-period (bright or dark) of each AC cycle of VLIQ. If switch 401 remains open, the voltage level on the integrating capacitor ncap408, and thus the output value of the ADC, fully decays and stays there. If instead switch 401 is closed, the integrating capacitor ncap408 is precharged again (to VPRE) and becomes ready for another measurement. Thus, switch 401 allows multiple data points to be collected for each sub-period (bright or dark) of each AC cycle. Such multiple measurements can allow higher resolution with a fixed ADC (e.g., 8-bit to 14-bit due to the greater number of measurements, which may be averaged). The multiple measurements can also provide kinetic information about the molecule threaded into the nanopore. The timing information can allow the determination of how long a threading takes place. This can also be used in helping to determine whether multiple nucleotides that are added to the nucleic acid strand are being sequenced.Attorney Docket No. P39049-WO

[0135] FIG. 5A shows example data points captured from a cell during a single alternating current (AC) modulation period and its respective dark and bright period. The voltage (VLIQ) applied to the counter electrode is at a constant level, such as, for example, about 500 to 1000m V, preferably about 600 to 900 mV. A voltage signal 510 (VPRE) applied to the working electrode is an AC signal shown as a square wave (or a repeated pattern of superimposed square waves or any other modulation know in the art), where the duty cycle (i.e. bright period pulse width divided by modulation cycle width) may be any suitable value, such as less than or equal to about 80%, preferably about 40 to 60%, more preferably about 60% (as exemplified) or about 50%. In some examples, and as exemplified in FIG. 5B, the voltage VLIQ may be modulated with an AC signal (e.g., a square wave or a repeated pattern of superimposed square waves or any other modulation known in the art) and applied to the bulk electrolyte in contact with the membrane in the nanopore cell (either with VPRE held at a constant value or with VPRE utilised in an AC mode).

[0136] During a bright period 530, the voltage signal 540 applied to the counter electrode by voltage source VLIQ 420 is lower than the voltage signal 510 applied to the working electrode by voltage source VPRE 405, such that a molecule (e.g. a tag or reporter code) may be forced into the barrel of the nanopore by the electric field caused by the different voltage levels applied at the working electrode and the counter electrode (e.g., due to the charge on the molecule and / or flow of the ions). When switch 401 is opened, the voltage at a node before the ADC (e.g., at an integrating capacitor) will decrease. After a voltage data point is captured (e.g., after a specified time period), switch 401 may be closed and the voltage at the measurement node will increase back to VPRE again. The process can repeat to measure multiple voltage data points. In this way, multiple data points may be captured during the bright period, for example 532 and 534. During a dark period 520, voltage signal 540 applied to the counter electrode is higher than the voltage signal 510 applied to the working electrode, such that any molecule (e.g. tag or reporter code) would be pushed out of the barrel of the nanopore. When switch 401 is opened, the voltage at the measurement node increases because the voltage level of voltage signal (VLIQ) is higher than VPRE 510. After a voltage data point is captured (e.g., after a specified time period), switch 401 may be closed and the voltage at the measurement node will decrease back to VPRE again. The process can repeat to measure multiple voltage data points. Thus, multiple data points may be captured during the dark period, including a first point delta 522 and subsequent data points e.g. 524. As described above, during the dark period, any molecule (e.g. tag or reporterAttorney Docket No. P39049-WO code) is pushed partially or totally out of the nanopore, and thus minimal information about such molecules is obtained, besides for, for example, in use in normalisation.

[0137] The circuitry described herein may be configured to apply AC signals with different phases to different groups of cells. As a result of the different phases of an AC signal, during a certain time period, a first group of cells may be in a dark period, while a second group of cells may be in a bright period. The techniques described herein can also be applied to systems that use alternating current voltages for the working electrode, the counter electrode or both the working electrode and the counter electrode, as well as periodically changing DC bias, which may also have a “dark” period when the electrode is recharged.

[0138] The voltage measured during a bright or dark period might be expected to be about the same for each measurement of a constant resistance of the nanopore (e.g., made during a bright mode of a given AC cycle while one tag or reporter code is in the nanopore), but this may not be the case when charge builds up on the membrane, which is schematically represent in the boxed portion of FIG. 4. This charge build-up can cause the time constant of the nanopore cell to change. As a result, the voltage level may be shifted, thereby causing the measured value to decrease for each data point in a cycle. Thus, within a cycle, the data points may change somewhat from data point to another data point, as shown in FIG. 5 A and FIG. 5B.

[0139] Further details regarding measurements can be found in, for example, U.S. Patent No. 9,557,294 entitled “Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. Patent No. 9,863,904 entitled “Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. Patent No. 10,155,979 entitled “Non-Destructive Bilayer Monitoring Using Measurement Of Bilayer Response To Electrical Stimulus,” and U.S. Patent No. 10,465,240 entitled “Electrical Enhancement Of Bilayer Formation,” the disclosures of which are incorporated by reference in their entirety for all purposes.

[0140] 4. Normalization and Base Calling

[0141] For each usable nanopore cell of the nanopore sensor chip, a production mode can be run to sequence nucleic acids. The ADC output data captured during the sequencing can be normalized to provide greater accuracy. Normalization can account for offset effects, such as cycle shape, gain drift, charge injection offset, and baseline shift. In some implementations, the signal values of a bright period cycle corresponding to a threading event can be flattened so that a single signal value is obtained for the cycle (e.g., an average) or adjustments can be made to the measured signal to reduce the intra-cycle decay (a type of cycle shape effect). Gain drift generally scales entire signal and changes on the order to 100sAttorney Docket No. P39049-WO to 1,000s of seconds. As examples, gain drift can be triggered by changes in solution (pore resistance) or changes in bilayer capacitance. The baseline shift occurs with a timescale of -100 ms and relates to a voltage offset at the working electrode. The baseline shift can be driven by changes in an effective rectification ratio from threading as a result of a need to maintain charge balance in the sequencing cell from the bright period to the dark period.

[0142] After normalization, embodiments can determine clusters of voltages for the threaded channels, where each cluster corresponds to a different tag species or reporter code, and thus a different nucleotide. The clusters can be used to determine probabilities of a given voltage corresponding to a given nucleotide. As another example, the clusters can be used to determine cutoff voltages for discriminating between different nucleotides (bases).

[0143] III. Compensating for Capacitance Changes in an Array of Nanopore Sequencing Cells

[0144] As discussed above, each complex of a nanopore and associated template or each Xpandomer molecule can be used to provide sequence information for a particular nucleic acid molecule of interest. However, capacitance changes in the membranes of the nanopore cells over time, due to charge build up on the membrane for example, can lead to changes in electrical measurements in those cells, which can ultimately lead to errors in base calling. The invention described herein provides a method for controlling the integration time prior to data collection to maintain signal quality in a system with variable integration time constant due to uncontrolled changes in the size of membrane capacitances contributing to total integration capacitance.

[0145] In nanopore measurement systems where the recorded signal is based on integration on a capacitor, the information quality of the signal obtained is typically a function of the voltage maintained across the nanopore and the degree of saturation of the integration capacitor. This includes signal -to-noise ratio (SNR) in single molecule measurements and error modes in sequencing systems. For example, if the rate of sequencer read errors such as deletions and insertions is a function of the instantaneous voltage across the nanopore, it is advantageous to maintain a known voltage, to some degree of tolerance, over the entire time of an experiment and all nanopores in an array. Likewise, maintaining a consistent separation between signals generated can simplify analysis algorithms such as event detection and in the example of sequencing applications, base calling.

[0146] In addition to the voltage maintained across the nanopore, the integration time of the sample measurement relative to the shape of the exponential decay curve influences the SNR, where the integration time is the period of time used to take aAttorney Docket No. P39049-WO measurement from the integrating capacitor. The shape of the integrated voltage signal response in time is an exponential function of the integration RC time constant (“tau” or “T”) which is determined by the conductance of the nanopore and sum of membrane capacitance and solid state integration capacitance of the sensor. While the solid state capacitor behavior is fixed, the membrane and membrane capacitance can change over time. If a fixed integration time is used, this can cause undesired changes in the recorded signal if the membrane capacitance changes. Mathematically this can be represented as:

[0147] Measured Signal = A * (1 - exp(- t_int / r)), where A is the magnitude of input signal, t int is the integration time of measurements, and r is the integration time constant, or RC time constant, as described above. If t int is fixed and tau changes due to uncontrolled drift in the integration capacitance, the magnitude of the Measured Signal will increase or decrease as the ratio of t int / tau becomes larger or smaller, respectively.

[0148] Therefore, in some embodiments, the nanopore sequencing system and method described herein maintains a target or constant ratio of the integration time (t int) to time constant (tau = RC) by adjusting the integration time in response to changes in the integration time constant throughout the duration of a time series of measurements. Adjustments to the integration time in this implementation are not expected to impact biophysical behaviors such as error rates or translocation of molecules through the nanopore; changes to integration time in this method is designed to ensure consistency in normalized signals throughout the duration of a series of measurements.

[0149] In some embodiments, the target ratio of t_int / tau is predetermined. In some embodiments, the target ratio is determined by measuring the integration time constant either before or at the beginning of the start of sequencing measurements. In some embodiments, after sequencing has started, the time constant is periodically measured across the cells in the array to determine an average time constant for the array of cells. During sequencing, some of the nanopores will be in an open channel state, while other nanopores will be filled with a molecule. In some embodiments, the time constant is measured using cells with nanopores in an open channel state. In some embodiments, the time constant is measured during the bright period of the sequencing waveform. In some embodiments, a diagnostic waveform or portions of another waveform, such as the sequencing waveform, can be used to determine the state of the nanopore (i.e., whether the nanopore is in an open channel state) and can also be used to measure the integration time constant.

[0150] FIG. 6 is a flow chart that illustrates an embodiment of a high level controller logic that has been programmed and / or configured to perform the adjustment of integrationAttorney Docket No. P39049-WO time based on an integration time constant measurement as described herein. In some embodiments, prior to initiating the sequencing run, the open channel integration time constant (OC RC) can be measured 600 and a target integration time to integration time constant ratio can be determined 602. In some embodiments, the ordering of steps 600 and 602 can be reversed, such that the target integration time to integration time constant ratio can be determined before the open channel integration time constant is measured. Either or both of these first two steps can be optional, and in some embodiments can be omitted. Sequencing can then be initiated 604, and then while sequencing, the OC RC can be measured 606 for cells that are in an open channel state. Next, the average OC RC for the cell array can be calculated 608. The integration time to average OC RC ratio can be calculated and compared with the target integration time to RC ratio 610. If the calculated ratio is greater than the target ratio 612 (plus an error amount such as within 5, 10, 15, 20 percent, for example), then the integration time is decreased so that the calculated ratio using the decreased integration time equals the target ratio 614. If the calculated ratio is less that the target ratio 616 (minus an error amount such as 5, 10, 15, 20 percent, for example), then the integration time is increased so that the calculated ratio using the increased integration time equals the target ratio 618. If the calculated ratio is about equal to the target ratio value or within the target ratio range, no adjustment of the integration time is needed, and the controller can check to see whether the sequencing time is finished 622 (which is also the next step after the integration time is adjusted in 614 or 618). If the sequencing time is not finished, a period of time is waited 624 and then the process begins again by measuring the OC RC 606. If the sequencing time is finished 622, then the sequencing data acquisition can be stopped and the routine can be exited 626.

[0151] In some embodiments, the integration time can be adjusted based on the average OC RC value. In this example, FIG. 6 would be modified by replacing steps 610- 620 with set t int = A * (avg OC RC value), where t int can be rounded to the nearest allowed value of t int.

[0152] IV. Compensating for Ion Inbalance Across a Nanopore

[0153] In the case of faradaic systems utilizing redox reactions under the application of an AC voltage, it has been observed that, under the same AC userwave, not all cells exhibit the same amount of electron transfer in a given direction over a given time, thus resulting in a variance of the concentration of oxidised and reduced species in individual cells in the array of cells. The variance is attributed to the stochastic nature of sequencing, to the variability of the physical and chemical characteristics of the biochemical circuit elements (for exampleAttorney Docket No. P39049-WO lipid bilayer membranes and nanopores) and more specifically to how much time a given nanopore is empty. For example, in the case of sequencing using SBX™ chemistry involving ferricyanide and ferrocyanide, an ion imbalance in the following redox reaction can be observed:

[0154] Fe(CN)63- + e- Fe(CN)64-

[0155] Such an imbalance may have negative effects on the impedance at the working electrode interface and the Nernst potential across the membrane of an individual cell, and thus depending on each working cell’s individual imbalance can result in variability in performance (e.g. electrical performance and accuracy of subsequent sequence reads) across the working cells. An example of indicative data that indicates that such an imbalance is occurring is a change in open channel voltage. Such a change may be observable in the raw data as a greater or smaller deflection from Vpre.

[0156] In some embodiments, one way of compensating for the effects caused by this ion imbalance is to adjust the recharge voltage that is applied during the dark period of the sequencing waveform. This can be used to reverse the redox reaction and reduce the buildup of the ion imbalance. In some embodiments, the amplitude of the recharge voltage can be increased or decreased. In other embodiments, the duration of the application of the recharge voltage can be increased or decreased. In yet other embodiments, a combination of modulating the amplitude and the duration of the recharge voltage can be used.

[0157] In some embodiments, a voltage measurement that corresponds to the voltage across the nanopore can be used as a target voltage. In some embodiments, the target open channel voltage can be determined prior to or at the beginning of a sequencing run. In some embodiments, this voltage measurement can be taken when the cell is in an open channel state. In some embodiments, the voltage measurement can be taken during the bright period of a sequencing waveform. In some embodiments, a diagnostic waveform or portions of another waveform, such as the sequencing waveform, can be used to determine the state of the nanopore (i.e., whether the nanopore is in an open channel state) and can also be used to measure the integration time constant and the voltage across the nanopore.

[0158] FIG. 7 is a flow chart that illustrates an embodiment of a high level controller logic that has been programmed and / or configured to perform the adjustment of the recharge voltage based on a voltage measurement that corresponds to the voltage across the nanopore as described herein. In some embodiments, prior to initiating the sequencing run, the open channel (OC) voltage can be measured 700 and a target OC voltage can be determined 702. Either or both of these first two steps can be optional, and in some embodiments can beAttorney Docket No. P39049-WO omitted. Sequencing can then be initiated 704, and then while sequencing, the OC voltage can be measured 706 for cells that are in an open channel state. Next, the average OC voltage for the cell array can be calculated 708. The average OC voltage can be compared with the target OC voltage 710. If the average OC voltage is greater than the target OC voltage 712 (plus an error amount such as within 5, 10, 15, 20 percent, for example), then the recharge voltage is decreased 714. If the average OC voltage is less that the target OC voltage 716 (minus an error amount such as 5, 10, 15, 20 percent, for example), then the recharge voltage is increased 718. If the average OC voltage is about equal to the target OC voltage or within the target OC voltage range, no adjustment of the recharge voltage is needed, and the controller can check to see whether the sequencing time is finished 722 (which is also the next step after the recharge voltage is adjusted in 714 or 718). If the sequencing time is not finished, a period of time is waited 724 and then the process begins again by measuring the OC voltage 706. If the sequencing time is finished 722, then the sequencing data acquisition can be stopped and the routine can be exited 726.

[0159] In some embodiments, the controller can adjust both the integration time and the recharge voltage to compensate for both membrane capacitance changes and ion imbalance changes. FIG. 8A illustrates the effect of adjusting the recharge voltage on base call levels, while FIG. 8B illustrates the effect of adjusting the integration time. As seen in FIG. 8B, adjusting the integration time results in steadier normalized base calls, which results in improved base call accuracy.

[0160] V. PID Control of Recharge Voltage

[0161] FIG. 9 illustrates another system and method for adjusting the recharge voltage that is similar to the system and method illustrated in FIG. 7, but with the use of a PID (proportional-integral-derivative) control function 912 to determine how the recharge voltage should be adjusted in order to maintain the OC voltage at the target OC voltage.

[0162] The method starts with the steps of measuring the OC voltage 900 and identifying a target OC voltage 902. Although FIG. 9 illustrates these steps as being performed in a certain order, these two steps can be performed in any order. For example, in some embodiments, the step of identifying the target OC voltage 902 can be performed before the step of measuring the OC voltage 900. The ordering of these two steps in the other embodiments of adjusting the recharge voltage, for example as shown in FIG. 7, can also be switched. In addition, either or both of these first two steps can be optional, and in some embodiments can be omitted. Sequencing can then be initiated 904, and then whileAttorney Docket No. P39049-WO sequencing, the OC voltage can be measured 906 for cells that are in an open channel state. Next, the average OC voltage for the cell array can be calculated 908.

[0163] The voltage error, E, can be calculated 910 as the difference between the average OC voltage and the target OC voltage. The voltage error E can be used by a PID control function 912 to determine the recharge voltage adjustment response, A, according to the following algorithm: A=Kp*E + Ki*(time integral of E) + Kd*(time derivative of E), where Kp, Ki, and Kd are predetermined or predefined parameters corresponding to the proportional, integral, and derivative response terms, respectively. The recharge voltage can then be adjusted by A 914, where the value of A can be greater than, less than, or equal to zero.

[0164] In some embodiments, Kp can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Kp can be less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Kp can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0165] In some embodiments, the adjustment response can be based only on the proportional component, Kp*E, with Ki and Kd set to zero. However, this may result in oscillations of the OC voltage around the target OC voltage due to overshooting the target. In some embodiments, the integral component, Ki*(time integral of E) and / or the derivative component, Kd*(time derivative of E), can be added to the proportional component to reduce the oscillations. In addition, the values of the K parameters, can be adjusted to decrease the time to reach the target OC voltage and to reduce the amount of oscillations. For example, a large Kp will decrease the time to reach the target OC voltage, but will increase the oscillations. The values of Kp, Ki, and Kd can be determined empirically based on desired performance characteristics, such as time to reach the target OC voltage and degree of oscillations.

[0166] In addition, the starting recharge voltage can have a large effect on how long it takes to reach the target OC voltage, particularly in the basic recharge voltage adjustment method shown in FIG. 7, but also in the other methods such as the PID method described herein. If the starting recharge voltage is too low and the step size is too small, it can take a very long time to reach the target OC voltage. In some embodiments, the starting recharge voltage can be about or at least about 0, -10, -20, -30, -40, -50, -60, -70, -80, -90, or -100 mV. In some embodiments, the starting recharge voltage can be less than about 0, -10, -20, -30, - 40, -50, -60, -70, -80, -90, or -100 mV.

[0167] Next, the controller can check whether the sequencing time is finished 922. If the sequencing time is not finished, a period of time is waited 924 and then the processAttorney Docket No. P39049-WO begins again by measuring the OC voltage 906. If the sequencing time is finished 922, then the sequencing data acquisition can be stopped and the routine can be exited 926.

[0168] The PID control adjustment shown in FIG. 9 and described above is an improvement over the stepwise adjustment shown in FIG. 7, and is able to make larger adjustments of the recharge voltage when needed, for example when the measured OC voltage is far from the target OC voltage, thereby more quickly arriving at the target OC voltage. In addition, as the measured OC voltage approaches the target OC voltage, the adjustments made using the PID control algorithm are smaller in order to prevent or reduce the chance that the adjustment overshoots the target OC voltage.

[0169] FIG. 10 illustrates another system and method for determining the recharge voltage based on using a look-up table. The method starts with the steps of measuring the OC voltage 1000 and identifying a target OC voltage 1002. Although FIG. 10 illustrates these steps as being performed in a certain order, these two steps can be performed in any order. For example, in some embodiments, the step of identifying the target OC voltage 1002 can be performed before the step of measuring the OC voltage 1000. The ordering of these two steps in the other embodiments of adjusting the recharge voltage, for example as shown in FIG. 7, can also be switched. In addition, either or both of these first two steps can be optional, and in some embodiments can be omitted. Sequencing can then be initiated 1004, and then while sequencing, the OC voltage can be measured 1006 for cells that are in an open channel state. Next, the average OC voltage for the cell array can be calculated 1008.

[0170] The recharge voltage can then be determined using a look-up table based on the average OC voltage and the target OC voltage 1010. The look-up table can be stored in memory, and in some embodiments, can be updated periodically or as desired, by for example downloading an updated look-up table from a website or server. In some embodiments, a voltage error, E, is determined, where the voltage error is the difference between the average OC voltage and the target OC voltage, and the voltage error is used to determine the recharge voltage using the look-up table.

[0171] Next, the controller can check whether the sequencing time is finished 1022. If the sequencing time is not finished, a period of time is waited 1024 and then the process begins again by measuring the OC voltage 1006. If the sequencing time is finished 1022, then the sequencing data acquisition can be stopped and the routine can be exited 1026.

[0172] FIG. 11 illustrates another system and method for determining the recharge voltage based on using an algebraic equation. The method starts with the steps of measuring the OC voltage 1100 and identifying a target OC voltage 1002. Although FIG. 11 illustratesAttorney Docket No. P39049-WO these steps as being performed in a certain order, these two steps can be performed in any order. For example, in some embodiments, the step of identifying the target OC voltage 1102 can be performed before the step of measuring the OC voltage 1100. The ordering of these two steps in the other embodiments of adjusting the recharge voltage, for example as shown in FIG. 7, can also be switched. In addition, either or both of these first two steps can be optional, and in some embodiments can be omitted. Sequencing can then be initiated 1104, and then while sequencing, the OC voltage can be measured 1106 for cells that are in an open channel state. Next, the average OC voltage for the cell array can be calculated 1108.

[0173] The recharge voltage can then be determined using an algebraic equation based on the average OC voltage and the target OC voltage 1110. One example of an algebraic equation is the PID controller implementation described herein when Ki and Kd are set to zero. Another example is (recharge voltage) = A*(measured open channel voltage) + B, where parameters A and B are predetermined based on sequencing parameters (e.g. reagents, desired OC voltage, XP load volume and / or concentration, voltage modulation period, or any other sequencing parameter discussed herein).

[0174] Next, the controller can check whether the sequencing time is finished 1122. If the sequencing time is not finished, a period of time is waited 1124 and then the process begins again by measuring the OC voltage 1106. If the sequencing time is finished 1122, then the sequencing data acquisition can be stopped and the routine can be exited 1126.

[0175] The systems and methods described herein are applicable to a wide range of dynamic control of sequencing voltage application and signal behaviors, including read voltage tuning (by recharge voltage or direct read voltage adjustment), pulse voltage magnitude and / or width tuning, integration time tuning, dynamic voltage ramp adjustment, eject signal behavior, etc. Read voltage can be adjusted based on average OC voltage and the target OC voltage. Pulse voltage magnitude and pulse width can be adjusted based on read error rates or other signals that indicate a de-tuned pulse response. Integration time tuning is described in detail herein. Voltage ramp can be adjusted based on the OC voltage drop during sequencing, such as within a bright cycle or another measurement time period. Eject signal can be adjusted based the need to clear the pore (e.g. stuck XP, unintentional movement or advancement of the XP through the pore) or not clear the pore (e.g., intentional movement of the XP back and forth through the pore). Although the term XP has been used frequently throughout the specification, it should be noted that in many cases another molecule to be sequenced by the nanopore and that is threaded through the nanopore could be substituted for the XP molecule, such as a nucleic acid molecule.Attorney Docket No. P39049-WO

[0176] Any of the computer systems mentioned herein can utilize any suitable number of subsystems. Examples of such subsystems are shown in FIG. 12 in computer system 1110. In some embodiments, a computer system includes a single computer apparatus, where the subsystems can be the components of the computer apparatus. In other embodiments, a computer system includes multiple computer apparatuses, each being a subsystem, with internal components. A computer system can include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0177] The subsystems shown in FIG. 12 are interconnected via a system bus 1280. Additional subsystems such as a printer 1274, keyboard 1278, storage device(s) 1279, monitor 1276 which is coupled to display adapter 1282, and others are shown. Peripherals and input / output (VO) devices, which couple to VO controller 1271, can be connected to the computer system by any number of means known in the art such as VO port 1277 (e.g., USB, FireWire®). For example, VO port 1277 or external interface 1281 (e.g., Ethernet, Wi-Fi, etc.) can be used to connect computer system 1210 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via system bus 1280 allows the central processor 1273 to communicate with each subsystem and to control the execution of a plurality of instructions from system memory 1272 or the storage device(s) 1279 (e.g., a fixed disk, such as a hard drive, or optical disk), as well as the exchange of information between subsystems. The system memory 1272 and / or the storage device(s) 1279 can embody a computer readable medium. Another subsystem is a data collection device 1275, such as a camera, microphone, accelerometer, and the like. Any of the data mentioned herein can be output from one component to another component and can be output to the user.

[0178] A computer system can include a plurality of the same components or subsystems, e.g., connected together by external interface 1281, by an internal interface, or via removable storage devices that can be connected and removed from one component to another component. In some embodiments, computer systems, subsystem, or apparatuses communicate over a network. In such instances, one computer can be considered a client and another computer a server, where each can be part of a same computer system. A client and a server can each include multiple systems, subsystems, or components.

[0179] Aspects of embodiments can be implemented in the form of control logic using hardware circuitry (e.g., an APSIC or FPGA) and / or using computer software with a generally programmable processor in a modular or integrated manner. As used herein, a processor can include a single-core processor, multi-core processor on a same integrated chip, or multiple processing units on a single circuit board or networked, as well as dedicatedAttorney Docket No. P39049-WO hardware. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will know and appreciate other ways and / or methods to implement embodiments of the present invention using hardware and a combination of hardware and software.

[0180] Any of the software components or functions described in this application can be implemented as software code to be executed by a processor using any suitable computer language such as, for example, Java, C, C++, C #, Objective-C, Swift, or scripting language such as Perl or Python using, for example, conventional or object-oriented techniques. The software code can be stored as a series of instructions or commands on a computer readable medium for storage and / or transmission. A suitable non-transitory computer readable medium can include random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard-drive or a floppy disk, or an optical medium such as a compact disk (CD) or DVD (digital versatile disk), flash memory, and the like. The computer readable medium can be any combination of such storage or transmission devices.

[0181] Such programs can also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and / or wireless networks conforming to a variety of protocols, including the Internet. As such, a computer readable medium can be created using a data signal encoded with such programs. Computer readable media encoded with the program code can be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer readable medium can reside on or within a single computer product (e.g., a hard drive, a CD, or an entire computer system), and can be present on or within different computer products within a system or network. A computer system can include a monitor, printer, or other suitable display for providing any of the results mentioned herein to a user.

[0182] Any of the methods described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps. Thus, embodiments can be directed to computer systems configured to perform the steps of any of the methods described herein, potentially with different components performing a respective step or a respective group of steps. Although presented as numbered steps, steps of methods herein can be performed at a same time or at different times or in a different order. Additionally, portions of these steps can be used with portions of other steps from other methods. Also, all or portions of a step can be optional. Additionally, any of the steps of any of the methods can be performed with modules, units, circuits, or other means of a system for performing these steps.Attorney Docket No. P39049-WO

[0183] The specific details of particular embodiments can be combined in any suitable manner without departing from the spirit and scope of embodiments of the invention. However, other embodiments of the invention can be directed to specific embodiments relating to each individual aspect, or specific combinations of these individual aspects.

[0184] Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, the invention is not limited to the details provided. There are many alternative ways of implementing the invention. The disclosed embodiments are illustrative and not restrictive. The above description of example embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the teaching above.

[0185] A recitation of “a”, “an” or “the” is intended to mean “one or more” unless specifically indicated to the contrary. The use of “or” is intended to mean an “inclusive or,” and not an “exclusive or” unless specifically indicated to the contrary. Reference to a “first” component does not necessarily require that a second component be provided. Moreover, reference to a “first” or a “second” component is merely to distinguish between components and does not limit the referenced components to a particular location or order unless expressly stated. The term “based on” is intended to mean “based at least in part on.”

[0186] All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for all purposes. None is admitted being prior art.

Claims

PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WOCLAIMSWhat is claimed is:

1. A system, comprising: an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides, the voltage levels measured over a period of time called an integration time; and a controller configured to: measure an average RC time constant for the array of sequencing cells; and adjust the integration time to achieve a target integration time to RC time constant ratio.

2. The system of claim 1, wherein the RC time constant is measured when the nanopore is in an open channel state.

3. The system of claim 1, wherein the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

4. The system of claim 1, wherein the target integration time to RC time constant ratio is determined by measuring the RC time constant before the sequencing read operations are initiated.

5. A method, comprising: measuring an average RC time constant for an array of sequencing cells, wherein each sequencing cell is configured to support a membrane comprising a nanopore, wherein each sequencing cell comprises a measuring circuit; and adjusting an integration time to achieve a target integration time to RC time constant ratio, wherein the integration time is a period of time that is used to measure a voltage level by the measuring circuit that corresponds to a base of a nucleotide.PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO6. The method of claim 5, wherein the RC time constant is measured when the nanopore is in an open channel state.

7. The method of claim 5, wherein the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

8. The method of claim 5, wherein the target integration time to RC time constant ratio is determined by measuring the RC time constant before the array of sequencing cells are used to sequence a molecule.

9. A system, comprising: an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, wherein the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and adjust the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

10. The system of claim 9, wherein the average voltage is measured when the nanopore is in an open channel state.

11. The system of claim 9, wherein the controller is configured to decrease the recharge voltage when the measured average voltage is greater than the target average voltage.PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO12. The system of claim 9, wherein the controller is configured to increase the recharge voltage when the measured average voltage is less than the target average voltage.

13. A method, comprising: applying a sequencing waveform across an array of sequencing cells, wherein the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, wherein the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; and adjusting the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

14. The method of claim 13, wherein the average voltage is measured when the sequencing cells are in an open channel state.

15. The method of claim 13, further comprising decreasing the recharge voltage when the measured average voltage is greater than the target average voltage.

16. The method of claim 13, further comprising increasing the recharge voltage when the measured average voltage is less than the target average voltage.

17. A system, comprising: an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides, the voltage levels measured over a period of time called an integration time; and a controller configured to: measure an average RC time constant for the array of sequencing cells; adjust the integration time to achieve a target integration time to RC time constant ratio;PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, wherein the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and adjust the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

18. The system of claim 17, wherein both the average voltage and the average RC time constant are measured when the nanopore is in an open channel state.

20. The system of claim 17, wherein the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

21. The system of claim 17, wherein the target integration time to RC time constant ratio is determined by measuring the RC time constant before the sequencing read operations are initiated.

22. The system of claim 17, wherein the controller is configured to decrease the recharge voltage when the measured average voltage is greater than the target average voltage.

23. The system of claim 17, wherein the controller is configured to increase the recharge voltage when the measured average voltage is less than the target average voltage.

24. A method, comprising: measuring an average RC time constant for an array of sequencing cells, wherein each sequencing cell is configured to support a membrane comprising a nanopore, wherein each sequencing cell comprises a measuring circuit; adjusting an integration time to achieve a target integration time to RC time constant ratio, wherein the integration time is a period of time that is used to measure a voltage level by the measuring circuit that corresponds to a base of a nucleotide;PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO applying a sequencing waveform across the array of sequencing cells, wherein the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, wherein the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; and adjusting the recharge voltage based on the measured average voltage to achieve a target average voltage for the array of sequencing cells.

25. The method of claim 24, wherein the RC time constant is measured when the nanopore is in an open channel state.

26. The method of claim 24, wherein the RC time constant is determined by measuring a voltage change that occurs across the capacitor over a measurement time period.

27. The method of claim 24, wherein the target integration time to RC time constant ratio is determined by measuring the RC time constant before the array of sequencing cells are used to sequence a molecule.

28. The method of claim 24, wherein the average voltage is measured when the sequencing cells are in an open channel state.

29. The method of claim 24, further comprising decreasing the recharge voltage when the measured average voltage is greater than the target average voltage.

30. The method of claim 24, further comprising increasing the recharge voltage when the measured average voltage is less than the target average voltage.

31. A system, comprising: an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; andPCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, wherein the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; determine a voltage error, wherein the voltage error comprises a difference between the average voltage and a target voltage; determine a recharge voltage adjustment based on a proportional-integral- derivative control function; and adjust a recharge voltage by the recharge voltage adjustment.

32. The system of claim 31, wherein the average voltage is measured when the nanopore is in an open channel state.

33. A method, comprising: applying a sequencing waveform across an array of sequencing cells, wherein the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, wherein the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, wherein the voltage error comprises a difference between the average voltage and a target voltage; determining a recharge voltage adjustment based on a proportional-integral-derivative control function; and adjusting a recharge voltage by the recharge voltage adjustment.

34. The method of claim 33, wherein the average voltage is measured when the nanopore is in an open channel state.

35. A system, comprising:PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, wherein the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and determine a recharge voltage using a look-up table based on the measured average voltage and a target voltage.

36. The system of claim 35, wherein the average voltage is measured when the nanopore is in an open channel state.

37. A method, comprising: applying a sequencing waveform across an array of sequencing cells, wherein the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, wherein the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, wherein the voltage error comprises a difference between the average voltage and a target voltage; and determining a recharge voltage using a look-up table based on the measured average voltage and a target voltage.

38. The method of claim 37, wherein the average voltage is measured when the nanopore is in an open channel state.

39. A system, comprising:PCT / US25 / 44282 29 August 2025 (29.08.2025)Attorney Docket No. P39049-WO an array of sequencing cells, each sequencing cell configured to support a membrane comprising a nanopore, each sequencing cell further comprising a working electrode in electrical communication with a capacitor; an analog to digital converter (A / D) coupled to the capacitor and configured to sample voltage levels of the capacitor during sequencing read operations through the nanopore, the voltage levels corresponding to bases of nucleotides; and a controller configured to: apply a sequencing waveform across the nanopore, the sequencing waveform comprising a bright period during which the voltage levels of the capacitor are sampled during sequencing read operations and a dark period during which a recharge voltage is applied, wherein the bright period and dark period have opposite polarities; measure an average voltage across the nanopores for the array of sequencing cells; and determine a recharge voltage using an algebraic equation based on the average voltage and the target voltage.

40. The system of claim 39, wherein the average voltage is measured when the nanopore is in an open channel state.

41. A method, comprising: applying a sequencing waveform across an array of sequencing cells, wherein the sequencing waveform comprises a bright period during which sequencing signals that correspond to bases of nucleotides are acquired and a dark period during which a recharge voltage is applied, wherein the bright period and the dark period have opposite polarities; measuring an average voltage across the array of sequencing cells; determining a voltage error, wherein the voltage error comprises a difference between the average voltage and a target voltage; and determining a recharge voltage using an algebraic equation based on the average voltage and the target voltage.

42. The method of claim 40, wherein the average voltage is measured when the nanopore is in an open channel state.

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