Dynamic control of spatial and temporal variability in nanopore sequencing

Abstract

A system and method for sequencing a molecule, where the system includes: a nanopore sensor chip (602) with an array (200) of cells, where each cell of the array of cells has a working electrode (202) and a switch (406). The system also has a flow channel configured to flow an analyte comprising the molecule across the array of cells; a counter electrode (210) configured to apply a voltage to the analyte; an alternating current voltage source (228) configured to apply an AC voltage to the working electrode (202); a storage device (612) configured to represent either an open state or a closed state for the switch (406); an analog-to-digital converter (410); and one or more processors (430). The one or more processors (430) are programmed to: (1) analyze output data from the analog-to-digital converter (410) to detect a first sub-set of cells, where each cell of the first sub-set cells comprises a membrane with a nanopore embedded therein; (2) analyze further output data from the analog-to-digital converter (410) for the first sub-set of cells to detect a second sub-set of cells, where the first sub-set of cells comprises the second sub-set of cells; and in response to this analysis of the further output data: (3) update the state of the storage device (612) of each cell of the second sub-set of cells to an open state, thereby opening the switch (406) of each cell of the second sub-set of cells so as to disconnect the working electrode (202) of each cell of the second sub-set of cells from the AC voltage source (228) for a period of time during a single AC modulation period.

Description

PATENTAtorney Docket No.: P39047-WODYNAMIC CONTROL OF SPATIAL AND TEMPORAL VARIABILITY IN SEQUENCINGCROSS REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Application No. 63 / 689559, filed August 30, 2024, the entire contents of which is herein incorporated by reference.FIELD OF THE INVENTION

[0002] The present disclosure relates to the field of nanopore sequencing and more specifically, but not exclusively, to nanopore systems and associated methods for the determination of nucleotide sequences in DNA strands.BACKGROUND

[0003] Traditional DNA sequencing techniques have undergone significant advancements in the last 40 years. Technologies such as Sanger sequencing and next-generation sequencing (NGS) have become integral tools in genomics research and increasingly have become integral tools for the diagnosis and / or treatment of a variety of diseases. More recently, nanopore sequencing has emerged as a powerful technique for sequencing polymers, including nucleic acid molecules, offering the ability to provide real-time sequencing, long read lengths and the potential for cost-effective devices.

[0004] The fundamentals of nanopore sequencing involve passing a polymer molecule, such as a DNA molecule, through a nanopore immersed in a conducting fluid. When such a molecule passes through the nanopore (translocation), it can partially or completely block the nanopore, causing a change in the resistance of the nanopore. 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, thereby allowing detection of which molecule is in the nanopore. In the case of DNA, each of the four bases (adenine, thymine, cytosine and guanine) create an ionic current blockade to a differing degree, giving rise to an unique signature in the electrical signal, thus allowing for the identification of the nucleotide sequence.PATENTAtorney Docket No.: P39047-WO

[0005] Despite its significant advantages, nanopore sequencing can, in some instances, experience challenges in achieving high levels of accuracy, primarily due to factors such as translocation speed and signal noise. The accuracy of nanopore sequencing is critical for applications such as genomic research and medical diagnosis and / or treatment, thus it would be advantageous to provide a system and method for reliably and accurately sequencing polymer molecules, such as DNA.BRIEF SUMMARY

[0006] Techniques described herein relate to dynamically adjusting the behavior of individual working cells of an array of cells in a nanopore sensor chip following a feedback signal from a working cell indicating that there is a drift in its behavior and / or that the working cell requires an intervention. The working cells that are exhibiting such a drift in behavior and / or require such an intervention are then targeted by disconnecting their respective working electrodes from the cell’s dry circuit elements (and thus the AC voltage source and accompanying AC userwave) for a period of time during a single AC modulation period, or over multiple single modulation periods, thus varying the effective voltage experienced by each targeted individual cell. The effect of sources of spatial and temporal variability on individual cells during the sequencing process can thus be advantageously minimized.

[0007] Accordingly, the present disclosure provides a system for sequencing a molecule, the system comprising: a nanopore sensor chip comprising an array of cells, wherein each cell of the array of cells comprises a working electrode and a switch; and wherein the system further comprises: a flow channel configured to flow an analyte comprising the molecule across the array of cells; a counter electrode configured to apply a voltage to the analyte; an alternating current (AC) voltage source configured to apply an AC voltage to the working electrode; a storage device configured to represent either an open state or a closed state for the switch; an analog-to-digital converter (ADC); and a processor (for example, one or more processors) programmed to (and / or configured to): analyze output data from the ADC to detect a first subset of cells, wherein each cell of the first sub-set of cells comprises a membrane with a nanopore embedded therein; analyze further output data from the ADC for the first sub-set of cells to detect a second sub-set of cells, wherein the first sub-set of cells comprises the second sub-set of cells; and in response to this analysis of the further output data: update the state of the storage device of each cell of the second sub-set of cells to an open state, thereby opening the switch of each cell of the second sub-set of cells so as to disconnect the working electrode of each cell of the second sub-set of cells from the AC voltage source for a period of time during a (i.e. onePATENTAttomev Docket No.: P39047-WO or more) single AC modulation period. In some embodiments, the first sub-set of cells may be considered as working cells of the array of cells and the second sub-set of cells may be considered as targeted working cells (or a sub-proportion or sub-population of the working cells). As such, the step of analyzing the output data from the ADC to detect a first sub-set of cells may be considered the analysis of output data to determine which cells of the array of cells are working cells. Further, the step of analyzing the further output data from the ADC for the first sub-set of cells to detect a second sub-set of cells, may be considered the analysis of output data (which may be different data to the data that indicated that the cells are working cells) from the working cells to detect a sub-proportion or sub-population of the working cells that require targeting, for example because of a drift in signal and / or because such cells require an intervention.

[0008] The period of time of disconnection of the second sub-set of cells may be less than a single AC modulation period, and is preferably ten to ninety percent of the single AC modulation period, more preferably less than fifty percent of the single AC modulation period (for example, within the dark period). Given that the period of time is a disconnection period, so as to vary the effective voltage experienced by each targeted individual working cell, the processor may be further programmed to update the state of the storage device of each cell of the second sub-set of cells to a closed state, thereby closing the switch at, for example, the end of the period of time. This allows the working electrode of each cell of the second sub-set of cells to be reconnected to the AC voltage source during the same single AC modulation period. This disconnection and reconnection may happen for one or more cells of the second sub-set of cells one or more times during a single AC modulation period. Accordingly, the processor may be further programmed to update the state of the storage device of one or more cells of the second sub-set of cells so as to disconnect and reconnect the working electrode of one or more cells of the second sub-set of cells for one or more further times during the same single AC modulation period. This advantageously allows further manipulation of the effective voltage experienced by one or more of the second sub-set of cells. In addition or alternatively, the processor may be further programmed to update the state of the storage device of one or more of the cells of the second sub-set of cells to disconnect the working electrode of one or more of the cells of the second sub-set of cells from the AC voltage source during a (i.e. one or more) further single AC modulation period and to update the state of the storage device to reconnect the working electrode of one or more of the cells of the second sub-set of cells to the AC voltage source during the same further AC modulation period. In other words, the processor may be programmed to disconnect and reconnect the targeted working cells in one or more differentPATENTAttorney Docket No.: P39047-WOAC modulation periods during the sequencing data acquisition phase, thus allowing the further targeted tuning of the AC voltage experienced by an individual working cell.

[0009] Preferably, the processor is programmed to update the state of the storage device of each cell of the second sub-set of cells using a virtual representation of the array of cells indicating the binary state of each cell of the array of cells, wherein the state is either enabled or disabled. In other words, preferably the processor is programmed to update the state of the storage device of each cell of the second sub-set of cells using an active bit map. and preferably an instructing active bit map (held in, for example, a FPGA). Advantageously the use of an active bit map provides for an accurate and efficient method of disconnecting and reconnecting one or more targeted working cells in the array of cells. The processor may be further programmed to: analyse the output data from the ADC for the first sub-set of cells (i.e. working cells) against a set of predefined operating parameters for each cell of the first sub-set of cells; update the virtual representation of the array of cells using such analysis to provide an updated virtual representation of the array of cells (in other words an updated instructing active bit map) and thereafter using the updated virtual representation of the array of cells to: update the state of the storage device of each cell of the second sub-set of cells so as to disconnect or reconnect the working electrode of each cell of the second sub-set of cells from the AC voltage source. The updated virtual representation of the array of cells may be updated by one or more further virtual representations (i.e. one or more further active bit maps) of the array of cells, wherein for each further representation of the array of the cells the binary state of each cell of the array of cells is indicated, wherein the state is either enabled or disabled. The one or more further virtual representations of the array of cells may be considered the new' active bit maps that are created after the analysis of the further data and are the active bit maps that are used to update the instructing active bit map. Advantageously this allows for an accurate and efficient method of continual and “on-the-fly’’ data analysis and subsequent control of the voltage schedule applied to individual w orking cells in the array of cells, thus providing the ability to control the distribution of cell characteristics or behaviours in addition to the population mean.

[0010] In a preferred example, the application of the virtual representation of the array of cells, or the updated virtual representation of the array of cells, to update the state of the storage device of each cell of the second sub-set of cells is synchronised with an event in an AC signal from the AC voltage source. Advantageously this allows certain events in an AC signal to be targeted to only the required and desired working cells.

[0011] The analysis of the further output data from the ADC may, in some instances, indicate a change in behaviour (for example a drift in behaviour and / or the requirement for anPATENTAtorney Docket No.: P39047-WO intervention) of each cell of the second sub-set of cells. The change in behavior may be, for example, a change of temperature, a drift in an electrochemical state, a drift in bilayer capacitance state, a blocking of the nanopore, an interruption in a translocation of the molecule, a translocation event, the identification of a specific molecule passing through the nanopore or any combination thereof. In such cases, the present invention may be utilised to rectify these changes in behaviour.

[0012] Preferably, the system further comprises a shared AC voltage line which electrically couples the working electrode of each cell of the second sub-set of cells to the AC voltage source and / or the system further comprises a capacitor, wherein the capacitor is electrically coupled to the working electrode and wherein each cell of the array of cells comprises a further switch that selectively couples the capacitor to the AC voltage source. The further switch may be considered the reset switch 401 in Figure 4. Preferably, the ADC of the present disclosure is electrically coupled to the capacitor.

[0013] Each cell of the array of cells may comprise its own storage device and the storage device may be a memory bit. Accordingly, the storage device may be considered an arrangement of transistors that operate in conjunction with the switch to set the switch to open or closed. The storage device may also be considered a bit of memory that can represent the state of the switch in an open or closed state.

[0014] The processor utilised in the present disclosure may be a field programmable gate array, an application specific integrated circuit, an artificial intelligence chip, a processor in memory device, a graphics processing unit and / or a central processing unit. The virtual representation of the array of cells and / or the updated representation of the array of cells of the present disclosure are preferably stored in a field programmable gate array and / or associated storage and the one or more further virtual representations of the array of cells is preferably stored in a graphics processing unit and / or associated storage.

[0015] The system may comprise a sequencing instrument and a removable consumable device and the nanopore sensor chip, flow channel, storage device, ADC and / or counter electrode may be housed in the removable consumable device. Alternatively, the system may comprise a sequencing instrument and a reusable, preferably removable, device and the nanopore sensor chip, flow channel, storage device, ADC and / or counter electrode may be housed in the reusable, preferably removable, device. The removable consumable device, or reusable, preferably removable, device may be copulable to the sequencing instrument. The system may further comprise a thermal control system configured to contact the nanopore sensor chip. The array of cells of the system of the present disclosure may comprise at leastPATENTAtorney Docket No.: P39047-WO two million cells, preferably at least eight million cells. The number of cells in the first sub-set of cells of the array of cells may be ninety’ eight percent or less of the number of cells in the array of cells and the number of cells in the second sub-set of cells may be five to ninety7five percent of the number of cells in the first sub-set of cells.

[0016] The analy te of the present disclosure comprises a molecule to be analy sed and may comprise an ionic solution and the ionic solution may comprise a redox couple comprising a first species of the redox couple and a second species of the redox couple. The molecule may be an expanded molecule derived from a nucleic acid molecule and the expanded molecule may comprise one or more reporter codes. In the other words, the molecule may be an Xpandomer molecule.

[0017] The present disclosure also provides a method of sequencing a molecule using a nanopore sensor chip, wherein the nanopore sensor chip comprises an array of cells, and yvherein each cell of the array of cells comprises a yvorking electrode and a syvitch, the method comprising: forming a membrane and embedding a nanopore therein for each cell of a first subset of cells of the array of cells; flowing an analyte comprising the molecule across the array of cells; applying a voltage to the analyte (a first voltage); applying an AC voltage (a second, different voltage, which is an AC voltage) to the working electrode of each cell of the first subset of cells; receiving output data for the first sub-set of cells at an analog-to-digital converter (ADC); analysing the output data from the ADC for the first sub-set of cells; and, in response to this analysis: updating the state of a storage device of each cell in a second sub-set of cells to an open state, and thereby opening the switch so as to disconnect the working electrode of each cell of the second sub-set of cells from the AC voltage for a period of time during a single AC modulation period, wherein the first sub-set of cells comprises the second sub-set of cells and wherein the storage device is configured to represent either an open state or a closed state for the switch.

[0018] The present disclosure also provides a computer program product comprising a plurality7of instructions to cause the system disclosed herein to execute the steps (or perform the operations) of the methods disclosed herein.

[0019] The present disclosure also provides a computer readable storage medium having stored therein the computer program product disclosed above.PATENTAttorney Docket No.: P39047-WOBRIEF DESCRIPTION OF THE FIGURES

[0020] For a beter understanding of the present invention, and to illustrate how it may be put into effect, reference is now made, by way of example only, to the accompanying drawings (not to scale) in which:

[0021] FIG. 1 is a schematic of Xpandomer molecules;

[0022] FIG. 2 is a cross-sectional (side) view of an array of cells in an example nanopore sensor chip;

[0023] FIG. 3 is a cross-sectional (side) view of two cells in an array of cells in an example nanopore sensor chip;

[0024] FIG. 4 is an example of a suitable circuit for use in a nanopore sensor chip;

[0025] FIG. 5A and FIG. 5B show example data points captured from a cell during a singleAC modulation period, and its respective bright period and dark period;

[0026] FIG. 6 is an example arrangement of processors, and associated memory’, for housing and processing active bit maps that may be implemented in the systems and methods of the present disclosure;

[0027] FIG. 7 is a flowchart of an example process for sequencing a target molecule;

[0028] FIG. 8 is a modelled heatmap of one flow channel of cells in an 8M nanopore sensor chip;

[0029] FIG. 9 is a second heatmap of one half of one flow channel of cells in a 8M nanopore sensor chip showing the deletion rate as a function of position in the flow channel;

[0030] FIG. 10 is an example computer system that may be implemented in the systems and methods of the present disclosure.DETAILED DESCRIPTIONTerms

[0031] 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 necessarily meant to limit the scope of the present invention. Abbreviations used herein have their conventional meaning within the chemical, biological and engineering arts.

[0032] The term "AC userwave'” generally refers to a pre-programmed series of digital values detailing the AC voltage waveform, including the shape, frequency and amplitudes, and thePATENTAtorney Docket No.: P39047-WO associated application timings, where those digital values are subsequently converted to analogue values and applied to the nanopore sensor chip.

[0033] The term “a single AC modulation period" generally refers to the time it takes for one complete cycle of the AC signal (for example, measured in seconds) and is one complete repetition of the wave pattern, as exemplified in FIG. 5 A and FIG. 5B.

[0034] The term “active bit map" generally refers to a virtual representation of the array of cells indicating the binary state of each cell, wherein the state is either enabled or disabled.

[0035] The term “bright period” generally refers to the time period of the AC waveform when output signals are sampled by suitable circuitry (e.g., an analog-to-digital converter (ADC)), and is typically, but not exclusively, associated with the time period of the AC waveform for which the applied voltage and corresponding electric field promote capture (“threading’’) of molecules into the nanopore and, for example, the subsequent translocation of the molecule through the nanopore The term “dark period” generally refers to the time period of the AC waveform when output signals are not sampled, or minimally sampled, by suitable circuitry (e.g., an ADC), and is typically, but not exclusively, associated with the time period of the AC waveform for which the applied voltage and corresponding electric field promote ejection of molecules from the nanopore and, for example in the case of SBX chemistry, includes reverse translocation of the molecule through the nanopore. One AC modulation period can include one bright period and one dark period.

[0036] The term “dry circuit elements” generally refers to the parts of the circuitry of an individual cell which are integrated into or onto the substrate and dielectric layers of the individual cell and which can include, but are not limited to, the memory bit, capacitor (e.g. integrating capacitor) and switch, whereas the term “wet circuit elements” generally refers to the parts of the closed circuitry of an individual cell that are in contact with the analyte and which can include the counter electrode, the membrane and the nanopore. The working electrode of a cell may be considered to be at the interface between the dry and wet circuit elements. The dry and wet circuit elements are exemplified in FIG. 3 by dotted and dashed lines.

[0037] The term “indicative data” generally refers to any raw data, analyzed raw data and / or signals originating or derived from a cell, e.g. from the ADC of a working cell, that serve as a sign, indication or suggestion that the cell is, or has been, exhibiting abnormal behaviour, exhibiting a persistent dysfunctional state, exhibiting a drift in normal behaviour and / or that the cell requires intervention. Typical examples of indicative data include deletion rate, insertion rate, open channel voltage and sequence motifs, or any combination thereof.PATENTAttorney Docket No.: P39047-WO

[0038] The term “primer” generally 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.

[0039] A "polymerase" generally 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.

[0040] A “nucleic acid' generally 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, phosphoramidites, 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.

[0041] The term “nucleotide” in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, can be generally 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.

[0042] The term “sequencing data acquisition phase” generally refers to the time period(s) in the sequencing workflow when an AC voltage is applied to the w orking electrodes of the w orking cells and data is acquired by one or more processors, preferably by way of an ADC, from the working cells, and includes the bright and dark periods.

[0043] The term “sequencing workflow” generally refers to the various phases involved in the sequencing process (and specific steps involved in those phases), including, but not limitedPATENTAtorney Docket No.: P39047-WO to, the initialisation phase, the membrane formation phase, the nanopore formation phase, the calibration phase (e.g.. to identify cells that are working and non-working), the AC voltage application phase, which includes the sequencing data acquisition phase, the recalibration phase (e.g. to identify cells that are performing as desired and / or to identify cells that are not performing as desired), the analysing phase and the cleaning and reset phase.

[0044] The term "tag" generally 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.

[0045] The term “TCE pulse” generally refers to a pulse of positive or negative polarity voltage designated within the AC userwave that functions to overcome the energetic barrier associated with a TCE of a Xpandomer molecule and ideally results in translocation past a single TCE barrier, thus moving the Xpandomer further into the pore in the forward direction by an amount of one “base” position or further out of the pore in the reverse direction by an amount of one “base” position.Sequencing

[0046] The present disclosure describes novel methods of nanopore sequencing on sequencing instruments and these methods can be implemented using different sequencing chemistries. For example, the methods can be implemented with Sequencing by Tagging (SBT), a sub-set of Sequencing by Synthesis (SBS). In general terms, SBT involves tags of different sizes and / or chemical structures being attached to different molecules in the polymer (e.g. nucleotides in a nucleic acid molecule) to be sequenced. In some examples, a complementary strand to a template of the nucleic acid molecule to be sequenced may be synthesised by hybridising 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.

[0047] The novel systems and methods of nanopore sequencing disclosed herein are also particularly advantageous when implemented with Sequencing By expansion (SBX™). SBX™ is described in WO 2020 / 236526 Al, “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing,” filed May 14, 2020, and US 7,939,259 B2, “High throughput nucleic acid sequencing by expansion,” filedPATENTAtorney Docket No.: P39047-WOJune 19, 2008, the entire contents of both of which are incorporated herein by reference for all purposes.

[0048] In general terms, SBX™ uses biochemical polymerization to transcribe the sequence of a DNA template onto a surrogate polymer (herein referred to as an “Xpandomer”). The Xpandomer is a measurable polymer and it is formed by template directed synthesis that preserves the original genetic information of the target nucleic acid, while also increasing linear separation of the individual elements of the sequence data.

[0049] Xpandomer synthesis is based on four easily differentiated units called X-NTPs (also called High Signal-to-Noise Reporters). Each unit includes a reporter code portion or portions. The reporter codes correspond to the different nucleotides (e.g., adenine (A), thymine (T), cytosine (C) and guanine (G)). The reporter codes generate different electrical signals in the nanopore and therefore allow identification of the nucleotide sequence. Different reporter codes can be sized to block ion flow through a nanopore at different measurable levels. The structural elements of the X-NTPs also may be adjusted for improving measurements within a nanopore. An X-NTP has two distinct functional regions; namely, a selectively cleavable phosphorami date bond, linking the 5’ a-phosphate to the nucleobase, and a symmetrically synthesized reporter tether (SSRT) that is attached within the nucleoside triphosphoramidate at positions that allow for controlled expansion by cleavage of the phosphoramidate bond. The SSRT includes linkers and are separated by the selectively cleavable phosphoramidate bond. Each linker attaches to one end of a reporter code.

[0050] Each unit further includes a translocation control element (TCE). To pass through the nanopore, the TCE requires a higher voltage to be applied as compared to the baseline voltage for driving the rest of the unit through the nanopore. TCEs are polymers produced by solidphase synthesis using the phosphoramidite method with suitable monomeric building blocks that terminate with a branched structure (i.e., the '‘brancher”). Branched phosphoramidites include both symmetrical and asymmetrical branchers. The TCE brancher may be a symmetrical branching CED phosphoramidite, wherein each arm of the brancher is linked to a reporter code. Exemplary symmetrical chemical branchers include 1,2,3 -O-tris- (phosphosphodiesterjpropane, l,3-bis-(5-O-phosphodiester-pentylamido)-2-O- phosphodiester-propane, and l,4,7-O-tris-(phosphodiester)-heptane. A UV chromophore may be attached to the end of TCE, which may allow for visualization or quantification. Reporter codes and other features can be designed by selecting a sequence of specific phosphoramidites. Spacers may be attached to these reporter codes, and these spacers may be polyethylene glycol (PEG) units, which may act to modulate the length traversed in a nanopore. Enhancers may bePATENTAtorney Docket No.: P39047-WO attached to the spacers and these enhancers may be positively charged spermine that facilitate polymerase incorporation. The nucleotide may be attached to these enhancers. The nucleotide may include a triphosphoramidate diester.

[0051] FIG. 1 illustrates these different components of Xpandomers. Section 102 shows primer-directed Xpandomer synthesis. X-NTP 104 is illustrated in the “constrained configuration”, characteristic of the X-NTP substrates and the daughter strand products of template-dependent polymerization. The constrained configuration of polymerized X-NTPs is the precursor to the expanded configuration, X-NTP 108. Section 106 illustrates cleaving to expand the Xpandomer. The transition from the constrained configuration to the expanded configuration occurs upon scission of the P— N bond of the phosphoramidate within the primarybackbone of the daughter strand.

[0052] During assembly, the monomeric X-NTP substrates (XATP, XCTP, XGTP and XTTP) are polymerised on the extendable terminus of a nascent daughter strand by a process of template-directed polymerization using a single-stranded template as a guide. Generally, this process is initiated from a primer and proceeds in the 5' to 3' direction. Generally, a DNA polymerase 110 or other polymerase is used to form the daughter strand, and conditions are selected so that a complimentary copy of the template strand is obtained. After the daughter strand is synthesised, the coupled SSRTs form the constrained Xpandomer that further forms the daughter strand. SSRTs in the daughter strand have the “constrained configuration” of the X-NTP substrates. The constrained configuration of the SSRT is the precursor to the expanded configuration.

[0053] In this example, once synthesis and expansion are complete, each monomeric X-NTP unit 112 in the Xpandomer contains two reporter codes 116a and 116b with a reporter code “level” corresponding to the base type it encodes, and a Translocation Control Element (TCE) 120. The TCE controls the rate of Xpandomer translocation through a nanopore through a combination of sterics, electrorepulsion, and / or preferential interaction with the nanopore. The resistance of the TCE to the driving force of the ion current when positioned at the nanopore aperture and the consequent increase in applied voltage (i.e., the voltage pulse) necessary to overcome the arrest and resume translocation, can be customized by modulating various properties of the TCE, (and in some examples, the reporter codes and other elements of the SSRT) e.g., the bulk, length, and / or charge density-.

[0054] Brancher 124 is the branched structure that terminates the TCE and links to reporter codes 116a and 116b. Enhancers 128a and 128b may aid in polymerase incorporation. Nucleotide 132 is attached to enhancer 128b and may include a cleavable linker 136. CleavablePATENTAtorney Docket No.: P39047-WO linker 136 may be a photocleavable linker. Cleavable linker 136 may be cleaved to result in the expansion shown in section 106.

[0055] The methods of sequencing disclosed herein (and associated systems) may involve utilising such, as described, SBX™ chemistry. Accordingly, the molecule to be sequenced may include a reporter code corresponding to a particular nucleotide, thus allowing detection of a nucleotide at a particular position of a nucleic acid. In other words, the molecule may be an expanded molecule transcribed from a nucleic acid molecule, wherein the expanded molecule comprises one or more reporter codes. The methods disclosed herein may further include one or more of the following methods steps (and the processors of the systems disclosed herein, or additional systems, may be programmed to carry out one or more of the following method steps): a) providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond; b) cleaving the at least one selectively cleavable bond to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid; and c) detecting the reporter elements of the Xpandomer.Sequencing Instrument and Consumable Device

[0056] In general terms, the consumable device engages with the sequencing instrument (for example, an High-Throughput Platform (HTP)), interfaces with the sequencing instrument, and functions together with the instrument system to perform a nanopore based biological assay, for example a nanopore based polymer (e.g. DNA or RNA) sequencing assay. The consumable device includes one or more nanopore sensor chips (which each include an array of cells on an integrated circuit), one or more flow channels and a means of interfacing with the sequencing instrument hardware and software, where such hardware and software function, at least to (a) apply a plurality of voltages (e.g. an AC userwave) to the array of cells and (b) retrieve data from the integrated circuit (and. more specifically, from the integrating capacitor(s) in the integrated circuit).

[0057] The consumable device is electrically connected with the sequencing instrument (preferably by way of one or more electrical connectors) and measurement data is read from the nanopore sensor chip of the consumable device and exported from the nanopore sensor chip of the consumable device to the sequencing instrument for storage and / or analysis (forPATENTAtorney Docket No.: P39047-WO example, data may be exported from the nanopore sensor chip to a Field Programmable Gate Array (FPGA), to a Graphics Processing Unit (GPU), to storage, to a Network Interface Card (NIC) and onto a local network and / or cloud storage). The consumable device captures and detects electrical signals generated during the movement of molecules through the nanopores (i.e. translocation) of the cells of the nanopore sensor chip, thus providing the raw data necessary for subsequent base-calling and sequence determination. The sequencing instrument may further comprise means for applying electrical waveforms to the nanopore sensor chip of the consumable device, such as a AC / DC conversion and regulation unit.

[0058] In addition, the consumable device is fluidically connected with the sequencing instrument so as to provide (preferably by way of the flow channel(s) of the consumable device) the necessary fluids or gases (e.g. reagents, analytes, cleaning agents) involved in the sequencing workflow. Accordingly, the sequencing instrument preferably includes a fluidic system including one or more pumps (e.g. a two-way syringe pump) for delivering and discharging (pushing / pulling) such fluids and gases into, and out of, one or more of the flow channels of the consumable device. The sequencing instrument, and fluidic system, may further include one or more valves (and respective actuators and motors) for delivering and discharging such fluids and gases. The sequencing instrument, and fluidic system, may further comprise a plurality7of ports, tubes, syringes and / or pipettes for transporting such fluids and gases. In addition, the sequencing instrument, and fluidic system, may further comprise one or more reservoirs / containers (such as reagent reservoirs and / or analyte reservoirs) for delivering / supplying the fluids and / or gases involved in the sequencing workflow. The sequencing instrument, and fluidic system, may also include one or more waste containers (such as a vented container, an expandable container, a one-way valve container and / or an absorbent material filled container) to retain waste substances, such as discarded / discharged fluids or gases (e.g. discarded reagents, discarded analytes, discarded cleaning agents). The sequencing instrument, and fluidic system, can also include one or more sensors (e.g. pressure sensors, flow sensors, temperature sensors).

[0059] In addition, the consumable device may be thermally connected to the sequencing instrument. In other words, the sequencing instrument may have one or more means for maintaining a stable temperature of the consumable device, so as to ensure the optimal performance of the membranes, nanopores, Xpandomers and other components involved in the sequencing workflow. One or more sub-units of the sequencing instrument may provide thermal control of the fluids or gases, the consumable device, the reservoirs / containers and any other substance or instrument unit. In other words, the sequencing instrument may comprisePATENTAttorney Docket No.: P39047-WO one or more means for heating and / or cooling so as to increase, lower or maintain the temperature of reagents, analytes and other substances used in the sequencing workflow. For example, the sequencing instrument may comprise one or more thermal control systems configured to contact the nanopore sensor chip, for example thermal pads, thermal blocks (e.g. a thermal electric cooler), cooling fans and / or heat sink assemblies, directly or indirectly contacting the nanopore sensor chip through, for example, any suitable thermal interfacing material. The consumable device may also have such thermal control means. Preferably, the sequencing instrument comprises a thermal block (e g. a thermal electric cooler) adjacent to (and thermally connected to, by way of athermal interfacing layer) the consumable device, and thus the flow cell and flow channels therein.

[0060] The consumable device may also be mechanically connected to the sequencing instrument, and may be configured by any suitable means for coupling, docking or loading the consumable device in, or on, the sequencing instrument. In other words, the consumable device may be configured to be coupled, docked or loaded into or onto the sequencing instrument. Accordingly, the sequencing instrument may be configured to receive the consumable device. For example, the sequencing instrument and / or consumable device may comprise one or more clamping mechanisms (for example with male and female connections). Such coupling, docking or loading means may function to initiate the electrical and / or thermal connections. For example, the consumable device may be configured to be attached or docked into a socket of the printed circuit board of the sequencing instrument.

[0061] The sequencing instrument may include one or more power sources and / or power supply modules (including, for example, an AC mains input and AC / DC conversion and regulation unit), robotic arms, gantries (e.g. moveable gantries), printed circuit board assemblies (PCBA). computer processors (e.g. one or more FPGAs, ASICs, artificial intelligence chips, PIM devices, GPUs and / or central processing unit (CPU)s), and memories, or any combination thereof, so as carry out the processes described herein. For example, the processor of the sequencing instrument may be configured to (or programmed to) control the fluidic system, the circuitry, the power sources and / or power supply modules, the robotic arms, the moveable gantries and / or the thermal means of the sequencing instrument.

[0062] The sequencing instrument may also provide one or more user interfaces (for example provided on a display screen or tablet, preferably as a touchscreen) for the customisation, initiation and monitoring of the sequencing workflow, along with any suitable means known in the art of providing connectivity to one or more computer processors and / or memories remote from the sequencing instrument.PATENTAttomev Docket No.: P39047-WO

[0063] The consumable device, which is configured to operate with the sequencing instrument, comprises one or more nanopore sensor chips and one or more flow channels (in other words, one or more fluidic channels or fluidic lanes). Preferably, the consumable device comprises one or more flow cells comprising the one or more flow channels.

[0064] The flow cell(s) may further comprise one or more counter electrodes, preferably one, that oppose the respective working electrode of the individual cells of the array of cells in the nanopore sensor chip(s). When the consumable device is engaged in the sequencing instrument, the counter electrode(s) may be positioned at the top of the flow channel, in other words opposite to the working electrode and the well, thus providing a ceiling to the flow cell and flow channels therein. The counter electrodes may also be positioned in alternative orientations. The consumable device (and more specifically, the flow cell of the consumable device) may comprise one or more substantially parallel, or parallel, flow channel(s), for example one, two, three, four, five, six, seven, eight, nine or ten, preferably two to four, more preferably four. Alternatively, or additionally, the consumable device may comprise one or more serpentine flow channels, preferably one or two. Each flow channel functions to direct fluids or gas to flow across the nanopore sensor chip (and thus the array of cells). In other words, the flow cell and / or flow channels therein allow liquids and gases to pass over and contact the cells (i.e. the individual sensors). Accordingly, each flow channel can be connected (directly or indirectly fluidically connected) to one or more inlets (e.g. inlet port), one or more outlets (e.g. outlet port) and one or more fluidic pumps. Preferably, each flow channel is connected to its own inlet and its own outlet. The flow cell and flow channel(s) therein can be fluidically sealed by any suitable means known in the art. The flow cell’s primary functions are to enable the fluidics across the nanopore sensor chip(s) in a sealed manner and to provide a connection to the counter electrode.

[0065] The consumable device may further comprise a printed circuit board, or equivalent, that functions to route the nanopore sensor chip(s) signals to the sequencing instrument, via electrical connectors. Accordingly, the nanopore sensor chip(s) may be embedded in, on and / or electrically connected to, the printed circuit board. The consumable device may further comprise electrical connections (e.g. leaf connections) from the counter electrode of the flow cell to the printed circuit board.

[0066] The consumable device is preferably removable from the sequencing instrument thus allowing for efficient replacement of the consumable device. The consumable may be in the form of a cartridge or disc. The removable consumable device is preferably copulable (i.e. able to be coupled) to the sequencing instrument and the nanopore sensor chip, flow channel.PATENTAtorney Docket No.: P39047-WO storage device, ADC and / or counter electrode are preferably housed in the removable consumable device.

[0067] As will be appreciated by a person of ordinary skill in the art, one or more of the components of the consumable device may alternatively be included on the sequencing instrument, and vice versa.

[0068] As will be further appreciated by a person of ordinary skill in the art, one or more of the components of the consumable device may alternatively be included in a reusable device. As such, the term consumable device can be replaced herein, in any and all instances, by reusable device.Nanopore Sensor Chip

[0069] The consumable device encloses or houses one or more nanopore sensor chips. A nanopore sensor chip has a large number (for example, thousands or millions) of autonomously operating sensor cells configured as an array. Each cell is configured to sequence a molecule, such as a polymer, e g. a polynucleotide or a polypeptide. The array of cells are made up of columns of cells (for example, numbered column 1 to column m) and rows of cells (for example, numbered row 1 to row n). In some examples, the nanopore sensor chip may have an array of 2M cells (2,097,152 cells) with, for example, 512 rows with 4096 columns of cells. In some examples, the nanopore sensor chip may have an array of 8M cells (8,388,608 cells) with, for example, 2048 rows with 4096 columns of cells. These 8M cells may be in the form of four parallel flow channels of 2M cells (512 rows with 4096 columns). Alternatively, the 8M cells may be in the form of one serpentine flow channel of 8M cells. Each cell in the individual flow channel may receive the same analyte for characterization (and the same reagents) as the other cells in the same flow channel. The cells in the individual flow channels may be separated by side walls so that each flow channel has the ability to receive a different analyte for characterization, without cross-contamination. Each analyte may comprise one or more patient samples for characterization. Each cell of the array is configured to have a membrane and a nanopore. As such, the cell can be considered a nanopore cell.

[0070] In some embodiments, all cells of the array of cells comprise a membrane, and all cells with their respective membranes comprise a nanopore embedded within that membrane. However, due to a plurality of reasons (which would be known to a person of ordinary skill in the art), not all cells of the array of cells may have a membrane and not all cells with a membrane may have a nanopore. For example, approximately less than 10%, approximately less than 5% or less than 2% of cells in the array of cells may not have a membrane or a nanopore. A cell with a membrane and a nanopore embedded in that membrane will be referredPATENTAtorney Docket No.: P39047-WO to herein as a “working cell”, whereas a cell without a membrane or a cell without a nanopore embedded in a membrane will be referred to herein as a “non-working cell”.

[0071] Accordingly, one of the steps in the sequencing workflow may involve establishing which individual cells in the array of cells are working cells and which individual cells are nonworking cells, using any suitable technique known in the art, for example using the technique as described in WO2017 / 167809A1.

[0072] An AC voltage waveform can be applied between the working electrode and the counter electrode and an electrical measurement can be periodically taken in response to monitor membrane formation and pore insertion. A variety of electrical measurements can be used to detect membrane formation. For example, the electrical measurement can be current, which will be highest when no membrane is present, lowest when a membrane covers the well, and an intermediate value when a pore is inserted into the membrane. Alternatively, the electrical measurement can be resistance, which is inversely correlated with current. Yet another electrical measurement that can be used is a voltage measurement (i.e., a voltage change) from an integrating capacitor as further described herein, which can be similarly correlated with membrane formation.

[0073] After the membrane has been formed over the well, nanopore insertion can be initiated. When the membrane covers the well without a nanopore, it blocks ion flow between the electrodes and thus causes a low current, or high resistance, or a low change in voltage across the integrating capacitor. Once a nanopore is successfully inserted into the membrane, ions can flow through the nanopore, which results in a higher current reading, or a lower resistance reading, or a greater change in voltage measured across the integrating capacitor. Electrical measurement thresholds (i.e., current, resistance, voltage, etc.) can be set using empirical data to identify the different states of the cell (i.e., uncovered, covered by membrane without a nanopore, or covered by a membrane with a nanopore).

[0074] So as to cany7out such analysis, and further data analysis and chip control, the array of cells of the nanopore sensor chip(s) are electrically connected to one or more of a memory', a GPU, a CPU, a FPGA, an application-specific integrated circuit (ASIC), a data converter and a high-speed I / O interface.

[0075] A further step in the sequencing workflow may involve switching off the nonworking cells from the voltage source(s) (e.g. the AC voltage source) and utilizing only the working cells in the subsequent sequencing data acquisition phase.CellsPATENTAtorney Docket No.: P39047-WO

[0076] Each cell of the array of cells can include a well containing a volume of electrolyte, a membrane formed over, and spanning across, the well and a sample chamber on the membrane and separated from the well by the membrane. The sample chamber may hold a bulk electrolyte (which may comprise a nanopore) and the analyte of interest. The cell can include a working electrode at the bottom of the well and a counter electrode disposed in the sample chamber. A signal source can apply a voltage signal between the working electrode and the counter electrode. The membrane includes a trans side (i.e. the side facing the working electrode) and a cis side (i.e. the side facing the counter electrode). The volumes enclosed on the opposite sides of the membrane can therefore be referred to as the cis well and the trans well, with species to be sequenced moving from cis to trans during detection. The trans well is generally the side of the membrane proximal to and electrically connected to the working electrode.

[0077] The cell can be formed on a substrate (e.g. a silicon substrate, such as a silicon wafer). One or more dielectric layers may be formed on the substrate and an electric circuit may be formed on or within the substrate and / or on or within the dielectric layer(s). The working electrode can be formed on the uppermost (i.e. the layer furthermost from the silicon substrate) dielectric layer and can form at least a part of the bottom of the well. The walls of the well (in other words, the walls surrounding the well) may be formed on the dielectric layer and may be formed of any suitable material, such as a dielectric.

[0078] Suitable dielectrics include glasses, oxides, nitrides (e.g. silicon mononitride (SiN), polyimides, or photoresist polymers e g. a hydrophobic photoresist polymers).

[0079] The well includes a volume of electrolyte above the working electrode. The volume of electrolyte may be buffered and may include one or more of the following: ammonium chloride (NEUCl), ammonium glutamate (C5H12N2O4). 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 (SrCk), manganese chloride (MnCh), and magnesium chloride (MgCh).

[0080] The membrane spanning across the well (and formed on top of the well walls) can be made from any suitable material known in the art, or any combination of suitable materials known in the art. The membrane can be an organic membrane, such as a lipid bilayer (for example, spanning across the opening of the well), or a synthetic membrane, such as a membrane formed of a polymeric material. The membrane may also include a lipid monolayer, optionally formed on top of hydrophobic layer (for example on top of the well walls).PATENTAttomev Docket No.: P39047-WO

[0081] The lipid monolayer or bilayer can comprise or consist of lipids, such as a phospholipid, for example, selected from diphy tanoyl-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(poly ethylene glycol)-350], 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-550], 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-750], 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-! 000], 1 ,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene 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. DSPA), 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. Alternatively, the membrane 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 examples, the membrane can be formed from a combination of lipid based materials and non-lipid based materials. In some examples, 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). Preferably, the membrane is either a phospholipid or a tri-block co-polymer or a mixture thereof.

[0082] One or more nanopores can be inserted into the membrane by any suitable process known in the art (e.g. electroporation). In a preferred arrangement, only one, single nanopore is embedded in the membrane for a given cell. Although, it is contemplated that more than one nanopore could be inserted into the membrane.PATENTAtorney Docket No.: P39047-WO

[0083] The nanopore can be considered a pore, hole, channel or passage formed (or otherwise provided) in a membrane (e.g. an electrically insulating membrane). The nanopore can be used as a single-molecule detector. A nanopore may be formed using a solid-state material, such as a semiconductor material or alternatively the nanopore may be formed using a biological material, such as a-hemolysin, MspA or other known alternatives. The nanopore may be a protein nanopore, in other words, the nanopore may be formed from a protein, such as a soluble protein nanopore transmembrane molecular complex (PNTMC) or other known alternatives. The nanopore has a pore size in the nanometre range and must be large enough for passing at least a portion of the analyte of interest and / or small ions (e.g., Na+, K+, Ca2+, CT) through the membrane. In some examples, a nanopore has a characteristic width or diameter (e.g. a mean internal width or diameter) in the order of 0.1 nanometers (nm) to about 1000 nm (preferably approximately 1 nm), as measured by scanning electron microscopy or crystallographic techniques.

[0084] Each cell in the array of cells can be considered an independent sequencing entity. Each cell produces a unique signal associated with the analyte of interest and specific to the individual membrane and nanopore of the cell.

[0085] The sample chamber over the membrane functions to hold a solution of the analyte of interest for characterisation. This solution preferably comprises an aqueous solution comprising bulk electrolyte, and may comprise an ionic solution, wherein the ionic solution comprises a redox couple comprising a first species of the redox couple and a second species of the redox couple, such as ferrocyanide and ferricyanide. The solution may comprise one or more of ammonium chloride (NEUCl), ammonium glutamate (C5H12N2O4), 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 (SrCb), manganese chloride (MnCh), and magnesium chloride (MgCh). The solution is further buffered to an optimum ion concentration and maintained at an optimum pH to keep the nanopore open. The nanopore crosses the membrane and embeds in the membrane, and thus provides a path for ionic flow from the bulk electrolyte to the working electrode. The volume of electrolyte in the well (above the working electrode) may be isolated from the solution of the analyte of interest (held in the sample chamber) by the membrane, which in comparison to the nanopore allows negligible transport of ions between the cis and trans sides of the membrane.

[0086] The working electrode of an individual cell is independent from the working electrode of other cells and thus can be referred to in the art as a cell electrode.PATENTAtorney Docket No.: P39047-WO

[0087] The counter electrode can be shared between individual cells of the array of cells and thus can be referred to in the art as a common electrode. The counter electrode may apply a common potential to the bulk electrolyte in contact with the nanopore. The counter electrode may be capable of forming an electrical circuit with the working electrode of each cell. The common electrode is preferably housed in the flow cell of the consumable device.

[0088] The working electrode and / or counter electrode may be a metal electrode, such as, for example, platinum, gold, titanium nitride, ruthenium nitride and graphite.

[0089] The counter electrode and working electrode may be coupled to one or more signal sources for providing electrical stimulus (e.g., voltage bias) across the membrane, and can be used for sensing electrical characteristics of the membrane (e g., resistance, capacitance, voltage and ionic current flow). Preferably, in the system of the present disclosure a voltage is applied between the working electrode and the counter electrode.

[0090] In some arrangements, the common potential and the common electrode can be common to all cells, or a proportion or grouping thereof.

[0091] Each cell of the array of cells has its own electrical circuit (e.g. made up of wet circuit and dry circuit elements) and may share this electrical circuit, or parts thereof, with one or more other cells. For example, one or more cells may share a storage device (e g. a memory bit), a capacitor (for example, shared across a row7or column of the array of cells), a voltage source and / or an ADC (all examples of dry circuit elements) and / or one or more cells may share the counter electrode of the wet circuit elements. In other words, each cell has, or shares, a respective control circuit / electrical circuit integrated on a substrate (e.g. silicon substrate) of the nanopore sensor chip and / or within one of the dielectric layers formed on the substrate. Such an electrical circuit controls electrical stimulation across the membrane of the cell and detects and processes the electrical signals from the cell. In other words, the electrical circuit of a cell (or one or more cells) detects and processes electrical patterns of a molecule passing through the nanopore embedded in the membrane of the cell. The electrical circuit may comprise a plurality of patterned metal layers, a plurality of active devices (e.g., transistors, amplifiers, integrators, analog-to-digital converters (ADC), noise filters, feedback control logic), a plurality of voltage sources and / or various other components. The electrical circuit can be coupled to one or more processors, where those one or more processors may be coupled to one or more memories, where the processor functions, amongst other things, to analyse the sequencing data to determine sequences of the polymer molecules.

[0092] FIG. 2 is a cross-sectional view of an array of cells 200 in an example nanopore sensor chip. FIG. 2 shows a plurality of cells in a row or a column of array of cells 200. AsPATENTAtorney Docket No.: P39047-WO described above, each cell includes an electric circuit 222 integrated on silicon substrate 230 and / or dielectric layer(s) 201 of the nanopore sensor chip. Each cell includes a respective well 205 formed by dielectric layers 201 and 204 and working electrode 202 at the bottom of well 205. Well 205 can hold the volume of electrolyte 206. A membrane 214 is formed on dielectric layer 204 and covers each well 205. Membrane 214 includes a nanopore 216 on top of each well 205. The nanopore 216 may be inserted on the cis side of the membrane (as exemplified) or alternatively inserted on the trans side of the membrane. Sample chamber 215 on top of membrane 214 is configured to hold bulk electrolyte 208, which includes the molecules to be analysed. A molecule 270 to be analysed is docked on nanopore 216. FIG. 2 also illustrates side walls 236 to separate groups of cells such that each group may receive a different sample for characterization and a cover plate 250 that encloses sample chamber 215.

[0093] Counter electrodes 210 from different cells are disposed in sample chamber 215 and are connected to voltage source 228 for applying a common VLIQ to the cells, or a proportion thereof. Counter electrodes 210 for different cells may be, and are preferably, physically connected to each other to form one common electrode / counter electrode (as exemplified in FIG. 3). Working electrodes 202 of different cells are connected, via one or more switches (not shown), to one or more common voltage sources for applying a precharging voltage VPRE (e.g. the AC voltage that is applied to the integrating capacitor for precharging the capacitor, as exemplified in FIG. 4). This voltage source may be a different voltage source (not shown) to voltage source 228. Electric circuits 222 of different cells are connected to a bus 260, and the voltage level on the capacitors (e.g. integrating capacitors) of the different cells are sequentially read out through bus 260 by sequentially selecting the different cells.

[0094] FIG. 3 is a cross-sectional view of two individual cells 301 and 302 in an array of cells in an example nanopore sensor chip. Each cell includes an electric circuit 222 (as exemplified in FIG. 2) which together form, along with other shared electrical components, a general integrated circuit 322 for all of the cells in the array of cells. Each cell includes a respective well 305 formed by cladding 320 (e.g. dielectric layers 201 and 204 of FIG. 2) and working electrode 303 at the bottom of well 305. Well 305 holds the volume of electrolyte 306. A membrane 314 spans across the opening of the well 305. A nanopore (i.e. pore) 316 is shown as embedded in the membrane 314 in a blowup of the nanopore region of the right hand working cell 302 of the figure, and an Xpandomer molecule 317 is illustrated as translocating through this nanopore 316. Sample chamber 315 located on top of membranes 314 holds the molecules to be analysed in solution, in this specific example, the molecules being Xpandomer molecules. A counter / common electrode 310 is disposed in sample chamber 315 and a potentialPATENTAtorney Docket No.: P39047-WO difference is applied between counter electrode 310 and working electrode 303. FIG. 3 further illustrates the parts of the wet circuit elements by way of the top dotted and dashed rectangle and parts of the dry circuit elements of cell 301 by way of the bottom dotted and dashed rectangle.Circuitry

[0095] FIG. 4 illustrates an example of an electrical circuit 400 for a cell, such as cells 200 as exemplified in FIG. 2 or cell 301 or 302 as exemplified in FIG. 3. This electrical circuit 400 may include portions or components of the electrical circuit discussed above with reference to FIG. 2 and FIG. 3. Electrical circuit 400 has a membrane (preferably a phospholipid and / or triblock co-poly mer membrane) 412 situated between the cell’s working electrode 414 and a counter electrode 416 (that is shared between a plurality of cells or all cells in the array of cells), such that a voltage is applied across the membrane 412. The membrane 412 is also in contact with a bulk liquid / electrolyte 418. Note that working electrode 414, membrane 412, and counter electrode 416 are drawn upside down as compared to the working electrode, membrane, and counter electrode in FIG. 2 and FIG. 3. The counter electrode is configured to apply a common potential to the bulk liquid in contact with the membranes in the cells by connecting the common electrode to a voltage source VLIQ 420. The common potential and the counter electrode are common to all of the cells.

[0096] Switch 406 is a transistor that can be used to connect or disconnect the membrane 412 and the working electrode 414 from the rest of the electric circuit 400. Switch 406 is controlled by a memory bit 402 to enable or disable a voltage stimulus to be applied across the membrane in the cell.

[0097] Electrical circuit 400 includes an on-chip capacitor e.g. integrating capacitor Cint 408 (map), which functions to measure the voltage of the working electrode. Integrating capacitor Cint 408 may be pre-charged by using a reset signal 403 to close switch 401 (the “further switch” of the present disclosure), such that integrating capacitor Cint 408 is connected to a voltage source VPRE 405. When switch 401 is closed, integrating capacitor Cint 408 may be pre-charged to a voltage level of voltage source VPRE 405. Switch 401 may be considered as a precharge switch that functions to make and break a connection to VPRE in order to selectively apply the AC voltage to pre-charge the integrating capacitor Cint 408. An additional switch (not shown) may be provided to selectively deliver the voltage on the integrating capacitor Cint 408 to the ADC. 410.

[0098] After integrating capacitor Cint 408 is pre-charged, reset signal 403 may be used to open switch 401 such that integrating capacitor Cint 408 is disconnected from voltage sourcePATENTAttorney Docket No.: P39047-WOVPRE 405. At this point, depending on the level of voltage source VLIQ, the potential of counter electrode 416 may be at a level higher than the potential of working electrode 414 (and integrating capacitor Cint 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 examples, integrating capacitor Cint 408 may 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 416 and working electrode 414. In other examples, the charging and discharging may occur in dark periods and bright periods, respectively.

[0099] Integrating capacitor Cint 408 may be charged or discharged for a fixed period of time, as chosen by the user and dependent on conditions. The period of time may be, for example, about 0. 1 to 2ms, preferably about 0.2 to 1ms, more preferably about 0.5 to 0.7ms. The voltage level may be sampled and converted by ADC 410 at the end of the integration period. A particular voltage level would correspond to a particular molecule (e.g. reporter code or tag species) in the nanopore, and thus correspond to the nucleotide at a current position on the template.

[0100] After being sampled by ADC 410, integrating capacitor Cint 408 may be pre-charged again by using a reset signal 403 to close switch 401, such that integrating capacitor Cint 408 is connected to voltage source VPRE 405 again. The steps of pre-charging integrating capacitor Cint 408, waiting for a fixed period of time for integrating capacitor Cint 408 to charge or discharge, and sampling and converting the voltage level of integrating capacitor by ADC 410 can be repeated in cycles throughout the sequencing process. The same steps of pre-charging integrating capacitor Cint 408, waiting for a fixed period of time for integrating capacitor Cint 408 to charge or discharge, and sampling and converting the voltage level of integrating capacitor by ADC 410 can also be applied when VPRE is provided as a constant voltage with VLIQ provided as an AC voltage or alternatively when both VPRE and VLIQ are provided as AC voltages. Additionally, the same steps can be utilized when the voltage is not a square wave (as exemplified in FIG. 5A and FIG. 5A) but may resemble a summation of square waves each of the same modulation period but which may have varied duty cycle and phase delay with respect to the start of the modulation period.PATENTAtorney Docket No.: P39047-WO

[0101] 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 cells into various data frames. In some examples, digital processor 430 can perform further downstream processing, such as base determination. Digital processor 430 can be any suitable hardware (e.g., a GPU, CPU, FPGA, ASIC). Digital processor 430 can be present in the nanopore sensor chip of the consumable device, in or on the printed circuit board of the sequencing instrument, in the sequencing instrument or may be held remote from the sequencing instrument.

[0102] 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 open-channel state when a molecule (e.g. a reporter code or tag species) is absent from the barrel of the nanopore. Other states of the nanopore can correspond to when tags or reporters are held in the barrel of the nanopore.

[0103] When the voltage level on integrating capacitor Cint 408 is measured after a fixed period of time, the different states of a nanopore may 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 Cint 408 (i.e., the steepness of the slope of a voltage on integrating capacitor Cint 408 versus time plot) depends on the nanopore resistance. More particularly, as the resistance associated with the nanopore in different states is different due to the molecules’ distinct chemical structures, different corresponding rates of voltage decay may be observed and may be used to identify the different states of the nanopore. The voltage decay curve may be an exponential curve with an RC time constant r = RC, where R is the resistance associated with the nanopore and C is the capacitance associated with the membrane, in parallel with R. A time constant of the cell can be, for example, about 200-500 milliseconds. The decay curve may not fit exactly to an exponential curve due to the detailed implementation of the membrane, but the decay curve may be similar to an exponential cune and is monotonic, thus allowing detection of molecules.

[0104] The rate of the decay of the voltage on integrating capacitor Cint 408 may be determined in different ways. As explained above, the rate of the voltage decay may be determined by measuring a voltage decay during a fixed time interval. For example, the voltage on integrating capacitor Cint 408 may be first measured by ADC 410 at time tl, and then the voltage is measured again by ADC 410 at time t2. The voltage difference is greater when the slope of the voltage on integrating capacitor Cint 408 versus time curve is steeper, and the voltage difference is smaller when the slope of the voltage curve is less steep. Thus, the voltagePATENTAttomev Docket No.: P39047-WO difference may be used as a metric for determining the rate of the decay of the voltage on integrating capacitor Cint 408, and thus the state of the cell.

[0105] 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 lOOOmV, 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).

[0106] 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, anyPATENTAtorney Docket No.: P39047-WO molecule (e.g. tag or reporter 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.

[0107] 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.Control of voltage across the nanopore on a cell-by-cell basis during the sequencing data acquisition phase

[0108] Due to the need for microminiaturisation of the nanopore sensor chips involved in the present disclosure, often each cell of the array of cells does not have its own AC voltage source, e.g. precharge voltage source VPRE, or its own individual voltage line to one or more AC voltage sources. Instead, the array of cells, or a sub-population thereof (e.g. sub-arrays, banks or subbanks), usually share one or more (preferably two) AC voltage sources (e.g. precharging voltage sources VPRE(S)). Further, each AC voltage source may have one or more (for example two) AC voltage lines (e.g. precharging voltage lines), where each AC voltage line is shared by sub-populations of the array of cells.

[0109] As such, the AC voltage supplied to each cell in the array of cells may be by way of one or more electrical lines (e.g. precharging voltage lines) that supply all cells of the array of cells, or a proportion thereof. One or more AC voltage lines can therefore be shared across sub-populations of cells in the array of cells (herein referred to as "‘global lines”). Accordingly, the system of the present disclosure (and associated method) preferably comprise one or more shared AC voltage line(s) which electrically couple(s) the working electrode of each cell of the second sub-set of cells (targeted working cells) to the AC voltage source. As a result, the AC userwave utilized in a specific sequencing data acquisition phase may be shared between one or more working cells (or sub-sets) of the array of cells.

[0110] However, given the fact that the circuits of the present disclosure employ biochemical circuit elements (for example lipid bilayer membranes and nanopores), and that sequencing processes in general are stochastic in nature, variability in electrical behaviors across the individual cells in the array of cells is common. This is because, as a sequencing run evolves, several important quantities or characteristics may change on a per cell basis largelyPATENTAttomev Docket No.: P39047-WO independently of one another, and in response to both shared and independent stimuli, which can then impact the voltage across the nanopore and electrochemical impedance of an individual cell. The impact of these changes may affect both the bright and dark periods, and can be problematic for the subsequent accurate base-calling and sequence determination. Additionally, the impact of these changes may lead to an undesired sequence of biophy sical events in that particular cell. Due to the variability in the biochemical circuit elements of each cell and the variability in the external stimuli that the individual cell may experience, when the array of cells is viewed in aggregate, both the mean and variance of the array’s set of characteristics, e.g. electrical characteristics, change. One adaptive protocol to partially compensate for cells’ behaviour drifts over time is to modify the AC userwave settings globally. With such an approach, the population’s mean value for a specific quantity or characteristic can be adjusted, however, it is generally not possible to tighten the distribution, prevent it from spreading further or to target specific characteristics or behaviours of individual cells.

[0111] However, the inventors have discovered a novel and advantageous mechanism of dynamically addressing the voltage needs of a working cell on an individual basis during the sequencing data acquisition phase of the sequencing workflow. In some embodiments, this dynamic process involves disconnecting (and subsequently reconnecting) the working electrode of each desired working cell from the global line(s) during a (one or more) single AC modulation period for a given period of time. Some embodiments thus allow for greater control over the voltage schedule applied to individual cells and thus provides the ability to control the distribution of cell characteristics or behaviours in addition to the population mean. For example, the distribution of cell behaviours may be shifted to target the upper third and / or the bottom third of a distribution curve of behaviours to bring them in line with the middle third of behaviours.

[0112] The time that the working electrode of each targeted working cell is disconnected from the AC voltage source during a single AC modulation period (i.e. the time between disconnection and reconnection to the global line(s)) can be any time that is less than a single AC modulation period. As a person of ordinary skill in the art would understand, the specific disconnection time will be dependent on the length of time of the single AC modulation period. Such single AC modulation periods may be any suitable time as set by the user and can typically range from about 1 second to 10 seconds. The disconnection time (i.e. the time between disconnection and reconnection) in a single AC modulation period may be any value between about 5 milliseconds (ms) to 2 seconds (s), preferably about 50ms to 1.5s, morePATENTAttorney Docket No.: P39047-WO preferably about 100 ms to Is. The disconnection time may be a percentage of the time of the single AC modulation period. For example, the working electrode of each cell in the targeted sub-set / sub-population may be switched off from the dry circuit elements and AC voltage source (e.g. VPRE) for about 1 to 99% of the single AC modulation period, preferably about 10 to 90% of the single AC modulation period, more preferably less than about 50% of the single AC modulation period. For example, the working electrode of each cell in the targeted sub-set may be switched off from the dry circuit elements and AC voltage source (e.g. VPRE) for about 1 to 50%, preferably about 2 to 20%, more preferably about 5 to 10% of the time of a single AC modulation period. In other words, the targeted sub-set of cells may only be active / enabled for about 50 to 99%, preferably about 80 to 98%, more preferably about 90 to 95% of a single AC modulation period. The rest of the working cells in the array of cells (i.e. the working cells not in the targeted sub-set) will receive a voltage to the working electrode about 100% of the time of the single AC modulation period or of the time of the sequencing data acquisition phase. Alternatively, the working electrode of each cell of the targeted sub-set of cells may be switched off from the dry circuit elements and the AC voltage source for about 1 to 99% of the dark period, for example about 1 to 80% of the dark period, preferably 1 to 50% of the dark period. The disconnection during the dark phase advantageously minimizes the disruption to useful sequencing data acquisition. Alternatively, the working electrode of each cell in the targeted sub-set may be switched off from the dry circuit elements and the AC voltage source for. for example, 1 to 20% of the time of the sequencing data acquisition phase. One or more of the targeted sub-set of working cells may be disconnected and reconnected more than once, for example for a plurality7of times, in a single AC modulation period. As such, the percentage of disconnection time may be considered the total time of disconnection for one or more disconnections in the same AC modulation period. Accordingly, the present disclosure also provides for the processor being further programmed to reconnect the working electrode of each cell of the second sub-set of cells to the AC voltage source during the same single AC modulation period, to disconnect and, subsequently, reconnect the working electrode of one or more cells of the second sub-set of cells for one or more further times during the same single AC modulation period and / or to disconnect the working electrode of one or more of the cells of the second sub-set of cells from the AC voltage source during a further single AC modulation period followed by reconnecting the working electrode of one or more of the cells of the second sub-set of cells to the AC voltage source during the same further AC modulation period.

[0113] Some embodiments of the present disclosure can thus be considered as involving the targeted tuning of the AC voltage experienced by an individual cell by superimposing a highPATENTAtorney Docket No.: P39047-WO frequency duty cycle (i.e. high frequency switching cycle), during one or more single AC modulation periods, for one or more individual working cells that share an AC voltage line and associated AC userwave.

[0114] Preferably, the disconnection (and subsequent reconnection) of the working electrode of each targeted working cell from a shared AC voltage source and global lines(s) during a single AC modulation period is carried out during the dark phase. This causes the desired, targeted, working cell to re-charge at a different amount (depending on disconnection time) to the working cells that have not been targeted and have thus remained connected to the global line(s) and AC userwave(s) for one hundred percent of the single AC modulation period. Advantageously, this allows a correction to be applied (e.g. by the processor and associated software) to the behaviour of each cell of the targeted working cells and / or an intervention to be enabled for each cell of the targeted working cells, whilst minimizing the disruption to useful sequencing data acquisition.

[0115] The decision of which w orking cells should be enabled or disabled, and for how long, may be made in real time or quasi real time by one or more processors, such as a FPGA and / or a GPU following analysis, by one or more processors, of each working cells’ (e.g. each targeted sub-set of working cells’) data.

[0116] The disconnection of a cell’s working electrode from the dry circuit elements and its AC voltage source is achieved by utilizing a switch (e.g. switch 406 of FIG. 4), which is controlled by a storage device, more specifically a state storage device such as one or more memory bits 402. The switch can be any suitable means of (or device for) disconnecting and reconnecting a cell’s w orking electrode from a voltage stimulus, such as an AC voltage source. Preferably, the switch is a transistor which is activated by the application of a high or low voltage to open or close so as to disconnect or connect, respectively, the working electrode from the dry circuit elements (and the AC voltage source). The activation of the switch is controlled by a storage device that can be in one of two stable states represented by a single binary value, usually a “0 or 1’' (alternatively a “no or yes’’ or “off or on”) Thus, in the context of the present disclosure, the storage device is configured to represent either an open state or a closed state for the switch. The storage device can thus be considered a bit of memory that can represent the state of the switch in an open or closed state. Preferably each cell of the array of cells has its own. dedicated, storage device. Preferably, the storage device is one or more memory bits, more preferably one memory bit.

[0117] A memory bit (e.g. memory bit 402) may be an arrangement of transistors that operate in conjunction with the switch (e.g. switch 406) to set the switch to open or closed. Preferably,PATENT Atorney Docket No.: P39047-WO the storage device (e.g. memory bit) is made up of four transistors, operating as two inverters “tied” in a circle, with two further current providing transistors that function to flip the state of the inverters. Accordingly, preferably each cell of the array of cells has a storage device comprising six transistors.

[0118] The storage device is preferably part of the cell’s individual circuitry and is further preferably located adjacent to the working electrode of the cell, i.e. within the integrated circuit sub-unit for each cell. Alternatively, the storage device may reside outside of each individual cell’s circuitry and instead reside within circuitry shared by one or more cells of the array of cells. Accordingly, one storage device may control one or more switches. This may advantageously provide for a higher packing density within the nanopore sensor chip.

[0119] The state of the storage device (e.g. memory bit) of each cell may be updated in accordance with a map of Boolean values (e.g. a “0 or 1”) sent by a processor, such as a FPGA or GPU, or any other suitable means for performing various digital functions and which can be programmed and reprogrammed.

[0120] The map of Boolean values, known herein as the Active Bit Map (ABM) (or alternatively active bit mask) provides the instructions for which cells should be connected to the dry circuit elements and the global line(s) and thus dictates, with the aid of the cell switch, which cells are connected, or disconnected, from the dry7circuit elements and respective AC voltage source (and associated AC voltage line(s)) at a given moment in time. The ABM may be viewed as a virtual representation of the physical array of cells of the nanopore sensor chip indicating the binary state of each cell of the array of cells, where the state is either enabled or disabled. In other words, the state of each cell in the ABM is virtually represented as either a 1 (enabled / activated) or a 0 (disabled / deactivated), which subsequently results in the phy sical cell either receiving an AC voltage or not receiving an AC voltage, respectively, as exemplified in Table 1.PATENTAtorney Docket No.: P39047-WOTable 1

[0121] The use of such an ABM allows the dynamic updating of the state of the storage devices in the array of cells in order to change the active status of working cells during the sequencing data acquisition phase and thus manipulate the voltage across the nanopores of individual, targeted cells within the array. Thus, preferably, the ABM is continually updated following analysis of each working cell’s data.

[0122] The system and method of the present disclosure may use one or more ABMs. For example, two or more ABMs may be utilized and switched between with the intention of affecting a change to a targeted sub-population of cells in the array of cells. For example, two or more ABMs may be switched between in a single AC modulation period so as to disconnect and subsequently reconnect the working electrodes of the targeted cells. In the same way, more than two ABMs (for example three to twenty) may be utilized to affect further disconnections and reconnections in the same single AC modulation period or in subsequent single AC modulation periods.

[0123] One or more ABMs may be applied during the bright period or dark period of the sequencing data acquisition phase. Preferably, one or more ABMs are applied during the dark period, advantageously minimizing the disruption to useful sequencing data acquisition. In one example, two or more ABMs can be utilized to adjust how long different working cells are exposed to the same dark period AC voltage. Alternatively, during the dark period, two or more ABMs may be utilized, and synchronized with the AC userwave, to specify segments of time where different AC voltages are applied globally, with different working cells connected to the global lines during those segments of time. The ABMs of the present disclosure may also be synchronized with one or more AC waveforms in an AC userwave that are applied to one or more sub-sets of working cells.

[0124] In a preferred example of the present disclosure, the ABM may be updated to provide a new, updated virtual representation of the array of cells following analysis of the data received from the ADC. Accordingly, the present disclosure may involve the use of an instructing ABM which is continually updated by one or more further ABMs. In such an arrangement, the one or more further ABMs may be considered as the ABMs that are created after analysis of the data being received from the working cells (more specifically, the ADC of the working cells)PATENTAttorney Docket No.: P39047-WO and that are used to update the instructing ABM. The instructing ABM may be considered as the ABM that instructs the storage devices (e.g. memory bits) of the targeted working cells (i.e. the second sub-set of cells). Preferably, the instructing ABM is continually updated by one or more further ABMs during the sequencing data acquisition phase.

[0125] These one or more further ABMs could be created during the sequencing process itself, based on, for example, indicative data acquired and processed during the sequencing data acquisition phase, for example earlier in the sequencing data acquisition phase or earlier in the sequencing workflow. One or more further ABMs could be created and applied in real time and “on-the-fly” In other words, the ABMs can be created following real-time analysis of the working cell data. Thus, the instructing ABM may be continually and iteratively updated from the analyzed data, and then subsequently updated by one or more further ABMs that have been created from this analyzed data. Advantageously, this provides for the ability of the instructing ABM to adapt in real time to measurements taken earlier on in the sequencing run, thus allowing the sequencing data acquisition phase to adapt to real time stimuli.

[0126] Alternatively, the one or more further ABMs may be pre-programmed ABMs, e.g. pre-programmed to take account of previous analysis of indicative data (for example, preprogrammed before the sequencing data acquisition phase begins or pre-programmed before a specific sequencing workflow). In other words, the one or more further ABMs may be predetermined spatial patterns and these fixed patterns can be applied in a regimented series of timings (for example by timing their application to specific points in the AC userwave). Advantageously, this allows the user to take account of known behaviours and known variabilities of the nanopore sensor chip.

[0127] The working cell indicative data may indicate that one or more working cells are drifting in their behaviour away from a threshold value or a set of predefined operating parameters and / or that one or more of the working cells needs some kind of intervention. The analysis of the data, e.g. indicative data, received from the ADC of the working cells may be carried out by any suitable processor, such as a GPU or FPGA, utilizing suitable algorithms or logic design. Such processors may be programmed to further analyse such data against a given threshold value or some other predefined operating parameter(s) or condition. The processor may be further programmed to identify any working cells that are operating above or below a threshold value or outside predefined operating parameters and thereafter target such identified cells for a particular intervention, for example to bring their behaviour back to the threshold value (or above or below it) or within the predefined operating parameters, and for example, back in line with the behaviours of the other working cells in the array of cells. Accordingly,PATENTAtorney Docket No.: P39047-WO the instructing ABM may be updated by the processor utilising the further one or more ABMs. For example, the processor may be programmed to undertake a delta analysis between the binary state of each virtual cell of the further ABM and the binary state of each respective virtual cell of the instructing ABM. When a delta is identified between the two respective states, the processor may be programmed to update the necessary binary state of the virtual cell in the instructing ABM. When a delta is not identified, then the processor may be programmed to take no action for that particular virtual cell in the instructing ABM (i.e. the binary state of the virtual cell in the instructing ABM stays the same).

[0128] As a specific example, if the processor identifies that virtual cell X (at e.g. virtual column 5 and row 200) in the further ABM should have a state of 0, for example after the data from that physical cell X (at physical column 5 and row 200) indicates that it requires an intervention and needs to be disconnected from the AC voltage source, and also identifies that the same virtual cell (virtual cell X (at virtual column 5 and row 200)) in the instructing ABM has a state of 1, then a delta is identified and a further action is taken. This further action involves changing the state of the virtual cell in the instructing ABM from 1 to 0. As such, the processor is programmed accordingly to action this change in state.

[0129] If, however, the processor identifies that virtual cell Y (at e g. virtual column 20 and row 30) in the further ABM should have a state of 0, for example after the data from that physical cell Y (at physical column 20 and row 30) indicates that it still requires an intervention and still needs to be disconnected from the AC voltage source, and also that the same virtual cell in the instructing ABM has a state of 0, then a delta is not identified. As such, the processor is programmed to not change the state of the virtual cell in the instructing ABM.

[0130] Such a delta calculation is carried out between all virtual working cells in the instructing and one or more further ABMs. The processor is programmed accordingly.

[0131] The new updated instructing ABM thereafter is utilized to update the state of the storage device of each working cell that requires a change in state, and thereafter these changes in state to the individual working cell storage devices affect a change in their respective switches thus disconnecting or reconnecting the necessary individual, targeted cells accordingly. In the case of the above-discussed specific example, the processor is programmed to utilise the instructing ABM to instruct the storage device of physical cell X (at physical column 5 and row 200) to instruct the switch to open, thus disconnecting the working electrode of physical cell X from the AC voltage source and AC global line (and respective AC userwave).PATENTAtorney Docket No.: P39047-WO

[0132] The timing of the disconnection and reconnection of individual working cells within a single AC modulation period is thus advantageously continuously managed by the updating of the instructing ABM.

[0133] Preferably, the updating of the ABM or instructing ABM is synchronized with the AC userwave and more preferably with an event in the AC signal from the AC voltage. Examples of such events include a change in amplitude in the AC signal, a change in frequency of the AC signal and / or a change in shape of the AC signal. Accordingly, the processor is further programmed to ensure that the updates to the instructing ABM (and subsequent updates to the states of the storage devices and switches) occur before any given change in the AC userwave voltage is applied (for example, a change to a different voltage amplitude). Advantageously, in this way, all necessary working cells are activated and are thus ready to receive, and be exposed to, a given voltage (or a given pattern of voltages). Additionally, working cells that do not need to be exposed to the AC userwave are disconnected in time, thus minimizing any associated disadvantageous effects, such as membrane rupture.

[0134] The use of a FPGA as the processor (either individually or with the use of a GPU to, for example, apply the AC userwave) is particularly advantageous in achieving such synchronization. For example, the FPGA may be programmed to store particular ABM patterns to be synchronized with the AC userwave. For example, a first further ABM pattern could be applied for a given time during a given phase of the AC userwave, then a second further ABM pattern applied for a given time during a different phase of the AC userwave.

[0135] Preferably, the updated, virtual representation of the array of cells (i.e. the updated instructing ABM) is applied during the dark period.

[0136] As a person of ordinary skill in the art would understand, suitable metadata management (e g. time stamp, applied AC userwave, specifics of capture), can be utilised by a processor (and accompanying algorithms) to subsequently analyse and interpret data output for feeding into the ABMs of the present disclosure, and associated algorithms for their formation.

[0137] The person of ordinary skill in the art would also readily understand the processor can be any suitable piece of hardw are e.g. a GPU, CPU, FPGA, ASIC, Al chip, PIM device. As a person of ordinary skill in the art would further understand, one or more (e.g. a plurality) of such processors may be utilised in the systems and methods of the present disclosure. A processor may be integrated into, or onto, the nanopore sensor chip, the consumable device, the sequencing instrument or a platform remote from the sequencing instrument and nanopore sensor chip, dependent on the type of processor utilised. Preferably, a FPGA is housed or located within or on the printed circuit board of the sequencing instrument (which may be partPATENTAtorney Docket No.: P39047-WO of the motherboard of the sequencing instrument) and is electrically connected via a socket to the consumable device. Alternatively, the FPGA may be housed or located within or on the integrated circuit of the consumable device. Preferably, a GPU is housed or located within the sequencing instrument or alternatively may be housed or located remote from the sequencing instrument.

[0138] Preferably, the processor is one or more of a FPGA, ASIC (for example designed for the specific use cases described herein), artificial intelligence (Al) chip. Processor in Memory (PIM) device, a graphics processing unit and / or a central processing unit.

[0139] One or more ABMs may be stored internally in a FPGA and / or preferably stored in (and retrieved from) associated external memory such as a Dynamic Random Access Memory (DRAM) or High Bandwidth Memory (HBM). Alternatively, or additionally, one more ABMs may be stored internally in a GPU and / or preferably stored in (and retrieved from) associated external memory. such as HBM. The instructing ABM may sit within a GPU and associated memory, or preferably within a FPGA and associated memory. The one or more further ABMs may sit within a FPGA and associated memory or preferably sits within a GPU and associate memory. Preferably, the virtual representation of the array of cells and / or the updated representation of the array of cells (the instructing ABMs) are stored in a FPGA and / or associated storage. Preferably, the one or more further virtual representations of the array of cells are stored in a graphics processing unit and / or associated storage.

[0140] The processors may be connected to one another and the nanopore sensor chip by any suitable interface or connection known in the art. For example, the processor (e.g. FPGA) may send commands to the nanopore sensor chip via a serial programming / peripheral interface (SPI) and / or a High Speed Interface (HSI). Data from the nanopore sensor chip (analogue voltages that are converted to digital values by way of the ADC and a multiplexer) may be transported back to the processor (e.g. FPGA) by way of any suitable means in the art, such as by way of a low-voltage differential signaling (LVDS) interface.

[0141] Preferably, the storage devices (e.g. memory bits) of the individual working cells are updated in groups of 2048 bits (preferably with the use of a HSI) or 32 bits (preferably with the use of SPI).

[0142] As a person of ordinary skill in the art would understand, any alternative device or mechanism could be utilized instead of a FPGA, to provide the same function. For example, an application specific integrating circuit (ASIC) could be utilized, which is particularly cost beneficial for high volume manufacture of nanopore systems. Alternatively, a CPU could be utilized when interfaced with a PCB arrangement. In the context of the present disclosure, aPATENTAttomev Docket No.: P39047-WOFPGA is preferred due to its superior timing and synchronisation capabilities, when compared to the CPU alternative.

[0143] FIG. 6 is an example arrangement of processors, and associated memory, for housing and processing active bit maps that may be implemented in the systems and methods of the present disclosure. The arrangement of FIG. 6 also shows a number of predetermined sequences of ABMs pre-scheduled through or with an AC userwave. The arrangement 600 shows a nanopore sensor chip (i.e. nanopore chip) 602 connected to a FPGA 604, CPU 606 and GPU 608. Each of these processors has its own associated storage / memory, DRAM 610, Storage 612 and HBM 614, respectively. An example process with this arrangement includes: Step 1: Cell data is sent from nanopore sensor chip 602 to FPGA 604 via high speed interface LVDS, then from the FPGA 604 to CPU 606 via high speed interface (e.g. PCIe or Quad Small Form-factor Pluggable (QSFP)) and then from CPU 606 to GPUs 608 via PCIe. Step 2: GPUs 608 process the cell data. Step 3: Based on data analysis, a new ABM is calculated in GPUs 608. Step 4: FPGA 604 receives ABM from GPUs 608 either directly via DMA over PCIe or coordinated through the CPU 606 via QSFP. FPGA 604 stores the new instructing ABM in internal memory or associated DRAM 610. Step 5: FPGA 604 compares the current instructing ABM with the new ABM and calculates which working cells in the virtual array of cells have changed in their states and updates the states of these working cells in the instructing ABM. Step 6: New instructing ABM used to update the state of the memory bit of the targeted working cells in the nanopore sensor chip 602 by sending data from FPGA 604 to nanopore sensor chip 602 via HSI or SPI high speed interface.

[0144] FIG. 7 is a flowchart of an example process 700 for sequencing a molecule. One or more of the process steps of FIG. 7 may be performed by arrangement 600 exemplified in FIG. 6 and system 1000 exemplified in FIG. 10, including detector 1002 and logic system 1003. A nanopore sequencing chip as described herein including array of cells 200, as shown in FIG. 2, and electrical circuit 400, as shown in FIG. 4, may further be used for the process of FIG. 7.

[0145] In step 701, data from the array of cells, and more specifically from an analogue to digital conversion of each cell of the array of cells, is analysed to determine which cells in the array of cells are working cells. In other words, data from the array of cells is analysed to determine which cells in the array of cells have an intact membrane and which cells with an intact membrane have a nanopore embedded within that intact membrane. The analysis can be performed with any known techniques in the art, for example determining the physical state of each cell by measuring a capacitance, a resistance or measuring a current flow when a bias voltage is applied. Alternatively, a delta voltage change at an integrating capacitor can bePATENTAtorney Docket No.: P39047-WO monitored in response to a delta voltage change applied to the bulk liquid in contact with the membrane, and as described in more detail in WO2017 / 167809A1, the entire contents of which are incorporated herein by reference for all purposes. In some examples, the data analysis is performed with GPU 608, FPGA 604 or CPU 606 of FIG. 6.

[0146] In step 702, the non-working cells (i.e. the cells that either do not have a membrane or do not have a nanopore) are switched off from the AC voltage source. This can be achieved by the processor instructing the memory bit 402 of each non-working cell to open the switch 406 so as to disconnect the working electrode from the AC global line.

[0147] In step 703, optionally an ABM is created to reflect the state (enabled or disabled) of the working cells and the non-working cells in the array of cells.

[0148] In step 704. an AC voltage waveform is applied to the working cells, via one or more global lines, and the sequencing data acquisition stage is started.

[0149] In step 705, the acquired analogue sequencing signals from the working cells are received at the respective ADCs and converted to digital values which are then preferably transported back to the processor (e.g. FPGA) by way of low-voltage differential signaling (LVDS) interface and preferably subsequently to a GPU by peripheral component interconnect express (PCIe) or QSFP. The processor (e g. FPGA and / or GPU) and necessary algorithms then process the data.

[0150] In step 706, the processor, and associated algorithms, determine that a sub-proportion of the working cells requires an intervention because a change in behavior has been observed, for example (i) a change of temperature, (ii) a drift in an electrochemical state, (iii) a drift in bilayer capacitance state, (iv) a blocking of the nanopore, (v) an interruption in a translocation of the molecule, (vi) a translocation event, (vii) the identification of a specific molecule, or a part thereof (such as a sample ID (SID) or a unique molecular identifier (UMI)) passing through the nanopore, or any combination thereof.

[0151] In step 707, optionally either a predetermined ABM pattern or set of patterns is applied to the instructing ABM created in step 703 (S707a) or one or more further ABMs are created to take account of the required interventions. These one or more further ABMs are then applied to the instructing ABM (S707b).

[0152] In step 708, the memory bits of the sub-population of working cells (the targeted cells) are updated to switch off the targeted cells for one or more periods of time in a single modulation period, or multiple single modulation periods. The process is then returned to step 705 and the process stopped when all necessary sequencing data has been obtained.PATENTAtorney Docket No.: P39047-WO

[0153] Process 700 may include additional implementations, such as any single implementation or any combination of implementations described herein and / or in connection with one or more other processes described elsewhere herein.

[0154] Although FIG.7 shows example blocks of process 700, in some implementations, process 700 may include additional blocks, fewer blocks, different blocks, or differently arranged blocks than those depicted in FIG. 7. Additionally, or alternatively, two or more of the blocks of process 700 may be performed in parallel.

[0155] In summary, the process exemplified in FIG. 7 (and discussed generally herein) involves changing the amount of time that each working cell’s working electrode is connected or disconnected from a global line (shared AC voltage line) or AC voltage source, so that the user has the ability to dynamically adjust the effective voltage experienced by an individual working cell in a nanopore sensor chip following a feedback signal from the working cell that some kind of intervention is required. There are significant benefits to being able to control the voltage across the nanopore and membrane on an individual working cell basis in such a manner. It can allow the user to target a population of working cells (a sub-population or a subset of cells of the array of cells) to be turned off and on rapidly in order to regulate the behaviour of those targeted sub-populations towards the behaviour of the other non-targeted cells in the array of cells. In other words, the user can finesse a sub-population of the array of cells by applying a duty / s witching cycle on top of the AC userwave, and more specifically on top of the AC modulation. The effect of sources of variability, e.g. spatial and / or temporal variability, on individual working cells during the sequencing process can thus be advantageously minimised.

[0156] One example of a source of variability' for individual working cells during the sequencing workflow, and which the present invention can advantageously help to minimize, is temperature variation across the nanopore sensor chip surface.

[0157] When the consumable device containing (e.g. enclosing) the nanopore sequencing chip is placed in the sequencing instrument, by, for example, mounting the nanopore sequencing chip onto the PCB of the sequencing instrument, the underside of the nanopore sequencing chip may be configured to contact a thermal control system that functions to cool the nanopore sequencing chip during one or more phases of the sequencing workflow.

[0158] Due to a combination of thin film material stresses in the nanopore sensor chip and mechanical forces exerted on the nanopore sensor chip (e.g. from clamping of the nanopore sensor chip into the PCB of the sequencing instrument), the nanopore sensor chip may be deformed such that there is a subtle bow (for example a micrometer-sized bow) across thePATENTAttomev Docket No.: P39047-WO nanopore sensor chip. The bow can affect contact between the nanopore sensor chip and the thermal control system, which is intended to cool it. In some cases, the center of the nanopore sensor chip may experience less bowing (and a more flush contact with the cooling block) than the edges. This can cause the center to be cooler than the edges.

[0159] FIG. 8 is a modelled heatmap of estimated local temperatures of one flow channel in an 8M nanopore sensor chip (with 2048 rows and 4096 columns of cells) in such a situation. The temperature estimations are based on observed indicative data (e.g. insertion and deletion rates) and corresponding computation of kinetic rates for single molecule processes. According to this heatmap, temperature varies by roughly 5°C across the columns of a flow channel, with the edges of the flow channel being hotter than the centre.

[0160] FIG. 9 is a deletion rate heatmap of one flow channel in an 8M nanopore sensor chip whilst sequencing utilizing SBX chemistry, and illustrates the deletion rate (an example of indicative data) as a function of position in the flow channel. The deletion rate has been derived or computed from a sequence produced from a nanopore and aligned to a known reference sequence. The heatmap shows that the deletion rates are lower in the center of the nanopore sensor chip and higher at the edges. This can be explained by the effect that temperature has on the probability of double translocations during sequencing. A double translocation occurs when two TCEs are passed through the nanopore within a single high voltage TCE pulse duration. When a double translocation occurs, then the reporter code in between the two TCEs in question is not read and thus cannot be counted as a base. The probability of double translocations is higher as temperature increases, and this shows up on the heatmap as an increased deletion rate near the edges of the flow channel. Conversely, lower temperature increases the probability of failed translocation attempts and thus lower deletion rates but higher insertion error rates. As such, FIG. 9 reflects the temperature variation across a flow channel in a nanopore sensor chip, with the temperature being cooler in the middle of the flow channel. Such a temperature variability can have an effect on the sequence detection process and on the accuracy and error profile of resulting reads.

[0161] Advantageously, the system and process of the present invention can help compensate for the effect of temperature variability by inducing nanopores to experience different average / effective voltages, for example, during the bright period, as a function of their position on the nanopore sensor chip.

[0162] For example, a predefmed / pre-programmed ABM could be utilized. This preprogrammed ABM could be created from historical data (e.g. historical indicative data) and knowledge of known behaviors regarding the temperature variability across the flow channelsPATENTAtorney Docket No.: P39047-WO in a nanopore sensor chip. Further, this pre-programmed ABM could be utilized to update the instructing ABM and could be applied during the bright or dark period of the sequencing data acquisition phase, preferably during the dark period, so as to minimize disruption to the acquisition of useful sequencing data. The pre-programmed ABM may involve designating certain cells as enabled or disabled - for example all cells in the edge columns (e.g. column 1 to X and column Y to 4096) of the flow channel designated as disabled, with a further predefined ABM being applied, in the same AC modulation period, subsequently to designate such cells in the edge columns as enabled. Accordingly, the sub-population is subjected to a high frequency disconnection from, and reconnection to, the AC voltage source in one or more single AC modulation periods, thus meaning that the effect of the AC userwave on each cell can be tailored.

[0163] Alternatively, one or more ABMs could be created during the sequencing data acquisition phase, based on, for example, indicative data acquired and processed during the sequencing workflow, for example earlier in the sequencing workflow or sequencing data acquisition phase. Such indicative data for temperature variation includes deletion rate. In other words, one or more further ABMs could be created and applied in real time and “on-the- fly ”. Thus, the instructing ABM may be continually and iteratively updated from the analysed data during the sequencing data acquisition phase.

[0164] Accordingly, the method of the present invention may be a method of reducing the effects of temperature variation during the sequencing a molecule.

[0165] Other examples of sources of variability for individual cells during the sequencing workflow, and which the present invention can advantageously help to minimise, are electrochemical state drift and bilayer capacitance state drift.

[0166] In the case of faradaic systems utilising 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 example lipid bilayer membranes and nanopores) and more specifically to how much time a given nanopore is empty7. 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:Fe(CN)63- + e FeiCNty4’PATENTAtorney Docket No.: P39047-WO

[0167] Such an imbalance may have negative effects on the impedance at the working electrode interface and the Nemst 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.

[0168] In addition, bilayer capacitance state drift may have a similar negative effect. Bilayer capacitance state drift is observed in non-faradaic systems (i.e. independent of a redox couple) with, for example, SBT chemistry. Bilayer capacitance state drift is also observed in faradaic systems. The effects of such an imbalance can be observed in the raw data signal, by, for example, changes in the magnitude of measurement circuit artifacts, following a sequence of voltages being applied to assess the bilayer capacitance state shift (in other words, a short sequence of voltages is applied in order to probe aspects of the wet circuit element characteristics).

[0169] In both cases, where it is desirable to even out the behavior of all of the working cells in the array of cells, the present invention can be utilized to target individual working cells to vary the effective voltage that they experience, by disconnecting the working electrodes of such cells from the global line(s) for a given period of time, for example for a given period of time in the dark period. Such a disconnection can be applied utilizing one or more ABMs. preferably two ABMs. For example, two or more ABMs may be utilized and rapidly switched between in a single AC modulation period (so as to disable and re-enable a targeted cell) with the intention of manipulating the application of the AC userwave experienced by an individual cell and thus the effective AC voltage experienced by that cell. Thus, all working cells exhibiting electrochemical state drift or bilayer capacitance state drift can be targeted to bring them back into line with the behaviors of the other, non-targeted, working cells in the array.

[0170] For example, a predefined / pre-programmed ABM (based on historical data (e.g. historical indicative data) and knowledge of known behaviors regarding the electrochemical state drift or bilayer capacitance state drift could be utilized. This pre-programmed ABM could be utilized to update the instructing ABM and could be applied during the bright or dark period of the sequencing data acquisition phase, preferably during the dark period, so as to minimize disruption to the acquisition of useful sequencing data. The pre-programmed ABM may involve designating certain cells as enabled or disabled, with a further predefined ABM being applied, in the same AC modulation period, subsequently to designate such cells as enabled.PATENTAttorney Docket No.: P39047-WOAccordingly, the sub-population is subjected to a high frequency disconnection from, and reconnection to, the AC voltage source in one or more single AC modulation periods, thus meaning that the effect of the AC userwave on each cell can be tailored.

[0171] Alternatively, one or more ABMs could be created during the sequencing data acquisition phase, based on, for example, indicative data acquired and processed during the sequencing workflow, for example earlier in the sequencing workflow or sequencing data acquisition phase. Such indicative data for electrochemical state drift and bilayer capacitance state drift includes, for example, measuring a change in a voltage measurement of a particular nanopore state (i.e., open channel measurement or measurement of the same molecule within the nanopore at two different times when subjected to the same voltage measurement waveform stimulus). In other words, one or more further ABMs could be created and applied in real time and “on-the-fly”. Thus, the instructing ABM may be continually and iteratively updated from the data analysed during the sequencing data acquisition phase.

[0172] Accordingly, the method of the present invention may be a method of reducing the effects of electrochemical state drift or bilayer capacitance state drift during the sequencing of a molecule.

[0173] The present invention also advantageously allows for targeted actions to be carried out on individual working cells during the sequencing data acquisition phase when indicative data (e.g. from the ADC of the working cells) indicates that an individual working cell requires an intervention, because, for example, it is in a dysfunctional state, such as, for example, a molecule is stuck in a nanopore, a molecule has been stuck in a nanopore for too long, the nanopore is clogged or because there has been an interruption in a translocation (i.e. ceasing to proceed along an Xpandomer). There are a number of indicators in the raw data acquired from the ADC that would indicate that such abnormal behavior or dysfunctional states are occurring. In the case of a stuck molecule, abnormal characteristics in the signal are observed. For example, the characteristic change in the measured electrical signal moving from one reporter element to the next is not observed. In other words, when stuck at a particular reporter element, there is no change in the electrical signal expected after applying the translocation voltage pulse. In addition, other dysfunctional states are often accompanied with noisy characteristics in the signal. Specific examples of targeted actions (i.e. interventions) in such scenarios include the application of cell clearing voltages or cell reverse translocation control to individual working cells.

[0174] In the case of sequencing by SBX™, in some cases the TCE or leader molecule of the Xpandomer, can get stuck while being pushed and / or pulled through the nanopore. InPATENTAttomev Docket No.: P39047-WO addition, not all of the expansions fully open or two or more Xpandomers can enter the nanopore at the same time, thus increasing the barrier to translocation and meaning that the applied voltages (e.g. the TCE pulses) in the AC userwave are insufficient to translocate the TCE and the Xpandomer further through the nanopore. Therefore, a clearing voltage needs to be applied in either the dark period or the bright period (preferably the dark period) to remove the Xpandomer wholly or partially from the nanopore. A clearing voltage is the application of a pulse of high voltage (i.e. higher than the measuring voltage) for a given period of time, and can be associated, in some instances, with negative side effects such as reduced membrane lifetime or single nanopore lifetime. The present invention advantageously allows the application of a clearing voltage to only the working cells that require it thus minimizing such disadvantageous effects.

[0175] For example, in one example, a certain window of time may be designated in the AC userwave during which a clearing voltage may be applied to all working cells attached to the AC voltage line applying that specific AC userwave. During that window of time, the state of the storage device (e.g. memory bit) for a cell would only be changed to a closed state (or maintained as a closed state) for those cells to which it was desired to apply a clearing voltage. Conversely, during that same window of time, the state of the storage device (e.g. memory bit) for a working cell would only be changed to an open state (or maintained as an open state) for those cells to which it was not desired to apply a clearing voltage. Furthermore, for an individual working cell, it would also be possible to connect that working cell to the clearing voltage (and the clearing voltage line) for either the entire window of time designated within the AC userwave, none of the time, or some fraction of the clearing voltage time window. One or more combinations of ABMs could be programmed accordingly.

[0176] Such ABMs could be created during the sequencing data acquisition phase, based on, for example, indicative data acquired and processed during the sequencing workflow, for example earlier in the sequencing workflow or sequencing data acquisition phase. In other words, one or more further ABMs could be created and applied in real time and “on-the-fly” based on which working cells require a clearing voltage. Thus, the instructing ABM may be continually and iteratively updated from the data analysed during the sequencing data acquisition phase.

[0177] Accordingly, the method of the present invention may be a method of reducing the disadvantageous effects of clearing voltages during the sequencing of a molecule.

[0178] In another case of sequencing by SBX™, and when carrying out the methods as described in WO2024 / 074412 Al, '‘Single Molecule Multi-Molecular Trace Methods andPATENTAttomev Docket No.: P39047-WOSystems;’ filed September 29, 2023, the entire contents of which are incorporated herein by reference for all purposes, Xpandomers may be passed forward and backward through a nanopore several times to allow for multiple reads. For example, during the bright period the Xpandomer is pushed through the nanopore whilst acquiring sequencing data, then the polarity of the AC voltage is reversed and the Xpandomer is reversed out (pulled out) of the nanopore whilst still acquiring sequencing data. The Xpandomer is pushed and pulled by the application of pulses of voltage that are higher than the measuring voltage but lower than a clearing voltage. Xpandomers may include one leader segment which functions to capture easily in the forward directly (i.e. has a relatively low energy’ barrier to entering the nanopore), but that in the reverse direction does not re-enter the nanopore easily (i.e. relatively high energy barrier for the leader to translocate through the nanopore in the reverse direction). This allows the Xpandomer molecule to be captured during the bright period, but not be immediately ejected during the first dark period. These leaders may, however, get stuck in the membrane on one side of the nanopore when backing the Xpandomers out of the nanopore.

[0179] The present invention advantageously allows targeted Xpandomer molecule capture and trapping. In one example, the processor is configured to monitor the Xpandomer molecule captures in real time during the sequencing data acquisition phase, for example during the bright period, and designate, record and store, a time stamp (time index) at each capture. Thereafter, the processor is programmed to utilise the time-stamped capture data to evaluate the number of TCE pulses (and in which direction) that have been applied during the sequencing data acquisition phase, for example in the bright period, on a per working cell basis. Such analysis is preferably carried out on the assumption, that under normal conditions, the Xpandomer molecule will have proceeded forward by as many X-NTP positions as TCE pulses that had been applied. The processor is programmed to, thereafter, apply the same number of reverse TCE pulses during the sequencing data acquisition phase, for example during the dark period, so as to drive the Xpandomer molecule backwards through the nanopore by that same number of X-NTP positions, but not to reverse it further than that amount. As such, the present invention can be used to control the number of reverse pulses, in essence, applying a duty cycle (i.e. the switching off and on of the working electrode from the AC userwave) to the reverse pulse period of the AC userwave to achieve this effect. This process can be applied on a per working cell basis by the utilization of one or more AB Ms.

[0180] Such AB Ms could be created during the sequencing data acquisition phase, based on, for example, indicative data acquired and processed during the sequencing workflow, for example earlier in the sequencing workflow or sequencing data acquisition phase. An examplePATENTAtorney Docket No.: P39047-WO of ty pical indicative data is the sequence of reporter code data. In other words, one or more further ABMs could be created and applied in real time and ‘rtn-the-fly?’ based on which working cells require reverse pulses. Thus, the instructing ABM may be continually and iteratively updated from the data analyzed during the sequencing data acquisition phase.

[0181] Accordingly, the method of the present invention may be a method of controlling the number of reverse translocations during the sequencing of a molecule.

[0182] Examples of the predefined operating parameters discussed herein may be a given temperature range, a given electrochemical state range and a given bilayer capacitance range. Examples of threshold values may be a given temperature, a given electrochemical state concentration and a given bilayer capacitance. Examples of abnormal behaviour or dysfunctional states discussed herein that indicate that an intervention may be required include a molecule is stuck in a nanopore, a molecule has been stuck in a nanopore for too long, a specific molecule is passing through the nanopore (for example with specific sequence content), the nanopore is clogged or there has been an interruption in a translocation. Examples of required interventions discussed herein include the application of a clearing voltage or the application of one or more reverse TCE pulses to affect one or more reverse translocations.

[0183] Accordingly, the processor of the system of the present disclosure may be programmed to analyze the further output data from the ADC to detect working cells that require targeting, where the analysis of such further output data may indicate a change in behavior of each cell of the targeted working cells, where examples of the change in behavior include one or more of (i) a change of temperature, (ii) a drift in an electrochemical state, (iii) a drift in bilayer capacitance state, (iv) a blocking of the nanopore, (v) an interruption in a translocation of the molecule, (vi) a translocation event, (vii) the identification of a specific molecule, or a part thereof (such as a sample ID (SID) or a unique molecular identifier (UMI)) passing through the nanopore, or any combination thereof.Systems

[0184] FIG. 10 illustrates a measurement system 1000. The system as shown includes a sample 1005 within a sample holder 1001, where sample 1005 can be contacted with an assay 1008 to provide a signal of a physical characteristic 1015. Assay 1008 may include sequencing by expansion with a nanopore. Physical characteristic 1015 (e.g., a voltage, a current, or other electrical characteristic), from the sample is detected by detector 1002. Detector 1002 (e.g. integrating capacitor Cint 408 of FIG. 4) can take a measurement at intervals (e.g., periodic intervals) to obtain data points that make up a data signal. An analog-to-digital converter converts an analog signal from the detector into digital form at a plurality of times. DetectorPATENTAtorney Docket No.: P39047-WO1002 may be a voltage or current measurement device. The sample holder, assay and detector may be housed in a consumable device. A data signal 1025 is sent from detector 1002 to logic system 1003. Data signal 1025 may be stored in a local memory' 1035, an external memory 1004, or a storage device 1045.

[0185] Logic system 1003 may be, or may include, one or more FPGAs, GPUs, CPUs, ASICs, Al chips, PIM devices, computer systems, microprocessors, etc. It may also include or be coupled with a display (e.g., monitor, LED display, etc.) and a user input device (e.g., mouse, keyboard, buttons, etc ). Logic system 1003 and the other components may be part of a standalone or network connected computer system, or they may be directly attached to or incorporated in a device (e.g., a sequencing instrument) and / or consumable device that includes detector 1002 and / or sample holder 1001. Logic system 1003 may also include software that executes in a processor 1020. Logic system 1003 may include a computer readable medium storing instructions for controlling system 1000 to perform any of the methods described herein. For example, logic system 1003 can provide commands to a system that includes sample holder 1001 such that sequencing or other physical operations are performed. Such physical operations can be performed in a particular order, e.g., with reagents being added and removed in a particular order. Such physical operations may be performed by a robotics system, e.g., including a robotic arm, as may be used to obtain a sample and perform an assay.

[0186] Any of the computer systems mentioned herein may utilise any suitable number of subsystems such as a printer, keyboard, storage device(s), monitors (e.g., a display screen, such as an LED). Peripherals and input / output (I / O) devices, which couple to a I / O controller, can be connected to the computer system by any number of means known in the art such as input / output (I / O) ports (e.g., USB. Thunderbolt, Lightning). For example, an I / O port or external interface (e.g. Ethernet, Wi-Fi, etc.) can be used to connect the computer system to a wide area network such as the Internet, a mouse input device, or a scanner. An interconnection via system bus allows the processor 1020 to communicate with each subsystem and to control the execution of a plurality of instructions from system memory 1035 or the storage device(s) 1045 (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 1035 and / or the storage device(s) 1045 may embody a computer readable medium.

[0187] Aspects of the methods of the present disclosure can be implemented in the form of control logic using hardware circuitry' (e.g., an ASIC 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.PATENTAttomev Docket No.: P39047-WO or multiple processing units on a single circuit board or networked, as well as dedicated 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.

[0188] Any of the software components or functions described in this application may 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 may 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) or Blu-ray disk, flash memory, and the like. The computer readable medium may be any combination of such storage or transmission devices.

[0189] 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, examples 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 may be used with portions of other steps from other methods. 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.

[0190] 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.

[0191] 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 does not limit the referenced component to a particular location unless expressly stated.

Claims

PATENTAtorney Docket No.: P39047-WOCLAIMS1. A system for sequencing a molecule, the system comprising: a nanopore sensor chip comprising an array of cells, wherein each cell of the array of cells comprises a working electrode and a switch; wherein the system further comprises: a flow channel configured to flow an analyte comprising the molecule across the array of cells; a counter electrode configured to apply a voltage to the analyte; an alternating current (AC) voltage source configured to apply an AC voltage to the working electrode; a storage device configured to represent either an open state or a closed state for the switch; an analog-to-digital converter (ADC); and one or more processors programmed to: analyze output data from the ADC to detect a first sub-set of cells, wherein each cell of the first sub-set cells comprises a membrane with a nanopore embedded therein; analyze further output data from the ADC for the first sub-set of cells to detect a second sub-set of cells, wherein the first sub-set of cells comprises the second sub-set of cells; and in response to this analysis of the further output data: update the state of the storage device of each cell of the second sub-set of cells to an open state, thereby opening the switch of each cell of the second subset of cells so as to disconnect the working electrode of each cell of the second subset of cells from the AC voltage source for a period of time during a single AC modulation period.

2. The system according to claim 1, wherein the period of time is less than the single AC modulation period.

3. The system according to any preceding claim, wherein the one or more processors are further programmed to update the state of the storage device of each cell of the second subset of cells to a closed state, thereby closing the switch so as to reconnect the working electrode of each cell of the second sub-set of cells to the AC voltage source during the same single AC modulation period.PATENTAtorney Docket No.: P39047-WO4. The system according to any preceding claim, wherein the one or more processors are further programmed to update the state of the storage device of one or more cells of the second sub-set of cells so as to disconnect and reconnect the working electrode of one or more cells of the second sub-set of cells for one or more further times during the same single AC modulation period.

5. The system according to any preceding claim, wherein the one or more processors are further programmed to update the state of the storage device of one or more of the cells of the second sub-set of cells to disconnect the working electrode of one or more of the cells of the second sub-set of cells from the AC voltage source during a further single AC modulation period and to update the state of the storage device to reconnect the working electrode of one or more of the cells of the second sub-set of cells to the AC voltage source during the same further AC modulation period.

6. The system according to any preceding claim, wherein the one or more processors are further programmed to update the state of the storage device of each cell of the second subset of cells using a virtual representation of the array of cells indicating the binary state of each cell of the array of cells, wherein the state is either enabled or disabled.

7. The system according to claim 6. wherein the one or more processors arefurther programmed to: analyse the output data from the ADC for the first sub-set of cells against a set of predefined operating parameters for each cell of the first sub-set of cells; and wherein the processor is further programmed to: update the virtual representation of the array of cells using such analysis to provide an updated virtual representation of the array of cells and thereafter using the updated virtual representation of the array of cells to: update the state of the storage device of each cell of the second sub-set of cells so as to disconnect or reconnect the working electrode of each cell of the second sub-set of cells from the AC voltage source.

8. The system according to claim 7, wherein the updated virtual representation of the array of cells is updated by one or more further virtual representations of the array of cells,PATENTAtorney Docket No.: P39047-WO wherein for each further representation of the array of the cells the binary state of each cell of the array of cells is indicated, wherein the state is either enabled or disabled.

9. The system according to any of claims 6 to 8, wherein the application of the virtual representation of the array of cells, or the updated virtual representation of the array of cells, to update the state of the storage device of each cell of the second sub-set of cells is with an event in an AC signal from the AC voltage source.

10. The system according to any preceding claim, wherein the analysis of the further output data from the ADC indicates a change in behavior of each cell of the second sub-set of cells.

11. The system according to claim 10, wherein the change in behavior is a change of temperature.

12. The system according to claim 10 or 11. wherein the change in behavior is a drift in an electrochemical state.

13. The system according to any of claims 10 to 12, wherein the change in behavior is a drift in bilayer capacitance state.

14. The system according to any of claims 10 to 13, wherein the change in behavior is a blocking of the nanopore.

15. The system according to any of claims 10 to 14, wherein the change of behavior is an interruption in a translocation of the molecule.

16. The system according to any of claims 10 to 15, wherein the change in behavior is a translocation event.

17. The system according to any of claims 10 to 16, wherein the change of behavior is the identification of a specific molecule passing through the nanopore.PATENTAtorney Docket No.: P39047-WO18. The system according to any preceding claim, wherein the system further comprises a shared AC voltage line which electrically couples the working electrode of each cell of the second sub-set of cells to the AC voltage source.

19. The system according to any preceding claim, wherein the system further comprises a capacitor and wherein the capacitor is electrically coupled to the working electrode and wherein each cell of the array of cells comprises a further switch that selectively couples the capacitor to the AC voltage source.

20. The system according to any preceding claim, wherein each cell of the array of cells comprises its own storage device and wherein the storage device is a memory bit.

21. The system according to any preceding claim, wherein the storage device is an arrangement of transistors that operate in conjunction with the switch to set the switch to open or closed.

22. The system according to any preceding claim, wherein the storage device is a bit of memory that can represent the state of the switch in an open or closed state.

23. The system according to any of claims 19 to 22, wherein the ADC is electrically coupled to the capacitor.

24. The system according to any preceding claim, wherein the processor is a field programmable gate array, an application specific integrated circuit, an artificial intelligence chip, a processor in memory device, a graphics processing unit and / or a central processing unit.

25. The system according to any of claims 8 to 24, wherein the virtual representation of the array of cells and / or the updated representation of the array of cells is stored in a field programmable gate array and / or associated storage and wherein the one or more further virtual representations of the array of cells are stored in a graphics processing unit and / or associated storage.

26. The system according to any preceding claim, wherein the system comprises a sequencing instrument and a removable consumable device and wherein the nanopore sensorPATENTAtorney Docket No.: P39047-WO chip, flow channel, storage device, ADC and / or counter electrode are housed in the removable consumable device.

27. The system according to any preceding claim, wherein the system further comprises a thermal control system configured to contact the nanopore sensor chip.

28. The system according to any preceding claim, wherein the analyte comprises an ionic solution, wherein the ionic solution comprises a redox couple comprising a first species of the redox couple and a second species of the redox couple.

29. The system according to any preceding claim, wherein the molecule is an expanded molecule derived from a nucleic acid molecule and wherein the expanded molecule comprises one or more reporter codes.

30. A method of sequencing a molecule using a nanopore sensor chip, wherein the nanopore sensor chip comprises an array of cells, and wherein each cell of the array of cells comprises a working electrode and a switch, the method comprising: forming a membrane and embedding a nanopore therein for each cell of a first sub-set of cells of the array of cells; flowing an analyte comprising the molecule across the array of cells; applying a voltage to the analyte; applying an AC voltage to the working electrode of each cell of the first sub-set of cells; receiving output data for the first sub-set of cells at an analog-to-digital converter (ADC); analyzing the output data from the ADC for the first sub-set of cells; and, in response to this analysis: updating the state of a storage device of each cell in a second sub-set of cells to an open state, and thereby opening the switch so as to disconnect the working electrode of each cell of the second sub-set of cells from the AC voltage for a period of time during a single AC modulation period, wherein the first sub-set of cells comprises the second sub-set of cells and wherein the storage device is configured to represent either an open state or a closed state for the switch.

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