Lipid composition and methods of use in a nanopore sequencing assay

Abstract

The disclosure describes sequencing workflows and reagents used in those workflows that are suitable for sequencing a target nucleic acid using a nanopore sequencing device. The disclosure also describes methods and compositions used to treat a device pre- and post-sequencing to enable reuse of a consumable device in a nanopore sequencer.

Description

Lipid Composition and Methods of Use in a Nanopore Sequencing Assay CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority of U.S. Application Serial No. 63 / 688,458, filed August 29, 2024. Reference is also made to U.S. Application Serial Nos.63 / 688,462, filed August 29, 2024, and 63 / 688,460, filed August 29, 2024. The disclosures of the referenced applications are incorporated herein by reference. FIELD OF THE DISCLOSURE

[0002] This application relates to optimized compositions and methods for nanopore- based nucleic acid detection techniques, such as nanopore sequencing. The compositions and methods described herein are useful for pre- and post-nanopore nucleic acid detection protocols, including bilayer formation, pore insertion, reagent loading, and pre- and post-sequencing cleaning protocols.

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. BACKGROUND

[0004] A nanopore based sequencing device is an analytical tool that can be used for DNA sequencing. These devices can incorporate a large number of sensor cells configured as an array. For example, a sequencing device can include an array of one million cells, with, for example, 1000 rows by 1000 columns of cells. Each cell of the array can include a membrane and a protein pore having a pore size on the order of one nanometer in internal diameter. Such nanopores have been shown to be effective in rapid nucleotide sequencing.

[0005] When a voltage potential is applied across a nanopore immersed in a conducting fluid, a small ion current attributed to the conduction of ions across the nanopore can exist. The presence of molecules of varying types and sizes can impactthe electrical environment in the pore and changes in the electrical signal can be used to identify different molecules or species positioned within the nanopore. The molecule can be a particular tag attached to a particular nucleotide, thereby allowing detection of a nucleotide at a particular position of a nucleic acid. Alternatively, the molecule can be a synthetic species with each subcomponent of the molecule being a proxy for a subcomponent of a natural product, e.g., a nucleic acid or protein. In this alternative embodiment, as the synthetic species translocates through the nanopore, the changes in the electronic environment within the pore are indicative of the specific subcomponent present in the pore at a given time and the changes can be used to deduce the structure of the corresponding natural product. In each of these embodiments, a voltage or other signal in a circuit including the nanopore can be measured (e.g., at an integrating capacitor) as a way of measuring the resistance of the molecule, thereby allowing detection of which molecule or subcomponent of a larger molecule is in the nanopore at a given time.

[0006] Nanopore-based sequencing techniques are burdened by having to resolve small changes in electrical signals against a significant background of noise in a micro-volume electrochemical cell. This measurement challenge is complicated by small changes in the materials and parameters affecting the electrochemical cell and the sequencing chemistry used in the system, including but not limited to, reagents and methods used before, during, and after sequencing.

[0007] There remains a need for improved compositions and protocols, including lipid compositions, bilayer formation, pore insertion, reagent loading, and pre- and post- sequencing cleaning protocols, that provide for more efficient and accurate electrochemical signal detection in a nanopore-based nucleic acid sequencing devices, systems, and methods. BRIEF SUMMARY

[0008] The disclosure provides a method of sequencing a target nucleic acid comprising: providing a nanopore sequencing device comprising a plurality ofelectrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution; preparing the device for sequencing by: forming a lipid bilayer on the plurality of electrochemical cells having a nanopore molecule inserted in the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore, wherein the lipid composition comprises a phospholipid, a bilayer additive, an annular solvent, a silicone oil, and a cosolvent; and perfusing the cis chambers with an XP buffer comprising XP molecules; applying an electrical stimulus to the plurality of electrochemical cells that causes at least a portion of the XP molecules to translocate through the nanopore; and detecting changes in an electrical signal in the cell as the XP molecules translocate through the nanopore, wherein the changes in the electrical signal are indicative of a sequence of a target nucleic acid.

[0009] The disclosure also provides a lipid bilayer composition comprising: a phospholipid; a bilayer additive; an annular solvent; silicone oil; and a cosolvent, wherein (i) a molar ratio of phospholipid to bilayer additive in the composition is between 1:6 to 1:10; and / or (ii) a molar ratio of silicone oil to cosolvent is between 6:1 to 12:1. In a specific embodiment, the disclosure provides a composition including one or more of the following: (a) the phospholipid is selected from the group consisting of DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DPhPy, DSPE, and mixtures thereof; (b) the bilayer additive comprises single-tail lipids, lauryl dimethylamine-N-oxide, octyl beta-D-glucopyranoside, sodium dodecyl sulfate, Triton X-100, CHAPS, Span20, Span60, Span80, lysophospholipids, sphingosine, monoglycerides, cationic lipids, nonionic surfactants, and combinations thereof; (c) the annular solvent comprises hexane, chloroform, dichloromethane, toluene, cyclohexane, benzene, diethyl ether, petroleum ether, isooctane, carbon tetrachloride, and mixtures thereof; (d) the silicone oil comprises PDM20, mineral oil, squalene, polyalphaolefins, medium-chain triglycerides, isopropyl myristate, caprylic / capric triglycerides, perfluoropolyether, hydrogenated polyisobutene, and mixtures thereof; and (e) the cosolvent comprises decane, tridecane, octadecane,squalene, cyclohexane, heptane, hexane, hexadecane, toluene, xylene, and mixtures thereof.

[0010] The disclosure also contemplates a method of preparing a nanopore sequencing device for use in a sequencing assay: providing the nanopore sequencing device comprising a plurality of electrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution, introducing a nanopore composition into the device, introducing a lipid composition into the device under conditions sufficient to form a lipid bilayer between the cis and trans chambers, subjecting the device to an electrical stimulus sufficient to initiate poration of the nanopore into the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore, perfusing the device with an osmolarity buffer, and perfusing the cis chambers with an XP buffer comprising XP molecules.

[0011] Moreover, the disclosure provides a method of sequencing a target nucleic acid comprising: providing a nanopore sequencing device including a plurality of electrochemical cells positioned in a flow cell, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution; preparing the device for sequencing by sequentially introducing a nanopore composition and a lipid composition into the device under conditions sufficient to (i) form a lipid bilayer between the cis and trans chambers, and (ii) operably connect the cis and trans chambers by the nanopore; perfusing the cis chambers with an initial volume of XP buffer comprising 1:3-1:6 XP molecules at a first time interval; initiating a sequencing run by applying an electrical stimulus to the plurality of electrochemical cells to cause at least a portion of the XP molecules to translocate through the nanopore, and during the applying step, introducing one or more additional volumes of XP buffer at a subsequent time during the sequencing run; and detecting changes in an electrical signal in the cell as the XP molecules translocate through the nanopore, wherein the changes in the electrical signal are indicative of a sequence of a target nucleic acid.

[0012] Still further, the disclosure describes a method of sequencing a target nucleic acid comprising: providing a nanopore sequencing device including a plurality of electrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution; subjecting the device to a pre-sequencing cleaning protocol comprising sequentially perfusing the device with the following solutions: (i) a detergent solution; (ii) one or more volumes of an acidic solution and / or a basic solution; (iii) a system fluid; and perfusing the device with air between an application of one or more of (i)-(iii); preparing the device for sequencing by: forming a lipid bilayer on the plurality of electrochemical cells having a nanopore molecule inserted in the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore; perfusing the cis chambers with an XP buffer comprising XP molecules; applying an electrical stimulus to the plurality of electrochemical cells that causes at least a portion of the XP molecules to translocate through the nanopore; and detecting changes in an electrical signal in the cell as the XP molecules translocate through the nanopore, wherein the changes in the electrical signal are indicative of a sequence of a target nucleic acid.

[0013] Finally, the disclosure provides a method of cleaning a nanopore sequencing device adapted for use in a nanopore sequencer comprising subjecting the device to a pre-sequencing cleaning protocol comprising perfusing the device with the following solutions: (i) a detergent solution; (ii) one or more volumes of an acidic solution and / or a basic solution; (iii) a system fluid; and perfusing the device with air between an application of one or more of (i)-(iii).

[0014] Another embodiment of a method of cleaning a nanopore sequencing device adapted for use in a nanopore sequencer is provided that includes subjecting the device to an end of run cleaning protocol comprises sequentially perfusing the device with the following solutions: (i) system fluid; (ii) detergent solution; and (ii) a system fluid; and suspending the device in system fluid for later use.

[0015] Also provided is a method of sequencing a target nucleic acid comprising: (a) providing a nanopore sequencing device including a plurality of electrochemicalcells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution; (b) subjecting the device to a pre- sequencing cleaning protocol comprising sequentially perfusing the device with the following solutions: (i) a detergent solution; (ii) optionally, one or more flow cell volumes of a cleaning solution, an acidic solution and / or a basic solution; (iii) a system fluid; and perfusing the device with air between an application of one or more of (i)- (iii); (c) preparing the device for sequencing by: (i) forming a lipid bilayer on the plurality of electrochemical cells having a nanopore molecule inserted in the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore; (ii) perfusing the cis chambers with an XP buffer comprising XP molecules; (d) applying an electrical stimulus to the plurality of electrochemical cells that causes at least a portion of the XP molecules to translocate through the nanopore; and (e) detecting changes in an electrical signal in the cell as the XP molecules translocate through the nanopore, wherein the changes in the electrical signal are indicative of a sequence of a target nucleic acid. In a specific example of step (b)(ii), optionally, one or more flow cell volumes of an acidic solution and / or a basic solution are introduced. Alternatively, step (b)(ii) includes one or more flow cell volumes of a cleaning solution.

[0016] Additionally, the disclosure contemplates a method of cleaning a nanopore sequencing device adapted for use in a nanopore sequencer comprising subjecting the device to a pre-sequencing cleaning protocol comprising perfusing the device with the following solutions: (i) a detergent solution; (ii) optionally, one or more flow cell volumes of a cleaning solution, an acidic solution and / or a basic solution; (iii) a system fluid; and perfusing the device with air between an application of one or more of (i)-(iii). In a specific embodiment, (ii) includes one or more flow cell volumes of an acidic and / or a basic solution. Alternatively, (ii) includes one or more flow cell volumes of a cleaning solution.

[0017] The disclosure includes a method of cleaning a nanopore sequencing device adapted for use in a nanopore sequencer comprising subjecting the device to an end of run cleaning protocol comprises sequentially perfusing the device with one ormore flow cell volumes of the following solutions: (i) detergent solution; (ii) basic solution; (iii) system fluid; (iv) cleaning solution; and (v) protective solution, and combinations thereof in any order, and perfusing the device with air between an application of one or more of the solutions. In a particular embodiment, the end of run cleaning protocol includes introducing one or more of the following: (i) detergent solution; (ii) basic solution; (iii) system fluid, and optionally, a cleaning solution and / or a protective solution are introduced.

[0018] Moreover, the disclosure provides a method of preparing a nanopore sequencing device for use in a sequencing assay: providing the nanopore sequencing device comprising a plurality of electrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution, and introducing a nanopore composition into the device using an iterative pulsed flow to increase uniform delivery of nanopore molecules across the fluidic path. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The features of the disclosure are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0020] FIG.1 includes a top view of an embodiment of a nanopore sensor device having an array of nanopore cells.

[0021] FIG.2 illustrates the elements of a nanopore sensor device.

[0022] FIGs.3A-3B depict an embodiment of a device performing nucleotide sequencing with the nanopore-enabled SBS technique (panel 3A), and an embodiment of a cell performing sequencing by expansion (SBX) (panel 3B), respectively.

[0023] FIG.4 illustrates one embodiment of a method of forming a nanopore sequencing device suitable for SBX sequencing.

[0024] FIGs.5A-5E illustrate the components of the lipid membrane and the function of each component in a nanopore sequencing device.

[0025] FIG.6 illustrates the components of the lipid composition and the interaction between components.

[0026] FIGs.7A-7B show different methods of loading XP molecules to the device prior to and / or during a sequencing run. FIG.7A shows a single load XP addition, whereas FIG.7B shows a multi-load XP addition.

[0027] FIG.8A illustrates an embodiment of an electric circuit in a nanopore cell. FIGS. 8B-8C show the implementation of voltage application and phasing. FIG.8D shows the beginning of the voltage application for an example bright phase illustrating the onset of translocation pulses.

[0028] FIG.9 is a graphical illustration of a computer system, according to certain aspects of the present disclosure.

[0029] FIGs.10A-10B show standard pre- and post-sequencing cleaning protocols. Panel 10A shows an initial run cleaning protocol and panel 10B shows a subsequent run cleaning protocol.

[0030] FIG.11 shows a cleaning protocol including an alternative pre-sequencing cleaning protocol and the subsequent use of the device in one or more sequencing runs.

[0031] FIGs.12A-12D show a nanopore sequencer protocol workflow (panel 12A), an example of a sequencing workflow and the conditions of each step (panel 12B), an exemplary sequencing waveform applied during a sequencing run (panel 12C), and the reuse of a nanopore device in multiple sequencing runs in the workflow (panel 12D).

[0032] FIGs.13A-13C show alternative cleaning protocols. Panel 13A shows an alternative initial cleaning protocol, panel 13B shows an alternative subsequent run cleaning protocol, and panel 13C shows an alternative cleaning protocol that can be used as a pre-sequencing protocol after a plurality of sequencing runs.

[0033] FIGs.14A-14G show an additional alternative sequencing and cleaning protocols. FIG.14A shows the complete sequencing workflow, FIG.14B shows thewaveform applied during sequencing, FIG.14C shows a reagent stacking method used in a pre-sequencing cleaning protocol and FIG.14D shows the pre-sequencing cleaning protocol workflow. FIGs.14E, 14F(i)-14F(ii) show a reagent stacking method used for an end of run cleaning protocol and FIG.14G shows the end of run cleaning protocol. DETAILED DESCRIPTION

[0034] A. Definitions

[0035] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Methods, devices, and materials similar or equivalent to those described herein can be used in the practice of disclosed techniques. The following terms are provided to facilitate understanding of certain terms used frequently and are not meant to limit the scope of the present disclosure. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0036] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected," “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected," “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature mayhave portions that overlap or underlie the adjacent feature.

[0037] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. For example, as used herein, the singular forms “a," “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ."

[0038] Spatially relative terms, such as “under," “below," “lower," “over," “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly," “downwardly," “vertical," “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0039] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, andsimilarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present disclosure.

[0040] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise," and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0041] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0042] For the descriptions herein and the appended claims, the singular forms “a,” and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0043] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding (i) either or (ii) both of those included limits are also included in the disclosure. For example, “1 to 50” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0044] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual(Fourth Edition), Vols.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011, and now available in journal format online as Current Protocols in Molecular Biology, Vols.00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library(hereinafter “Ausubel”).

[0045] A "nanopore" refers to a pore, channel or passage formed or otherwise provided in a membrane. A membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed of a polymeric material or mixtures thereof. The nanopore can be disposed adjacent or in proximity to a sensing circuit or an electrode coupled to a sensing circuit, such as, for example, a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. In some examples, a nanopore has a characteristic width or diameter on the order of 0.1 nanometers (nm) to about 1000 nm. In some implementations, a nanopore may be a protein.

[0046] A “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, 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.

[0047] The term “nucleotide,” in addition to referring to the naturally occurring ribonucleotide or deoxyribonucleotide monomers, can be understood to refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.

[0048] The term "tag" refers to a detectable moiety that can be atoms or molecules, or a collection of atoms or molecules. A tag can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature, which signature can be detected with the aid of a nanopore. Typically, when a nucleotide is attached to the tag it is called a "tagged nucleotide." The tag can be attached to the nucleotide via the phosphate moiety.

[0049] The term “template” refers to a single stranded nucleic acid molecule that is copied into a complementary strand of DNA nucleotides for DNA synthesis. In some cases, a template can refer to the sequence of DNA that is copied during the synthesis of mRNA.

[0050] “Xpandomer” or “XP” refers to a polymer synthesized by transcription of the sequence of a nucleic acid template. The transcribed sequence is encoded along the XP backbone in high signal-to-noise reporters that are separated by ~10 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of XPs relative to natural DNA. XPs are used in to carry out sequencing- by- expansion (“SBX”) and the building blocks of XPs are XNTPs, which are non-natural nucleotide analogs used in XP synthesis to transcribe the sequence of a nucleic acid template. XNTPs are expandable, 5' triphosphate modified non-natural nucleotideanalogs compatible with template dependent enzymatic polymerization.

[0051] The term “signal value” refers to a value of the sequencing signal output from a sequencing cell. According to certain embodiments, the sequencing signal is an electrical signal that is measured and / or output from a point in a circuit of one or more sequencing cells e.g., the signal value is (or represents) a voltage or a current. The signal value can represent the results of a direct measurement of voltage and / or current and / or may represent an indirect measurement, e.g., the signal value can be a measured duration of time for which it takes a voltage or current to reach a specified value. A signal value can represent any measurable quantity that correlates with the resistivity of a nanopore and from which the resistivity and / or conductance of the nanopore (threaded and / or unthreaded) can be derived. As another example, the signal value can correspond to a light intensity, e.g., from a fluorophore attached to a nucleotide being added to a nucleic acid with a polymerase.

[0052] The term “osmolarity,” also known as osmotic concentration, refers to a measure of solute concentration. Osmolarity measures the number of osmoles of solute particles per unit volume of solution. An osmole is a measure of the number of moles of solute that contribute to the osmotic pressure of a solution. Osmolarity allows the measurement of the osmotic pressure of a solution and the determination of how the solvent will diffuse across a semipermeable membrane (osmosis) separating two solutions of different osmotic concentration. The term “osmolyte” refers to any soluble compound that when dissolved into a solution increases the osmolarity of that solution.

[0053] Exemplary nanopore systems, circuitry, and sequencing operations are described below, as well as methods of using such systems and components in a sequencing workflow. Embodiments of the disclosure can be implemented in numerous ways, including as a process, a system, and a computer program product embodied on a computer readable storage medium and / or a processor, such as a processor configured to execute instructions stored on and / or provided by a memory coupled to the processor.

[0054] B. Nanopore-based devices

[0055] Nanopore-based devices for detecting nucleic acids have been developed for rapid sequencing and various designs and methods of use are known in the art. See e.g., US9494554B2, US9567630B2, US9557294B2, US9605309B2, each of which hereby incorporated by reference herein. These devices generally include a plurality of electrochemical cells and each electrochemical cell is in fluid communication with the cells in the array (or plurality) and comprises a chamber containing a nanopore embedded in a membrane. The membrane acts to separate the cell chamber into two sub-chambers, referred to as the cis and trans sides of the cell, each of which contain an electrode.

[0056] Within the electrochemical cell, the nanopore embedded in the membrane is disposed in proximity to an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit. When a voltage potential is applied (via the electrodes) across a nanopore immersed in a conducting fluid, a small current attributed to the flow of ions through the nanopore can be observed. This ion flow is sensitive to the pore size, and thus, molecules entering the pore affect the ion flow and the voltage measured through this sensor circuit.

[0057] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a massively parallel fashion in which thousands of such cells are configured as an array in a single device often referred to as a chip (or device). A typically nanopore-based sequencing device device incorporates an array of one million or more electrochemical cells, and may include 1000 rows by 1000 columns of such cells (see e.g., devices fabricated by Roche Sequencing Solutions, Santa Clara, CA, USA). Methods for fabricating and using such nanopore array devices can also be found in U.S. Patent Application Publication Nos.2013 / 0244340 A1, US 2013 / 0264207 A1, US2014 / 0134616 A1, 2015 / 0368710 A1, and 2018 / 0057870 A1, and published International Application WO 2019 / 166457 A1, each of which is hereby incorporated by reference herein. Each well in the array is manufactured using asemiconductor manufacturing process that provides surface modifications that allow for constant contact with biological reagents and conductive salts. Each well can support a phospholipid bilayer membrane with a nanopore-polymerase conjugate embedded therein. The electrode at each well is individually addressable by computer interface. All reagents used are introduced into a simple flow cell above the array device using a computer-controlled syringe pump. The device supports analog to digital conversion and reports electrical measurements from all electrodes independently at a rate of over 200 points per second, e.g., over 500 points per second, and in a specific embodiment, over 1000 points per second. Nanopore measurements can be made asynchronously at each of 8 M addressable nanopore- containing membranes in the array at least once every millisecond (msec) and recorded on the interfaced computer. Further description of exemplary electrochemical cells useful for nanopore-based nucleic acid assays, such as sequencing, including chamber and electrode materials, buffer solutions, sensing circuitry, array devices, and their use in various applications is provided below.

[0058] FIG.1 is a top view of an embodiment of a nanopore sensor device 100 having an array 140 of nanopore cells 150. Each nanopore cell 150 includes a control circuit integrated on a silicon substrate of nanopore sensor device 100. In some embodiments, side walls 136 are included in array 140 to separate groups of nanopore cells 150 so that each group can receive a different sample for characterization. Each nanopore cell can be used to sequence a nucleic acid. In some embodiments, nanopore sensor device 100 includes a cover plate 130. In some embodiments, nanopore sensor device 100 also includes a plurality of electrical contacts 110 (e.g., pins, wires, or solder bumps) for interfacing with other circuits, such as a computer processor.

[0059] In some embodiments, nanopore sensor device 100 includes multiple devices in the same package, such as, for example, a Multi-device Module or System-in- Package. The devices can include, for example, a memory, a processor, a field- programmable gate array (FPGA), an application-specific integrated circuit (ASIC),data converters, a high-speed I / O interface, etc.

[0060] In some embodiments, nanopore sensor device 100 is coupled to (e.g., docked to) a workstation 120, which can include various components for carrying out (e.g., automatically carrying out) various embodiments of the processes disclosed herein. These processes can include, for example, analyte delivery mechanisms, such as pipettes for delivering lipid suspension or other membrane structure suspension, analyte solution, and / or other liquids, suspension or solids. The workstation components can further include robotic arms, one or more computer processors, and / or memory. A plurality of analytes of interest can be detected on array 140 of nanopore cells 150. In some embodiments, each nanopore cell 150 is individually addressable.

[0061] Nanopore cells 150 in nanopore sensor device 100 can be implemented in many different ways. For example, in some embodiments, tags of different sizes and / or chemical structures are attached to different nucleotides in a nucleic acid molecule to be sequenced. In some embodiments, a complementary strand to a template of the nucleic acid molecule to be sequenced may be synthesized by hybridizing differently polymer-tagged nucleotides with the template. In some implementations, the nucleic acid molecule and the attached tags both move through the nanopore, and an ion current passing through the nanopore can indicate the nucleotide that is in the nanopore because of the particular size and / or structure of the tag attached to the nucleotide. In some implementations, only the tags are moved into the nanopore. There can also be many different ways to detect the different tags in the nanopores.

[0062] In a specific embodiment, the nanopore cells are used for SBX protocols and the compositions and methods described herein are optimized for SBX chemistry, as described in more detail herein.

[0063] FIG.2 illustrates an embodiment of an example nanopore cell 200 in a nanopore sensor device, such as nanopore cell 150 in nanopore sensor device 100 of FIG.1, that can be used to characterize a xpandomer, polynucleotide or apolypeptide. Nanopore cell 200 can include a well 205 formed of dielectric layers 201 and 204; a membrane, such as a lipid bilayer 214 formed over well 205; and a sample chamber 215 on lipid bilayer 214 and separated from well 205 by lipid bilayer 214. Well 205 can contain a volume of electrolyte 206 containing a nanopore, and sample chamber 215 can hold bulk electrolyte 208 containing a nanopore, e.g., a soluble protein nanopore transmembrane molecular complexes (PNTMC), and the analyte of interest (e.g., a nucleic acid molecule to be sequenced).

[0064] Nanopore cell 200 can include a working electrode 202 at the bottom of well 205 and a counter electrode 210 disposed in sample chamber 215. A signal source 228 can apply a voltage signal between working electrode 202 and counter electrode 210. A single nanopore (e.g., a PNTMC) can be inserted into lipid bilayer 214 by an electroporation process caused by the voltage signal, thereby forming a nanopore 216 in lipid bilayer 214. The individual membranes (e.g., lipid bilayers 214 or other membrane structures) in the array can be neither chemically nor electrically connected to each other. Thus, each nanopore cell in the array can be an independent sequencing machine, producing data unique to the single polymer molecule associated with the nanopore that operates on the analyte of interest and modulates the conductance of the otherwise low conductivity lipid bilayer.

[0065] As shown in FIG.2, nanopore cell 200 can be formed on a substrate 230, such as a silicon substrate. Dielectric layer 201 can be formed on substrate 230. Dielectric material used to form dielectric layer 201 can include, for example, glass, oxides, nitrides, and the like. An electric circuit 222 for controlling electrical stimulation and for processing the signal detected from nanopore cell 200 can be formed on substrate 230 and / or within dielectric layer 201. For example, a plurality of patterned metal layers (e.g., metal 1 to metal 6) can be formed in dielectric layer 201, and a plurality of active devices (e.g., transistors) can be fabricated on substrate 230. In some embodiments, signal source 228 is included as a part of electric circuit 222. Electric circuit 222 can include, for example, amplifiers, integrators, analog-to-digital converters, noise filters, feedback control logic, and / or various other components.Electric circuit 222 can be further coupled to a processor 224 that is coupled to a memory 226, where processor 224 can analyze the sequencing data to determine sequences of the polymer molecules that have been sequenced in the array.

[0066] Working electrode 202 can be formed on dielectric layer 201 and can form at least a part of the bottom of well 205. In some embodiments, working electrode 202 is a metal electrode. For non-faradaic conduction, working electrode 202 can be made of metals or other materials that are resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, working electrode 202 can be a platinum electrode with electroplated platinum. In another example, working electrode 202 can be a titanium nitride (TiN) working electrode. Working electrode 202 can be porous, thereby increasing its surface area and a resulting capacitance associated with working electrode 202. Because the working electrode of a nanopore cell can be independent from the working electrode of another nanopore cell, the working electrode can be referred to as cell electrode in this disclosure.

[0067] Dielectric layer 204 can be formed above dielectric layer 201. Dielectric layer 204 forms the walls surrounding well 205. Dielectric material used to form dielectric layer 204 can include, for example, glass, oxide, silicon mononitride (SiN), polyimide, or other suitable hydrophobic insulating material. The top surface of dielectric layer 204 can be silanized. The silanization can form a hydrophobic layer 220 above the top surface of dielectric layer 204. In some embodiments, hydrophobic layer 220 has a thickness of about 1.5 nanometer (nm).

[0068] Well 205 formed by the dielectric layer walls 204 includes volume of electrolyte 206 above working electrode 202. Volume of electrolyte 206 can be buffered and can include one or more of the following: ammonium chloride, lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). In some embodiments, volume ofelectrolyte 206 has a thickness of about two microns (μm).

[0069] As also shown in FIG.2, a membrane can be formed on top of dielectric layer 204 and spanning across well 205. In some embodiments, the membrane includes a lipid monolayer 218 formed on top of hydrophobic layer 220. As the membrane reaches the opening of well 205, lipid monolayer 208 can transition to lipid bilayer 214 that spans across the opening of well 205.

[0070] As shown, lipid bilayer 214 is embedded with a single nanopore 216, e.g., formed by a single PNTMC. As described above, nanopore 216 can be formed by inserting a single PNTMC into lipid bilayer 214 by electroporation. Nanopore 216 can be large enough for passing at least a portion of the analyte of interest and / or small ions (e.g., Na+, K+, Ca2+, CI-) between the two sides of lipid bilayer 214.

[0071] Sample chamber 215 is over lipid bilayer 214 and can hold a solution of the analyte of interest for characterization. The solution can be an aqueous solution containing bulk electrolyte 208 and buffered to an optimum ion concentration and maintained at an optimum pH to keep the nanopore 216 open. Nanopore 216 crosses lipid bilayer 214 and provides the only path for ionic flow from bulk electrolyte 208 to working electrode 202. In addition to nanopores (e.g., PNTMCs) and the analyte of interest, bulk electrolyte 208 can further include one or more of the following: ammonium chloride, lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2).

[0072] Counter electrode (CE) 210 can be an electrochemical potential sensor. In some embodiments, counter electrode 210 is shared between a plurality of nanopore cells and can therefore be referred to as a common electrode. In some cases, the common potential and the common electrode can be common to all nanopore cells, or at least all nanopore cells within a particular grouping. The common electrode can be configured to apply a common potential to the bulk electrolyte 208 in contact with the nanopore 216. Counter electrode 210 and working electrode 202 can be coupledto signal source 228 for providing electrical stimulus (e.g., voltage bias) across lipid bilayer 214, and can be used for sensing electrical characteristics of lipid bilayer 214 (e.g., resistance, capacitance, and ionic current flow). In some embodiments, nanopore cell 200 can also include a reference electrode 212.

[0073] In some embodiments, various checks are made during creation of the nanopore cell as part of calibration. Once a nanopore cell is created, further calibration steps can be performed, e.g., to identify nanopore cells that are performing as desired (e.g., one nanopore in the cell). Such calibration checks can include physical checks, voltage calibration, open channel calibration, and identification of cells with a single nanopore.

[0074] Nanopore cells, e.g., cells 150 in nanopore sensor device 100, can enable parallel sequencing using a single molecule nanopore based sequencing by synthesis (Nano-SBS) technique, described below.

[0075] C. Exemplary Nanopore Sequencing Chemistry

[0076] Nanopore-based sequencing-by-synthesis (“SBS”) uses a polymerase (or other strand-extending enzyme) covalently linked to a nanopore to synthesize a DNA strand complementary to a target sequence template (i.e., a copy strand). The nanopore embedded in a membrane in an electrochemical cell is used to concurrently detect the identity of each nucleotide monomer as it is added to that growing strand. See e.g., US Pat. Publ. Nos.2013 / 0244340 A1, 2013 / 0264207 A1, 2014 / 0134616 A1, 2015 / 0368710 A1, and 2018 / 0057870 A1, and published International Application WO 2019 / 166457 A1. Each added nucleotide monomer is detected by monitoring signals due to changes in ion flow through the nanopore as a tag moiety attached to each added nucleotide monomer enters the nanopore and alters the ion flow. For optimal performance, the tag moiety should reside in the nanopore for a sufficient amount of time to provide for a detectable, identifiable, and reproducible signal associated with altering ion flow through the nanopore (relative to the baseline “open current” flow), such that the specific nucleotide associated with the tag can be distinguished unambiguously from the other tagged nucleotides in the SBS solution.

[0077] FIG.3A illustrates an embodiment of a nanopore cell 300 performing nucleotide sequencing using the Nano-SBS technique. In the Nano-SBS technique, a template 332 to be sequenced (e.g., a nucleotide acid molecule or another analyte of interest) and a primer can be introduced into bulk electrolyte 308 in the sample chamber of nanopore cell 300. As examples, template 332 can be circular or linear. A nucleic acid primer can be hybridized to a portion of template 332 to which four differently polymer-tagged nucleotides 338 can be added.

[0078] In some embodiments, an enzyme (e.g., a polymerase 334, such as a DNA polymerase) is associated with nanopore 316 for use in synthesizing a complementary strand to template 332. For example, polymerase 334 can be covalently attached to nanopore 316. Polymerase 334 can catalyze the incorporation of nucleotides 338 onto the primer using a single stranded nucleic acid molecule as the template. Nucleotides 338 can comprise tag species (“tags”) with the nucleotide being one of four different types: A, T, G, or C. When a tagged nucleotide is correctly complexed with polymerase 334, the tag can be pulled (e.g., loaded) into the nanopore by an electrical force, such as a force generated in the presence of an electric field generated by a voltage applied across lipid bilayer 314 and / or nanopore 316. The tail of the tag can be positioned in the barrel of nanopore 316. The tag held in the barrel of nanopore 316 can generate a unique ionic blockade signal 340 due to the tag’s distinct chemical structure and / or size, thereby electronically identifying the added base to which the tag is attached.

[0079] Alternatively, the nanopore devices contemplated by the disclosure are used for Sequencing by Expansion ("SBX") as shown in FIG.3B, a nanopore-based nucleic acid sequencing method that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer molecule referred to as an xpandomer (“XP molecule” or “XP”). See e.g., U.S. Pat. No.7,939,259, entitled, “High Throughput Nucleic Acid Sequencing by Expansion;” and PCT publication WO2020236526A1, entitled “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing.” In the SBX process, a targetnucleic acid sequence is encoded along the backbone XP sequence with reporter constructs that are separated by ~10 nm that are designed to provide high signal-to- noise, well-differentiated response signals during nanopore translocation. The enhanced signal-to-noise provided by the different response signals provides significantly increased sequence read efficiency and accuracy of XPs relative to native nucleic acid molecules.

[0080] SBX chemistry sequences nucleic acids by creating an XP from a nucleic acid template. This is achieved by encoding the nucleic acid information on a surrogate polymer of extended length which is easier to detect. The surrogate polymer, i.e., XP, is formed by template directed synthesis which preserves the original genetic information of the target nucleic acid, while also increasing linear separation of the individual elements of the sequence data.

[0081] In one embodiment, a method is disclosed for sequencing a target nucleic acid, comprising: 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 XP of a length longer than the plurality of the subunits of the daughter strand, the XP 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 XP.

[0082] In more specific embodiments, the reporter elements for parsing the genetic information may be associated with the tethers of the XP, with the daughter strand prior to cleavage of the at least one selectively cleavable bond, and / or with the XP after cleavage of the at least one selectively cleavable bond. The XP may further comprise all or a portion of the at least one probe or nucleobase residue, and the reporter elements for parsing the genetic information may be associated with the atleast one probe or nucleobase residue or may be the probe or nucleobase residues themselves. Further, the selectively cleavable bond may be a covalent bond, an intra- tether bond, a bond between or within probes or nucleobase residues of the daughter strand, and / or a bond between the probes or nucleobase residues of the daughter strand and a target template.

[0083] In further embodiments, oligomer substrate constructs for use in a template directed synthesis for sequencing a target nucleic acid are disclosed. Oligomer substrate constructs comprise a first probe moiety joined to a second probe moiety, each of the first and second probe moieties having an end group suitable for the template directed synthesis, and a tether having a first end and a second end with at least the first end of the tether joined to at least one of the first and second probe moieties, wherein the oligomer substrate construct when used in the template directed synthesis is capable of forming a daughter strand comprising a constrained XP and having a plurality of subunits coupled in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, the first and second probe moieties and at least one selectively cleavable bond.

[0084] In another embodiment, monomer substrate constructs for use in a template directed synthesis for sequencing a target nucleic acid are disclosed. Monomer substrate constructs comprise a nucleobase residue with end groups suitable for the template directed synthesis, and a tether having a first end and a second end with at least the first end of the tether joined to the nucleobase residue, wherein the monomer substrate construct when used in the template directed synthesis is capable of forming a daughter strand comprising a constrained XP and having a plurality of subunits coupled in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, the nucleobase residue and at least one selectively cleavable bond.

[0085] In yet further embodiments, template-daughter strand duplexes are disclosedcomprising a daughter strand duplexed with a template strand, as well as to methods for forming the same from the template strand and the oligomer or monomer substrate constructs.

[0086] In the embodiment shown in Fig.3B, the XP molecule, 345, includes a translocational control element (TCE, 350) which serves to arrest XP translocation through a nanopore (347). The TCE (350) is surrounded by reporter codes 351 and 352. In this embodiment, TCE (350) has a larger physical bulk relative to that of the reporter codes 351 and 352. XP translocation through the nanopore 347 is arrested when TCE 350 encounters the pore aperture 353. In certain embodiments, both the bulk of the TCE and the charge densities of the reporter codes (i.e., the local electric field at the arrest site) contribute to translocation arrest. During the pause, reporter code 354 is held in the barrel of the nanopore and blocks the flow of current through the pore in a characteristic and detectable manner. To overcome the arrest, a voltage pulse is applied to the system, which forces the TCE to enter and pass through the pore. Translocation then resumes until the next TCE encounters the pore aperture. In one embodiment, SBX chemistry is facilitated by the inclusion of additives that, e.g., enhance the translocation rate of XP molecules through a pore, including but not limited to, stabilizers such as EDTA and redox reagents. In a specific embodiment, concentrations from about 10mM to about 300mM of one or more organic and inorganic redox-capable species are included, e.g., ferri / ferro- cyanide, metal bipyridine compounds such as iron tris-bipyridine or cobalt tris-bipyridine, and modified ferrocenes.

[0087] In the SBS and SBX processes, an electrical signal, e.g., resistance or conductance, of the nanopore including the loaded (threaded) tag or XP can be measured via a signal value (e.g., voltage or a current passing through the nanopore), thereby providing an identification of the species and thus the nucleotide at the position of the template nucleic acid. In some embodiments, a direct current (DC) signal is applied to the nanopore cell (e.g., so that the direction in which the species moves through the nanopore is not reversed). However, operating a nanopore sensorfor long periods of time using a direct current can change the composition of the electrode, unbalance the ion concentrations across the nanopore, and have other undesirable effects that can affect the lifetime of the nanopore cell. Applying an alternating current (AC) waveform can reduce the electro-migration to avoid these undesirable effects and have certain advantages as described below. The nucleic acid sequencing methods described herein are fully compatible with applied AC voltages, and therefore an AC waveform can be used to achieve these advantages.

[0088] D. Methods of Preparing a Nanopore Device for a Sequencing Run

[0089] FIG.4 illustrates an exemplary method to prepare the nanopore device for a sequencing run. Before use, the nanopore device may be provided in a suitable product package with a dry surface or with the cis and trans chambers filled with an appropriate fluid, e.g., system fluid. The nanopore device is prepared as follows: introducing a nanopore composition into the device, introducing a lipid composition into the device under conditions sufficient to form a lipid bilayer between the cis and trans chambers, subjecting the device to an electrical stimulus sufficient to initiate poration of the nanopore into the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore; perfusing the device with an osmolarity buffer; and perfusing the cis chambers with an XP buffer comprising XP molecules.

[0090] The nanopore composition includes a naturally-occurring pore-forming protein, such as -hemolysin from S. aureus, non-naturally occurring mutant or variant of a wild-type pore-forming protein. Alpha-hemolysin is a self-assembling toxin which forms an aqueous channel in the membrane of a host cell. It has many advantageous properties including high stability, self-assembly and a pore diameter which is wide enough to accommodate single stranded DNA but not double stranded DNA (Kasianowicz et al., 1996). A range of naturally and non-naturally occurring nanopores having varying pore-sizes and properties are known in the art. See e.g., US10351908B2, US10934582B2, US10227645B2, US10752948B2, US10683331B2, US20210269870, US10308918B2, US10968438B2, US10947516B2, US10851353B2, US10590480B2, US20200385433, US10752658B2, and US12043648B2.

[0091] In a specific embodiment, the nanopore composition includes up to 1-2nM hemolysin suspended in a pore dilution buffer (denoted in FIG.4 as “P”). The nanopore composition can include up to 500pM, e.g., up to 250pM, up to 150 pM, and more specifically, up to 100pM hemolysin. For example, up to 75 pM hemolysin is suspended in the pore dilution buffer and more specifically, between about 25-75 pM hemolysin, e.g., 50 pM. In a particular embodiment, the nanopore composition comprises wildtype hemolysin, and more specifically, the composition includes wildtype, heptameric hemolysin. Staphylococcus aureus alpha hemolysin wild type sequences are known in the art, see, e.g., National Center for Bioinformatics or GenBank Accession Numbers M90536 and AAA26598. In a specific embodiment, the composition includes a hemolysin molecule comprising 80, 85, 90, 95, 98% or more sequence identity to the nucleic acid coding region and / or the protein coding region of the wild-type alpha hemolysin sequence. Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul, et al., 1990 and Altschul, et al., 1997.

[0092] The pore dilution buffer further comprises ammonium chloride and detergent, e.g., Tween20 or Triton X-100, in a buffered solution, including e.g., HEPES, MOPS, MES, PIPES, PBS, or Tris-HCl, etc. Without wishing to be bound by any theory, it is believed that the presence of excess ammonium chloride and detergent stabilizes pore molecules and enhances pore insertion. Alternatives to ammonium chloride include, but are not limited to, sodium chloride, ammonium sulfate, potassium chloride, ammonium acetate, ammonium bicarbonate, and / or glycine, and any of these alternatives may be used alone or in combination with one or more of the listed alternatives and / or in combination with ammonium chloride. In addition, the detergent can include non-ionic surfactants, such as Tween 80, Tween 20, Triton X- 100, Brij 35, Pluronic F-127, ionic surfactants, such as SDS, and zwitterionic surfactants, such as CHAPS.

[0093] In a specific embodiment, the pore dilution buffer includes up to 3M NH4Cl, e.g., between 1.5-3M, and more specifically, up to 2M NH4Cl. In a particular embodiment, the pore dilution buffer includes up to 2M NH4Cl, 0.5-1.0M urea, 100- 200mM K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), 50-150mM HEPES, pH 6-8. In a particular embodiment, the nanopore composition comprises 25-75 pM wildtype, heptameric hemolysin, 2M NH4Cl, 0.8M urea, 150 mM K3(Fe(CN)6), 150 mM K4Fe(CN)6, 0.001% Tween20, and 100 mM HEPES, pH 6-8.

[0094] The following exemplary pore dilution buffers are provided for illustrative purposes: (a) Alpha-hemolysin: 1.5 nM; ammonium chloride: 2.8 M; urea: 0.8 M; K3(Fe(CN)6): 150 mM; K4(Fe(CN)6): 175 mM; HEPES: 100 mM, pH 7.2; (b) Alpha- hemolysin: 2.0 nM; ammonium chloride: 2.5 M; urea: 0.6 M; K3(Fe(CN)6): 125 mM; K4(Fe(CN)6): 125 mM; HEPES: 120 mM; pH 7.8; (c) Alpha-hemolysin: 1.2 nM; ammonium chloride: 1.9 M; urea: 1.0 M; K3(Fe(CN)6): 200 mM; K4(Fe(CN)6): 100 mM; HEPES: 80 mM; pH 6.5; (d) Alpha-hemolysin: 1.8 nM; ammonium chloride: 3.0 M; urea: 0.5 M; K3(Fe(CN)6): 160 mM; K4(Fe(CN)6): 140 mM; HEPES: 70 mM; pH 6.9; (e) Alpha- hemolysin: 50 pM; ammonium chloride: 2.0 M; urea: 0.8 M; K3(Fe(CN)6): 150 mM; K4(Fe(CN)6): 150 mM; HEPES: 100 mM; 0.001% Tween20; pH 7.4; (f) Alpha-hemolysin: 1.0 nM; ammonium chloride: 1.5 M; urea: 0.9 M; K3(Fe(CN)6): 180 mM; K4(Fe(CN)6): 125 mM; HEPES: 140 mM; pH 7.5; (g) Alpha-hemolysin: 2.0 nM; ammonium chloride: 2.2 M; urea: 0.7 M, K3(Fe(CN)6): 140 mM; K4(Fe(CN)6): 190 mM; HEPES: 100 mM; pH 7.0; (h) Alpha-hemolysin: 75 pM; ammonium chloride: 2.2 M; urea: 0.85 M, K3(Fe(CN)6): 140 mM; K4(Fe(CN)6): 140 mM; HEPES: 100 mM; pH 7.0; (i) Alpha-hemolysin: 65 pM; ammonium chloride: 2.0 M; urea: 0.7 M, K3(Fe(CN)6): 150 mM; K4(Fe(CN)6): 150 mM; HEPES: 100 mM; pH 7.2; (j) Alpha-hemolysin: 1.4 nM; ammonium chloride: 2.0 M; urea: 0.55 M; K3(Fe(CN)6): 110 mM; K4(Fe(CN)6): 160 mM; HEPES: 150 mM; pH 6.7; (k) Alpha- hemolysin: 1.9 nM; ammonium chloride: 1.7 M; urea: 0.9 M; K3(Fe(CN)6): 190 mM; K4(Fe(CN)6): 110 mM; Tween-20: 0.0007%; HEPES: 90 mM; pH 7.3; (l) Alpha-hemolysin: 150 pM; ammonium chloride: 2 M; urea: 0.8 M; K3(Fe(CN)6): 150 mM; K4(Fe(CN)6): 150 mM; Tween-20: 0.001%; HEPES: 100 mM; pH 8.2; (m) Alpha-hemolysin: 1.6 nM;ammonium chloride: 1.8 M; urea: 0.75 M; K3(Fe(CN)6): 130 mM; K4(Fe(CN)6): 200 mM; Tween-20: 0.0009%; HEPES: 75 mM; pH 6.8; (n) Alpha-hemolysin: 2.0 nM; ammonium chloride: 2.0 M; urea: 0.6 M; K3(Fe(CN)6): 170 mM; K4(Fe(CN)6): 140 mM; Tween-20: 0.0010%; HEPES: 110 mM; pH 7.6.

[0095] As shown in Table 1 below, in one embodiment, each step of the protocol may use a decreasing amount of ammonium chloride in the buffer. Table 1. Buffer Composition Pore dilution buffer Hemolysin, High NH4Cl, 0.5-1.0M urea, 100-200mM (P) K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), detergent, 50-150mM HEPES, pH 6-8 Run buffer (R) Moderate NH4Cl, 0.5-1.0M urea, 100-200mM K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), 50-150mM HEPES, pH 6-8 Osmo buffer (O) Lower NH4Cl, 0.5-1.0M urea, 100-200mM K3(Fe(CN)6), 100- 200mM K4(Fe(CN)6), 100mM MES, pH 5-8 XP buffer (X) Lowest NH4Cl, 0.5-1.0M urea, 100-200mM K3(Fe(CN)6), 100- 200mM K4(Fe(CN)6), 2.5-7.5% w / v PEG, 5-15mM MES, up to 3% Acetonitrile, 0.1-0.3% trehalose, 1-3mM HEPES, pH 5-8

[0096] As shown in FIG.4(C), once the pore molecules are introduced into the chambers of the plurality of electrochemical cells, a lipid composition is introduced into the device under conditions sufficient to form a lipid bilayer between the cis and trans chambers. In a specific embodiment, the lipid composition comprises a phospholipid, a bilayer additive, an annular solvent, a silicone oil, and a cosolvent. Without wishing to be bound by any specific theory, each element of the lipid composition contributes to the process of lipid bilayer formation and maintaining the stability of the lipid bilayer during sequencing in a different and complementary manner. The elements of the lipid composition are illustrated in FIGS.5-6 and the relative contribution of each element is listed in Table 2 (below).Table 2. Lipid Composition Components Lipid Composition Purpose Component Phospholipid, e.g., two Allows for planar bilayer formation, attracts XP via charge, tail lipid (FIG.5A) and enables concentrator effect Bilayer additive, e.g., Fills in bilayer gaps or weak points (FIG.5C), supports single tail lipid bilayer curvature at the pore interface (FIG.5D), and dominates the lipid composition in the bilayer Annular solvent, e.g., Stabilizes and supports the bilayer-surface interface (FIG. hydrophobic solvent 5C and 5E) Silicone oil Improves lipid deposition on the CD surface Cosolvent Provides uniform lipid application across the device surface, homogenizes opposing components of the lipid mix

[0097] The lipid composition can comprise a phospholipid, for example, selected from diphytanoyl-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero- 3-phosphocholine, 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methylester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn- glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 1000], 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. In some embodiments, the lipid composition 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.

[0098] In FIG.5A, the phospholipid is shown, preferably a two tail phospholipid. For example, the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DPhPy, DSPE, or mixture thereof. In a specific embodiment, the phospholipid is DPhPE.

[0099] Optionally, the lipid composition also includes a triblock co-polymer, e.g., as described in PCT / EP2024 / 062014 and PCT / EP2024 / 062013, the disclosures of which are incorporated herein by reference in their entireties. Typically, the amount of the triblock copolymer is about 2 % to about 40 % of mass of solids in the mixture with phospholipid that forms the bilayer. Generally, the desired composition of phospholipid and TBC is prepared as a mixture in a solution of silicone oil and hexadecane. The triblock copolymer used in the mixture can have a molecular weight of about 3500 Daltons to about 6500 Daltons. The triblock copolymer typically also has a polydispersity of about 1.1 to about 1.8. It is contemplated, however, that a wider range of polydispersity can be useful in various hybrid membrane applications.

[0100] As described in PCT / EP2024 / 062014 and PCT / EP2024 / 062013, a wide range of exemplary hybrid membranes are based on a lipid bilayer composition using a poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2- oxazoline) triblock copolymer, also referred to herein as a (PMOXA)m-(PDMS)n-(PMOXA)m triblock copolymer. Although triblock copolymers of the structural formula (PMOXA)m-(PDMS)n-(PMOXA)m are known in the art, compositions incorporating such TBCs in lipid bilayers with embedded nanopores, and the use of these hybrid membrane structures in nanopore-based sequencing methods are not.

[0101] TBCs useful in the lipid bilayer compositions of the present disclosure include the TBCs having the following formula: R1-(PMOXA)m-(PDMS)n-(PMOXA)m-R2, where R1 is a terminal headgroup comprising a triazole moiety and R2 is a terminal headgroup comprising a triazole moiety or a hydroxyl group. In this formula “(PMOXA)” is the poly(2-methyl-2-oxazoline) subunit of the polymeric structure. Two (PMOXA) polymers each of m subunits flank and are covalently attached to a central poly(dimethylsiloxane) (or “PDMS”) polymer of n subunits. The terminal headgroups (or “HG”) at R1, and optionally, R2, are each a chemical group covalently linked to a terminal (PMOXA) subunit. In at least one embodiment, the TBC is symmetric, with both R1 and R2 being the same terminal HG. In at least one embodiment, the TBC is asymmetric, with R1 and R2 being different terminal HGs. In at least one embodiment, the TBC is asymmetric, and the R2 group is a terminal hydroxyl group (or “-OH”) and the R1 group a terminal HG.

[0102] In a particular embodiment, the phospholipid includes a small zwitterionic head group. It has been observed that the relative size and concentration of the phospholipid head group has an impact on the function of the concentrator component in the XP. As described in U.S. Patent No.9,670,526, which is incorporated herein by reference in its entirety, by presenting a thin film of sample across a large nanopore sensor array, the target molecules can diffuse to the sensor surface in reasonable time periods. If the sensor surface is primarily a hydrophobic domain, target molecules modified with at least one hydrophobic capture element associate and diffuse along the surface, thereby increasing the likelihood of being detected. For example, it has been found that a phospholipid head group such as a PE head group with a primary amine showed 3-5x higher throughput than a PC head group with a quaternary amine. Although the concentrator functions when a PC headgroup is used, it is not as effective as a phospholipid including PE headgroups. It was also observed that increasing the length of the concentrator compensates for the PC head group's ability to interface as effectively with the concentrator. It was found that lipid compositions including mixtures of PE and PC head groups were able to maintain the concentrator effect. For example, 50:50 mixtures were comparable to 100:0 PE:PC mixtures. Optionally, the lipid composition includes between 100:0 to 25:75 PE:PC. In a specific embodiment, the lipid composition includes 50:50 PE:PC, and in a more specific embodiment, the lipid composition includes 25:75 PE:PC.Additional components of the phospholipid include one or more of the following: adding bridging species that span PE and PC molecules, such as methyl and dimethyl bridging species, increasing the number of positively charged species in the phospholipid structure, e.g., adding one or more additional amine species, to aid in concentrating XP at the lipid surface, and including chaotropic reagents, such as imidazole and guanidine, to leverage known interactions between XPs and chaotropes and improve XP solubility at the lipid surface.

[0103] FIG.6 illustrates the interaction of the various components in the lipid composition. The bilayer additive increases the presence of sequencing pores and is immiscible with oils. The bilayer additive includes, but is not limited to, single-tail lipids or single-chain lipids, lauryl dimethylamine-N-oxide, octyl beta-D- glucopyranoside, sodium dodecyl sulfate, Triton X-100, CHAPS, Span 20, Span 60, Span 80, lysophospholipids (LPC or LPE), sphingosine, sphinganine, monoglycerides, cationic lipids (e.g., CTAB or DTAC), nonionic surfactants, and combinations thereof. In a specific embodiment, the bilayer additive is a single-tail lipid, such as Span 80.

[0104] The cosolvent solubilizes the bilayer additive with other lipid components and provides uniform deposition to the device surface. For example, a suitable cosolvent is e.g., branched or unbranched, saturated or unsaturated hydrocarbons, such as decane, tridecane, octadecane, pentadecane, tetradecane, dodecane, undecane, squalene, cyclohexane, heptane, hexane, hexadecane, as well as aromatic solvents, such as toluene and xylene. In a specific embodiment, thecosolvent is hexadecane, alone or in combination with one or more additional branched or unbranched hydrocarbons, including but not limited to decane, tridecane, octadecane, pentadecane, tetradecane, dodecane, and undecane.

[0105] The annular solvent stabilizes and supports the bilayer-surface interface and reduces solution viscosity for deposition. In a specific embodiment the annular solvent is a hydrophobic solvent such as hexane, chloroform, dichloromethane, toluene, cyclohexane, benzene, diethyl ether, petroleum ether, isooctane, octanol, and / or carbon tetrachloride. In a more specific embodiment, the annular solvent is hexane. The annular solvent can also be mixed with additional solvent components, e.g., tert-butanol, n-butanol, sec-butanol, isobutanol, butan-2- ol, ethanol, isopropanol, acetone, ethyl acetate, either alone or in combination. In one embodiment, the annular solvent is a mixture of hexane and tert-butanol.

[0106] Finally, silicone oil improves lipid deposition on the device surface. Suitable silicone oil includes PDM20, mineral oil, squalene, polyalphaolefins, medium- chain triglycerides, isopropyl myristate, caprylic / capric triglycerides, perfluoropolyether, and / or hydrogenated polyisobutene. In a specific embodiment, the silicone oil is PDM20.

[0107] The ratios of various components in the lipid composition can be adjusted to optimize the interactions between components. For example, in one embodiment, the amount of bilayer additive is at least about twice as much as the amount of phospholipid, e.g., at least four times as much, specifically, at least six times as much, and more specifically, at least eight times as much. Likewise, the amount of silicone oil in the composition is at least twice as much as the amount of cosolvent, e.g., at least four times as much, specifically, at least six times as much, and more specifically, at least eight times as much. In a specific embodiment, the molar ratio of phospholipid to bilayer additive is about 1:8, and the molar ratio of silicone oil to cosolvent is about 9:1. The following is a list of exemplary lipid compositions: (a) DPhPC, 10 mM; Triton X-100, 80 mM; Hexadecane, 2 mM; Cyclohexane, 15 mM; Mineral Oil, 18 mM; (b) DOPE, 6 mM; Lysophosphatidylcholine (LPC), 48 mM; Decane, 1 mM;Hexane, 12 mM; Squalene, 9 mM; (c) DPPC, 8 mM; CHAPS, 64 mM; Dodecane, 1.5 mM; Toluene, 13 mM; PDM20, 13.5 mM; (d) DMPE, 5 mM; Cationic Lipid (CTAB), 40 mM; Tetradecane, 1.2 mM; Chloroform, 10 mM; Hydrogenated Polyisobutene, 10.8 mM; (e) DSPE, 7 mM; Span 80, 56 mM; Undecane, 2.2 mM; Dichloromethane, 14 mM; Medium- Chain Triglycerides, 19.8 mM; (f) DPhPE, 9 mM; Sphingosine, 72 mM; Octadecane, 3 mM; Isooctane, 20 mM; Mineral Oil, 27 mM; (g) DPPE, 4 mM; Monoglycerides, 32 mM; Tridecane, 0.6 mM; Benzene, 6 mM; Squalene, 5.4 mM; (h) DOPhPC, 12 mM; Lauryl Dimethylamine-N-Oxide, 96 mM; Pentadecane, 4 mM; Diethyl Ether, 25 mM; Polyalphaolefins, 36 mM (i) DMPC, 15 mM; Sodium Dodecyl Sulfate (SDS), 120 mM ; Decane, 5 mM; Cyclohexane, 30 mM; Isopropyl Myristate, 45 mM; (j) DPhPC, 10 mM; Nonionic Surfactant (Span 60), 80 mM; Hexadecane, 2 mM; Chloroform, 10 mM; Perfluoropolyether, 18 mM.

[0108] In a specific embodiment, the lipid composition includes one or more of the following components: (a) at least 1 mg / ml phospholipid; (b) at least 6 mg / ml bilayer additive, (c) between 5-10% annular solvent, (d) between 8-12% cosolvent, and (e) between 75-85% silicone oil. In a further specific embodiment, the lipid composition includes at least 1.5 mg / ml DPhPE, at least 5 mg / ml Span 80, between 3- 10% hexane, between 3-12% hexadecane, and between 75-85% PDM20. For example, the lipid composition includes about 8.1mg / ml Span 80, 1.9 mg / ml DPhPE, 6.5% hexane, 83.4% PDM20, 9.3% hexadecane.

[0109] Alternatively, the lipid composition includes about 5-6 mg / mL Span80, 1.25mg / mL DPhPE, 4-6% hexane, up to 10% tert-butanol, about 75-85% PDM20, 4-6% hexadecane, 40-60% of each of the following: pendadecane, tetradecane, tridecane, dodecane, undecane, and decane. Other alternatives include: (a) Span 80: 5.5 mg / mL; DPhPE: 1.25 mg / mL; Hexane: 5%; Tert-butanol: 8%; PDM20: 80%; Hexadecane: 5%; 40- 60% of each of the following: Pentadecane, Tetradecane, Tridecane, Dodecane, Undecane, Decane; (b) Span 80: 6 mg / mL; DPhPE: 1.25 mg / mL; Hexane: 4.5%; Tert- butanol: 9%; PDM20: 85%; Hexadecane: 6%; 40-60% of each of the following: Pentadecane, Tetradecane, Tridecane, Dodecane, Undecane, Decane; (c) Span 80: 5mg / mL; DPhPE: 1.25 mg / mL; Hexane: 6%; Tert-butanol: 10%; PDM20: 75%; Hexadecane: 4.5%; 40-60% of each of the following: Pentadecane, Tetradecane, Tridecane, Dodecane, Undecane, Decane; (d) Span 80: 5.8 mg / mL; DPhPE: 1.25 mg / mL; Hexane: 4%; Tert-butanol: 7.5%; PDM20: 80%; Hexadecane: 4.8%; 40-60% of each of the following: Pentadecane, Tetradecane, Tridecane, Dodecane, Undecane, Decane; (e) Span 80: 5.2 mg / mL; DPhPE: 1.25 mg / mL; Hexane: 5.2%; Tert-butanol: 6%; PDM20: 78%; Hexadecane: 5.5%; 40-60% of each of the following: Pentadecane, Tetradecane, Tridecane, Dodecane, Undecane, Decane.

[0110] The lipid composition can be introduced to the device in a single volume or multiple smaller volumes at a flow rate of up to 20uL / s, and up to a 3 minute pause in between each successive volume of lipid introduced to the device. The lipid composition may be followed by a volume of run buffer, formulated alone or in combination with a solvent or detergent suitable to thin excess lipid deposited on the device surface. Once the lipid composition is introduced to the cell, a lipid bilayer is formed (see, e.g., U.S. Patent No.12,000,822, which is incorporated herein by reference in its entirety). In one embodiment, the bilayer is formed by applying multiple volumes of run buffer, e.g., up to 8, 125 uL volumes of run buffer introduced at a rate of 20-120uL / s with a pause between volumes, with or without the application of a waveform. When lipids are first deposited into the cells to form the lipid bilayers, bilayers may form spontaneously and other cells merely have a thick lipid membrane spanning across the wells of the cells, wherein multiple layers of lipid molecules and solvent are combined together. Various techniques may be employed to initiate lipid bilayer formation, e.g., mechanical, electrical, or physical stimuli may be applied. Those of ordinary skill in the art will appreciate that various stimuli may be suitable for use with the methods and compositions described herein. One or more types of lipid bilayer initiating stimuli may be applied simultaneously, or in different order and the one or more types of stimuli may be applied multiple times, see., e.g., U.S. Patent Publication No.20170283867A1, which is incorporated herein by reference in its entirety.

[0111] Excess lipid composition is removed by the addition of a run buffer (“R” in FIG.4). In one embodiment, the run buffer comprises up to 2M NH4Cl, 0.5-1.0M urea, 0-200mM K3(Fe(CN)6), 0-200mM K4(Fe(CN)6), 50-150mM HEPES, pH 6-9. Suitable run buffer compositions include the following: (a) 2M NH4Cl, 0.5-1.0M urea, 100-200mM K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), 50-150mM HEPES, pH 6-9; (b) 1.5M NH4Cl, 0.7M urea, 150mM K3(Fe(CN)6), 175mM K4(Fe(CN)6), 120mM HEPES, pH 7.2; (c) 2M NH4Cl, 0.9M urea, 180mM K3(Fe(CN)6), 110mM K4(Fe(CN)6), 50mM HEPES, pH 6.8; (d) 1.3M NH4Cl, 0.6M urea, 100mM K3(Fe(CN)6), 130mM K4(Fe(CN)6), 150mM HEPES, pH 7.5; (e) 1.8M NH4Cl, 1.0M urea, 140mM K3(Fe(CN)6), 200mM K4(Fe(CN)6), 70mM HEPES, pH 7.0; (f) 2.0M NH4Cl, 0.75M urea, 120mM K3(Fe(CN)6), 160mM K4(Fe(CN)6), 90mM HEPES, pH 6.5; (g) 1.7M NH4Cl, 0.85M urea, 170mM K3(Fe(CN)6), 120mM K4(Fe(CN)6), 60mM HEPES, pH 7.8; (h) 1.4M NH4Cl, 0.5M urea, 200mM K3(Fe(CN)6), 140mM K4(Fe(CN)6), 75mM HEPES, pH 6.7; (i) 2M NH4Cl, 0.8M urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM HEPES, pH 8.2; (j) 1.2M NH4Cl, 0.95M urea, 130mM K3(Fe(CN)6), 180mM K4(Fe(CN)6), 140mM HEPES, pH 7.4; (k) 1.9M NH4Cl, 0.63M urea, 160mM K3(Fe(CN)6), 190mM K4(Fe(CN)6), 75mM HEPES, pH 7.1; (l) 1.6M NH4Cl, 0.52M urea, 110mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM HEPES, pH 6.9.

[0112] Referring once more to FIG.4, after bilayer is formed between the walls spanning the well of the cell, the device is subjected to an electrical stimulus sufficient to initiate poration of the nanopore into the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore. Nanopores can be inserted into the lipid bilayer a number of different ways. For example, if relying on force of pressure in the device to randomly diffuse the pores into the membranes, then the proportion would be governed by binomial distribution. In such a situation, many cells would have zero nanopores, some would have one, some would have two, and the majority would not have just one. However, it is preferable to have a single nanopore for each cell. According to certain embodiments, electroporation can be used to insert the nanopores into the bilayer. In some embodiments, electroporation applies a square wave across the bilayer to stress it. Too high a voltage would pop the lipidlayer. But, a suitable voltage can provide a tear where the nanopore can be inserted more easily. In order to ensure that a single nanopore is inserted into the bilayer a diagnostic measurement can be taken for each cell before, during, and after the electroporation signal is applied, e.g., a voltage value akin to an open channel measurement. The measured value can then be then analyzed to determine whether the measured value corresponds to a value that would be expected for a cell having only one nanopore. A single nanopore may be detected by tracking voltage changes during the electroporation process, and if the voltage changes significantly then it is assumed that poration has successfully completed. When a nanopore is observed to have been added to a cell, the electroporation process can be stopped for that well. This can be done independently for each well. For those cells that do not have any pores after the first electroporation step, the electroporation may be repeated. See, e.g., U.S. Patent No.10,816,537, which is incorporated herein by reference in its entirety. After a pore is inserted into a membrane of a cell, the voltage across the membrane begins to drop rapidly due to the relatively high conductance of the pore. The decrease in voltage across the membrane reduces the driving force for additional pore insertion in the membrane.

[0113] The methods described herein may be used alone or in combination with other methods to optimize pore insertion, e.g., adaptive poration and self-limiting poration, as described in U.S. Patent Publication No.20230105456A1, the disclosure of which is incorporated herein by reference in its entirety.

[0114] Achieving a high yield of the single insertion of correctly oriented protein nanopores into the suspended membrane in the array is important to high throughput sequencing. As shown in FIG.4, protein pores (e.g. alpha-hemolysin (aHL)) are dispensed into the flow channel prior to membrane formation. The protein pores are notionally trapped below the membranes (trans side), in the well structures of the array. The protein pores are then inserted into the membrane through the application of voltage with an approach designed to singly insert the pores and minimize the occurrence of multiply porated membranes (“multipores”).

[0115] In addition, it is desirable to reuse the device. However, it was found that positions of the array located farther from the inlet of the flow path result in unporated membrane over the extended use. Atomic Force Microscopy (AFM) imaging revealed that the farther from the inlet of the flow path, the less the number of adsorbed protein pores. When the adsorbed protein pores are below a certain threshold, the probability to insert a pore into the membrane decreases. Without wishing to be bound by any particular theory, this phenomenon may be due to the loss of protein pores when a single bolus of protein pore containing solution is continuously dispensed into the lane at a slow volumetric flow rate. The loss happens when protein pores from the bulk solution diffuse and adsorb onto the surface.

[0116] It was found that the surface concentration of protein on the active surface of the flow channel can be made uniform along the flow path. Specifically, protein pore (e.g. aHL) with the volume of the microfluidic channel is dispensed at a sufficiently high volumetric flow rate. This prevents the loss of protein pores (depletion of the bulk concentration) by surface adsorption along the flow path during continuously slow flow rate. After the flow, there is a controlled amount of incubation (reduced or no flow) time necessary for protein pores to diffuse to the surface. This fast flow and subsequent incubation may be repeated multiple times. This allows for a surface adsorption process that is uniform across the surface in the flow path, as can be measured by AFM and inferred from local pore insertion event rates.

[0117] Therefore, the nanopore composition can be introduced into the device using an iterative pulsed flow to increase uniform delivery of nanopore molecules across the fluidic path. For example, the iterative pulsed flow comprises two or more applications of a portion of the nanopore composition at a flow rate of between 1-500 uL / sec, e.g., 5-100 uL / sec, e.g., 15-75 uL / sec, 20-50 uL / sec, 20-30 uL / sec, or 25 uL / sec. Each application of the portion of nanopore composition can be followed by a waiting period of up to 2 minutes before an additional application is introduced.

[0118] In one embodiment, the iterative pulsed flow comprises 2 to 20applications of the portion of nanopore composition. For example, the pulsed flow rate comprises between 2 to 15, 2 to 10, 2 to 7, or 3 to 5 applications of the portion of nanopore composition.

[0119] The waiting period between the two or more applications of the portion of the nanopore composition is between is about up to 10 minutes, e.g., up to 5 minutes, or up to 3 minutes, or up to 2 minutes, or 30-90 seconds, or 60 seconds.

[0120] Alternatively, the flow rate is an iterative ramped flow rate, e.g., over a total nanopore application time of e.g., up to 60 minutes, at the end of which the total nanopore composition is introduced, the flow rate is adjusted from a high flow rate to a lower flow rate iteratively. The higher flow rate introduces a volume of nanopore composition, e.g., at a flow rate of between 1-500 uL / sec, e.g., 5-100 uL / sec, e.g., 15- 75 uL / sec, 20-50 uL / sec, 20-30 uL / sec, or 25 uL / sec, and the flow rate is subsequently adjusted to a lower flow rate, e.g., up to 5 uL / sec, or up to 2 uL / sec, or up to 1 uL / sec for a period of time equivalent to the waiting period, e.g., about up to 10 minutes, e.g., up to 5 minutes, or up to 3 minutes, or up to 2 minutes, or 30-90 seconds, or 60 seconds. Thereafter, the flow rate is readjusted to the higher flow rate, which is the same or different from the initial high flow rate and followed by a lower flow rate. This iterative ramped process is repeated until the total nanopore composition is introduced.

[0121] In one embodiment, the coefficient of variation (CV) of surface adsorption of nanopore molecules across the fluidic path is below 50%, e.g., below 30%. In a specific embodiment, the incidence of multipore insertion events is reduced relative to a constant flow.

[0122] The nanopore composition can be introduced to the device via reagent stacking, as described herein. For example, each sub-volume of nanopore composition can be drawn into the fluidic interface, with a volume of air between each sub-volume, such that the total nanopore composition is drawn into the fluidic interface at once. This process is illustrated in FIGs 14E-14F for pre- and post- sequencing cleaning protocols, but the process can also be used to introduce thenanopore composition to reduce time.

[0123] As shown in FIG.4, after pores are inserted into the lipid bilayer, the osmolarity imbalance across the membrane can be adjusted by introducing an osmolarity buffer. Manipulating the osmolarity imbalance across the membrane is described, e.g., in International Patent Publication No. WO2018001925, which is incorporated herein by reference in its entirety. Each of the pore dilution and run buffers includes osmolytes, i.e., soluble compounds that increase the osmolality of that solution and osmolytes affect osmosis across the lipid bilayer. Suitable osmolytes include, without limitation, ionic salts such as lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), ammonium chloride, lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2); urea; polyols and sugars such as glycerol, erythritol, arabitol, sorbitol, mannitol, xylitol, mannose mannitol, glycosyl glycerol, glucose, fructose, sucrose, trehalose, inositol, and isofluoroside; polymers such as dextrans, levans, and polyethylene glycol; and some amino acids and derivatives thereof such as glycine, trimethylglycine, alanine, alpha-alanine, arginine, proline, taurine, betaine, octopine, glutamate, sarcosine, y- aminobutyric acid, ectoine, and trimethylamine N-oxide ("TMAO") (see also e.g., Fisher et al. U.S. 20110053795, incorporated herein by reference in its entirety).

[0124] In one embodiment, the present disclosure utilizes a solution comprising an osmolyte, wherein the osmolyte is an ionic salt. Those of ordinary skill in the art will appreciate other compounds that are suitable osmolytes for use in the methods and compositions described herein, and optionally solutions including two or more different osmolytes are also contemplated. In a specific embodiment, the osmolyte is ammonium chloride (NH4Cl).

[0125] In one embodiment, the osmolarity buffer includes a salt / electrolyte buffer solution with a lower osmolarity / osmotic concentration than the osmolarity of the salt buffer solution below the lipid membrane in order to introduce an osmoticimbalance between the salt buffer solution above and below the lipid membrane, which causes the lipid solvent membrane to bow upwards. With the lipid membrane pushed outward from the well, a greater contact surface area of the lipid membrane is exposed to the flow of the salt buffer solution and, as a result, the flow of the salt buffer solution can more effectively remove any excess lipid solvent, such that the thick lipid membrane can be thinned out and transitioned into a lipid bilayer more efficiently. This technique has many advantages, including reducing the time to form lipid bilayers and increasing the efficiency and yield of the nanopore based sequencing device.

[0126] In the method illustrated in FIG.4, the concentration of osmolyte in the buffer below the lipid membrane in step (C) is up to 2M. Therefore, in order to adjust the osmolarity of the buffer solution below the membrane, an osmolarity buffer (“O” in Fig.4) is introduced with a concentration of osmolyte below 2M, e.g., up to 1.75M or between 1.5-1.75M. In a specific embodiment, the osmolarity buffer includes between 1.5-2.0M NH4Cl, 0.2-1.2M urea, 100-200mM K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), 50- 150mM MES, pH 5.5-6.0. The following is a list of suitable alternative osmolarity buffer compositions: (a) 6M NH4Cl, 0.8M urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100 mM MES, pH 5.8; (b) 1.8M NH4Cl, 0.9M urea, 150mM K3(Fe(CN)6), 180mM K4(Fe(CN)6), 120 mM MES, pH 6.7; (c) 1.5M NH4Cl, 0.6M urea, 120mM K3(Fe(CN)6), 140mM K4(Fe(CN)6), 95 mM MES, pH 7.5; (d) 1.7M NH4Cl, 0.7M urea, 100mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 80 mM MES, pH 6.9; (e) 1.95M NH4Cl, 1.0M urea, 135mM K3(Fe(CN)6), 200mM K4(Fe(CN)6), 110 mM MES, pH 7.2; (f) 1.7M NH4Cl, 0.85M urea, 190mM K3(Fe(CN)6), 130mM K4(Fe(CN)6), 140 mM MES, pH 7.8; (g) 2.0M NH4Cl, 0.95M urea, 180mM K3(Fe(CN)6), 160mM K4(Fe(CN)6), 50mM MES, pH 7.0; (h) 1.6M NH4Cl, 0.8M urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100 mM MES, pH 5-6.

[0127] Finally, as shown in FIG.4, the cis chamber of the cell is perfused with XP buffer comprising XP molecules in order to introduce XP molecules to the surface of the cell for a sequencing run. The XP buffer includes between 1:2 to 1:15 XP molecules:buffer, i.e., one part XP molecule to two parts buffer and up to 1 part XPmolecule to 15 parts buffer. In one embodiment, the XP buffer includes between 1:2 to 1:10 XP molecules:buffer, and more specifically 1:3 to 1:6 XP molecules to buffer. In a specific embodiment, the XP buffer comprises between 1:3 to 1:6 XP molecules suspended in up to 1.0M NH4Cl, 2.5-7.5% PEG8k; 0.2-1.2M urea, 100-200mM K3(Fe(CN)6), 100-200mM K4(Fe(CN)6), 5-15mM MES, up to 3% acetonitrile, 0.1-0.3% trehalose, 1-3mM HEPES, final pH 5-8. For example, the XP buffer comprises up to 1.0M NH4Cl, 800 mM urea, 150 mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 5% w / v PEG8000, 10mM MES (pH 5), adjusted to a final pH of 5.95, 2.8% acetonitrile, 0.2% trehalose, 2mM HEPES.

[0128] Suitable alternatively XP buffer compositions include the following: (a) up to 1.0M NH4Cl, 800 mM urea, 150 mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 5% w / v PEG8000, 10mM MES (pH 5), adjusted to a final pH of 5.95, 2.2-3.2% acetonitrile, 0.2% trehalose, 2mM HEPES; (b) 0.8M NH4Cl, 700 mM urea, 150 mM K3(Fe(CN)6), 180mM K4(Fe(CN)6), 5% w / v PEG8000, 10mM MES (pH 5), 1.2% acetonitrile, 0.2% trehalose, 2mM HEPES, pH 6.7; (c) 0.5M NH4Cl, 900 mM urea, 175 mM K3(Fe(CN)6), 140mM K4(Fe(CN)6), 6.8% w / v PEG8000, 12.5mM MES (pH 5), 2.5% acetonitrile, 0.3% trehalose, 1.5mM HEPES, pH 7.2; (d) 0.9M NH4Cl, 600 mM urea, 120 mM K3(Fe(CN)6), 125mM K4(Fe(CN)6), 7% w / v PEG8000, 5mM MES (pH 5), 0.8% acetonitrile, 0.1% trehalose, 2.8mM HEPES, pH 6.2; (e) 0.4M NH4Cl, 1000 mM urea, 110 mM K3(Fe(CN)6), 200mM K4(Fe(CN)6), 3.5% w / v PEG8000, 8.5mM MES (pH 5), 2.3% acetonitrile, 0.25% trehalose, 1.0mM HEPES, pH 7.8; (f) 0.6M NH4Cl, 850 mM urea, 140 mM K3(Fe(CN)6), 115mM K4(Fe(CN)6), 7.5% w / v PEG8000, 15mM MES (pH 5), 0.9% acetonitrile, 0.15% trehalose, 2.5mM HEPES, pH 7.0; (g) 1.0M NH4Cl, 800 mM urea, 150 mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 5% w / v PEG8000, 100mM MES (pH 5), 2.8% acetonitrile, 0.2% trehalose, 2.0mM HEPES; (h) 0.3M NH4Cl, 950 mM urea, 180 mM K3(Fe(CN)6), 160mM K4(Fe(CN)6), 5.3% w / v PEG8000, 11mM MES (pH 5), 1.8% acetonitrile, 0.22% trehalose, 2.2mM HEPES, pH 6.8; (i) 0.7M NH4Cl, 500 mM urea, 100 mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 3.7% w / v PEG8000, 9mM MES (pH 5), 2.9% acetonitrile, 0.3% trehalose, 3.0mM HEPES, pH 6.0.

[0129] SBX requires judicious application of XP molecules to the device. XP is typically produced in picomole quantities but the success of SBX hinges on the ability to capture a sufficient number of XP molecules in protein nanopores associated with individual sensors in the array, and subsequently read the base-representative information, in a timely manner. There is a potential trade-off between efficiency of XP use (and implicitly therefore also input sample DNA material) and quantity of XP read per unit time.

[0130] Allowing flexibility in the quantity of XP delivered and the observation time before additional volumes of XP are introduced to the device enables expediency and efficiency. For example, while it may be acceptable to deliver a large volume of high concentration of XP (representing a large quantity of input sample DNA) to obtain the necessary number of sequence reads in a short amount of time when the sample DNA and XP are not limiting factors, certain other applications of SBX may require greater efficiency of XP use (and thus input sample DNA) to maximize the information extracted, regardless of the observation times.

[0131] In one embodiment, an initial volume of XP buffer is added to the device before a sequencing run is initiated, followed by one or more additional volumes of XP buffer during sequencing. This method maximizes throughput by driving XP presentation to the bilayer at the start of the sequencing run and replenishing the bilayer with a fresh quantity of XP throughout the sequencing run. This method also reduces the time XP molecules are stored in a higher salt environment, reducing the likelihood of degradation.

[0132] The initial volume of XP buffer is up to twice the volume of the flow cell and each additional volume can be equivalent or smaller volumes. The volume of the flow cell may vary depending on the configuration of the device. For example, the flow cell volume may be up to 100uL, e.g., 25-75uL, and more specifically, between 25-35uL. In a specific embodiment, the first and second volumes of XP buffer are each about twice the volume of the flow cell and each subsequent volume is equal to or less than the volume of the flow cell. The amount of time between each volume of XPbuffer is approximately equal to the mixing time needed on the system, e.g., between about 2-4 minutes between volumes.

[0133] FIG.7A illustrates a method of introducing XP buffer to the device in a single application. By contrast, FIG.7B shows a multi-load approach, as described above. In the multi-load approach, the cis chambers are perfused with an initial volume of XP buffer comprising, e.g., 1:3-1:6 XP molecules suspended in the XP buffer, at a first time interval. Optionally, sequencing starts after an initial waiting period of up to 5 minutes, e.g., 2-4 minutes, or approximately 2 minutes. A sequencing run is initiated by applying an electrical stimulus to the plurality of electrochemical cells to cause at least a portion of the XP molecules to translocate through the nanopore, and while the electrical stimulus is being applied to the device, one or more additional volumes of XP buffer are added at a subsequent time during the sequencing run. In one exemplary embodiment, the first load of XP is approximately 15-20% of the total amount of XP, and each subsequent load (the one or more middle loads and the final last load) is the same or different than the first load. The amounts and timing of introducing each XP load can be adjusted by the skilled artisan without departing from the spirit or scope of the invention.

[0134] In one embodiment, the duration of a sequencing run is up to 180 minutes and one or more additional volumes of XP buffer are introduced at (180-n) minutes, where n is greater than or equal to 1. The one or more additional volumes of XP buffer can be introduced at a predetermined time interval over the duration of a sequencing run, e.g., up to every 1, 2, 5, 10, 20, 30, or 60 minutes over the duration of a 180 minute sequencing run, e.g., until all XP buffer has been introduced.

[0135] Alternatively, the one or more additional volumes of XP buffer can be introduced at a rate that maintains a relatively constant concentration of XP buffer present during the sequencing run to maximize sequencing throughput. In this alternative approach, the sequencing run can be monitored to determine the rate of XP reads throughout the sequencing run and one or more additional volumes of XP buffer can be introduced when the rate of XP reads during the sequencing run startsto decline or falls below a steady state.

[0136] The initial volume and one or more additional volumes of XP buffer can be introduced at variable volumes and flow rates over the course of a sequencing run or at consistent and stable volumes and flow rates. In one embodiment, the initial and subsequent volumes of XP buffer are introduced at a flow rate of up to 30uL / second, up to 15uL / second, or up to 10 uL / second. Alternatively, the initial and subsequent volumes of XP buffer are introduced at a flow rate of up to 10uL / second, e.g., up to 5uL / second, or more specifically, up to 1uL / second. The flow rate of the initial volume of XP buffer may differ from the flow rate of the subsequent volumes of XP buffer, e.g., the initial volume is introduced at a flow rate of up to 10uL / second and subsequent volumes are introduced at a slower rate, e.g., up to 5uL / second or up to 1uL / second. In one embodiment, at least the initial volume is introduced at a faster flow rate than the subsequent volumes, e.g., the initial volume is introduced at a flow rate of up to 30uL / second, up to 15uL / second, or up to 10 uL / second, and the subsequent volumes are introduced at a slower rate than the initial volume, e.g., up to 5uL / second or up to 1uL / second. Alternatively, the flow rate of the one or more additional volumes may steadily decline relative to the initial volume, e.g., the initial volume is introduced at a flow rate of up to 30uL / second, and the flow rate of introduction of the one or more additional volumes decreases by up to 50% of the flow rate of the previous volume, such that if the initial volume is introduced at a flow rate of 30uL / second, the second volume is introduced at a flow rate of about 15uL / second, the third volume is introduced at a flow rate of about 7.5uL / second, the fourth volume is introduced at flow rate of about 3.25uL / second, the fifth volume is introduced at a flow rate of about 1.63uL / second, and the sixth volume is introduced at a flow rate of about 0.8uL / second. In a specific embodiment, the one or more additional volumes include up to 10 additional volumes of XP buffer, e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional volumes of XP buffer. For example, the one or more additional volumes include up to 8 additional volumes and more specifically, up to 6 additional volumes.

[0137] In one embodiment, if the sequencing run is about 180 minutes, theinitial and one or more additional volumes of XP buffer are introduced over a defined dispensing time period, e.g., up to one hour, up to 45, 30, 20, 15, 10, or 5 minutes, and the dispensing period is followed by a gap during which no XP buffer is flowed. The gap can be less than or equal to the duration of the dispensing time period, e.g., equal to the dispensing time period, up to 75% of the dispensing time period, up to 50% of the dispensing time period, up to 25% of the dispensing time period, or up to 10% of the dispensing time period. In one embodiment, the dispensing time period is about 20-40 minutes and the gap is about 10-20 minutes, e.g., the dispensing time period is about 30 minutes and the gap is about 15 minutes of a 180 minute sequencing run that includes an initial volume and at least one additional volume of XP buffer.

[0138] The sequencing run can be initiated immediately after the initial volume of XP buffer is introduced or up to 5 minutes, up to 4, 3, 2, or 1 minute after the initial volume of XP buffer is added.

[0139] In a specific embodiment, the one or more additional volumes of XP buffer are introduced at a predetermined time interval during the sequencing run, e.g., up to 5 minutes, or specifically between 2-4 minutes after the previous volume of XP buffer is introduced until all of the XP buffer is added to the device.

[0140] Reagents can be introduced to the sequencing device in a stacked manner, i.e., if multiple reagents are used in a portion of the sequencing workflow, such as in PSP cleaning, lipid application, XP loading, sequencing, and / or end of run cleaning, each volume of reagent used in that portion of the workflow can be drawn into the fluidic introduction port, optionally separated by a volume of air, and then sequentially introduced to the device over the course of that portion of the workflow to reduce time and / or decrease potential cross-contamination of reagents. For example, if reagent stacking is used for lipid application and the order of introduction of reagents to the device are (1) lipid, and (2) run buffer, then the run buffer volume is first drawn into the fluidic introduction port (as it will be the last reagent introduced to the device), optionally followed by a volume of system fluid or running buffer, and then the volume of lipid is drawn into the port, optionally followed by another volumeof system fluid or running buffer. Therefore, all of the reagents needed for lipid application are contained in the fluidic introduction port at once and available for introduction to the device in the order in which each should be applied to the device.

[0141] Likewise, if reagent stacking is used for XP loading and a multi-load approach is taken, then each volume of XP is drawn into the fluidic interface, optionally with a volume of system fluid, running buffer, or other suitable buffer between each volume of XP.

[0142] E. Sequencing

[0143] Suitable conditions for measuring changes in an electrical signal passing through the nanopores are known in the art and examples are provided herein. The measurement can be carried out with a voltage applied across the membrane and pore. In some embodiments, the voltage used ranges from -400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from - 400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0 mV, and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. The voltage used can be in the range from 50 mV to 450 mV. It is possible to increase discrimination between different nucleotides and / or subcomponents of an XP molecule by a nanopore using an increased applied potential.

[0144] Sequencing nucleic acids using AC waveforms and tagged nucleotides is described in US Patent Publication No. US 2014 / 0134616, which is herein incorporated by reference in its entirety, and the methods described in the referenced published patent application apply to sequencing XP molecules as well. A specific embodiment of conditions for measuring changes in an electrical signal passing through the nanopores are described in PCT / EP2021 / 065252, filed June 8, 2021, the disclosure of which is incorporated herein by reference.

[0145] An embodiment of a circuit and its use in a nanopore sequencing method are shown in FIGS.8A-8C (see also, U.S. Patent No.10,215,731, which is incorporated herein by reference in its entirety) FIG.8A illustrates an example of anelectrical circuit 800 for a cell, such as cells 200 as exemplified in FIG.2 or cell 300 as exemplified in FIG.3. This electrical circuit 800 may include portions or components of the electrical circuit discussed above with reference to FIG.2 and FIG.3. Electrical circuit 800 has a membrane, as described hereinabove, 812 situated between the cell’s working electrode 814 and a counter electrode 816 (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 812. The membrane 812 is also in contact with a bulk liquid / electrolyte 818. 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 820. The common potential and the counter electrode are common to all of the cells.

[0146] Switch 806 is a transistor that can be used to connect or disconnect the membrane 812 and the working electrode 814 from electric circuit 800. Switch 806 is controlled by a memory bit 802 to enable or disable a voltage stimulus to be applied across the membrane in the cell. Electrical circuit 800 includes an on-chip capacitor e.g. integrating capacitor Cint 808 (ncap), which functions to measure the voltage on / across the capacitor. Integrating capacitor Cint 808 may be pre-charged by using a reset signal 803 to close switch 801, such that integrating capacitor Cint 808 is connected to a voltage source VPRE 805. When switch 801 is closed, integrating capacitor Cint 808 may be pre-charged to a voltage level of voltage source VPRE 805. Switch 801 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 808. An additional switch (not shown) may be provided to selectively deliver the voltage on the integrating capacitor Cint 808 to the ADC, 810.

[0147] After integrating capacitor Cint 808 is pre-charged, reset signal 803 may be used to open switch 801 such that integrating capacitor Cint 808 is disconnected from voltage source VPRE 805. At this point, depending on the level of voltage source VLIQ, the potential of counter electrode 816 may be at a level higherthan the potential of working electrode 814 (and integrating capacitor Cint 808), 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 814 is at a level higher than the potential of counter electrode 816. 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 814 is at a level lower than the potential of counter electrode 616. Thus, in some examples, integrating capacitor Cint 808 may be further charged during the bright period from the pre-charged voltage level of voltage source VPRE 805 to a higher level, and discharged during the dark period to a lower level, due to the potential difference between counter electrode 816 and working electrode 814. In other examples, charging and discharging may occur in dark periods and bright periods, respectively.

[0148] Integrating capacitor Cint 808 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, specifically about 0.2 to 1ms, more specifically about 0.5 to 0.7ms. The voltage level may be sampled and converted by ADC 810 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.

[0149] After being sampled by ADC 810, integrating capacitor Cint 808 may be pre-charged again by using a reset signal 803 to close switch 801, such that integrating capacitor Cint 808 is connected to voltage source VPRE 805 again. The steps of pre-charging integrating capacitor Cint 808, waiting for a fixed period of time for integrating capacitor Cint 808 to charge or discharge, and sampling and converting the voltage level of integrating capacitor by ADC 810 can be repeated in cycles throughout the sequencing process. The same steps of pre-charging integrating capacitor Cint 808, waiting for a fixed period of time for integrating capacitor Cint 808 to charge or discharge, and sampling and converting the voltagelevel of integrating capacitor by ADC 810 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 AC current is not a square wave or rectangular wave (as exemplified in FIG.8B-8C) but may resemble a summation of square or rectangular 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.

[0150] A digital processor 830 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 830 can perform further downstream processing, such as base determination. Digital processor 830 can be implemented as hardware (e.g., in a GPU, FPGA, ASIC) or as a combination of hardware and software. Digital processor 830 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.

[0151] 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.

[0152] When the voltage level on integrating capacitor Cint 808 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 808 (i.e., the steepness of the slope of a voltage on integrating capacitor Cint 808 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 voltagedecay 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 = 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 ms. 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 curve and is monotonic, thus allowing detection of molecules.

[0153] The rate of the decay of the voltage on integrating capacitor Cint 808 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 808 may be first measured by ADC 810 at time t1, and then the voltage is measured again by ADC 810 at time t2. The voltage difference is greater when the slope of the voltage on integrating capacitor Cint 808 versus time curve is steeper, and the voltage difference is smaller when the slope of the voltage curve is less steep. Thus, the voltage difference may be used as a metric for determining the rate of the decay of the voltage on integrating capacitor Cint 808, and thus the state of the cell.

[0154] FIG.8B-8C shows example data points captured from a cell during a single alternating current (AC) modulation period and its respective dark and bright period. The array is phased such that one half is in the “bright” phase of the voltage application and collecting sequencing data while the other half is in the “dark” phase of the voltage application and not collecting data. The bright phase consists of a brief period of diagnostic data collection followed by a pulse train superimposed on the “read” voltage application. In this case, the pulse voltage refers to the nominal value applied during the pulse. At the end of the “bright cycle,” the cells for which sequencing data is being recorded cease to record data for the remainder of the total modulation period, and those cells that were previously in the dark phase enter their bright phase. In the dark phase, a voltage intended to eject partially translocatedmolecules is applied to the cells, followed by a period of modest voltage to recharge the working electrode and reagents within the well. The illustrated implementation of voltage application and phasing is one example. There are other embodiments in which all cells may be in the same phase. FIG.8D shows the beginning of the voltage application for an example bright phase illustrating the onset of translocation pulses (831 and 832).

[0155] 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.

[0156] Further details regarding measurements can be found in, for example, U.S. Patent Publication No.2016 / 0178577 entitled “Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. Patent Publication No.2016 / 0178554 entitled “Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. Patent Application No.15 / 085,700 entitled “Non-Destructive Bilayer Monitoring Using Measurement Of Bilayer Response To Electrical Stimulus,” and U.S. Patent Application No.15 / 085,713 entitled “Electrical Enhancement Of Bilayer Formation,” the disclosures of which are incorporated by reference in their entirety for all purposes. Examples of nanopore sequencing systems and methods that thread the sample molecule through the pore during sequencing include sequencers from Oxford Nanopore (i.e. U.S. Patent Nos.9,758,823 and 10,416,117, each of which is incorporated by reference in its entirety) and Stratos Genomics (i.e. U.S. Patent Nos. 7,939,259 and 9,771,614, each of which is incorporated by reference in its entirety).

[0157] When the sequencing step is initiated, the device is perfused with a sequencing buffer comprising 0.5-2.5M ammonium chloride, 500-2000mM urea, up to250mM K3(Fe(CN)6), up to 250mM K4(Fe(CN)6), 5-7% w / v PEG8000, 50-200mM MES, pH 5-7. For example, the sequencing buffer is: (a) 1.7 M NH4Cl, 1000 mM Urea, 150mMK [Fe(CN) ], 50mM K [Fe(CN) ], PEG8000: 6% w / v, 75mM MES, pH: adjusted to 6.5; (b)1.25M NH4Cl, 1000mM Urea, 187.5mM K3(Fe(CN)6), 187.5mM K4(Fe(CN)6), 6.25% w / v PEG8000, 125mM MES (pH5), final pH adjusted 5.95; (c) 2.0 M NH4Cl, 1500mM Urea,50mM K [Fe(CN) ], 100mM K [Fe(CN) ], PEG8000: 7% w / v, 150mM MES, pH: adjusted to5.5; (d) 2.5 M NH4Cl, 2000mM Urea, 200mM K [Fe(CN) ], 50mM K [Fe(CN) ], PEG8000:5.5% w / v, 50mM MES, pH: Adjusted to 6.0; (e) 1.0 M NH4Cl, 800mM Urea, 100mMK [Fe(CN) ], 25mM K [Fe(CN) ], PEG8000: 6.5% w / v, 200mM MES, pH: Adjusted to 5.0; (f)0.5 M NH4Cl, 750mM Urea, 200mM K [Fe(CN) ], 200mM K [Fe(CN) ], PEG8000: 7% w / v,100mM MES, pH: Adjusted to 6.8; (g) 2.3 M NH4Cl, 1200mM Urea, 100mM K [Fe(CN) ],100mM K [Fe(CN) ], PEG8000: 5% w / v, 150mM MES, pH: Adjusted to 6.2; (h) 1.5 MNH4Cl, 1000 mM urea, 20mM K [Fe(CN) ], 250mM K [Fe(CN) ], PEG8000: 6.8% w / v,50mM MES, pH: Adjusted to 6.0.

[0158] F. Computer components used in a sequencing workflow

[0159] One or more of the processes described herein may be performed by a computer system operably connected to a nanopore sequencer. The computer system can utilize any suitable number of subsystems, many of which may be optional. Examples of such subsystems are shown in FIG.9 in computer system 1110. In some embodiments, a computer system includes a single computer apparatus, where the subsystems can be the components of the computer apparatus. In other embodiments, a computer system includes multiple computer apparatuses, each being a subsystem, with internal components. A computer system can include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0160] The subsystems shown in FIG.9 are interconnected via a system bus 1180. Additional subsystems such as a printer 1174, keyboard 1178, storage device(s) 1179, monitor 1176 which is coupled to display adapter 1182, and others are shown. Peripherals and input / output (I / O) devices, which couple to I / O controller 1171, can be connected to the computer system by any number of means known in the art suchas I / O port 1177 (e.g., USB, FireWire®). For example, I / O port 1177 or external interface 1181 (e.g. Ethernet, Wi-Fi, etc.) can be used to connect computer system 1110 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via system bus 1180 allows the central processor 1173 to communicate with each subsystem and to control the execution of a plurality of instructions from system memory 1172 or the storage device(s) 1179 (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 1172 and / or the storage device(s) 1179 can embody a computer readable medium. Another subsystem is a data collection device 1175, such as a camera, microphone, accelerometer, or other sensor and the like. Any of the data mentioned herein can be output from one component to another component and can be output to the user.

[0161] A computer system can include a plurality of the same components or subsystems, e.g., connected together by external interface 1181, by an internal interface, or via removable storage devices that can be connected and removed from one component to another component. In some embodiments, computer systems, subsystem, or apparatuses communicate over a network. In such instances, one computer can be considered a client and another computer a server, where each can be part of the same computer system. A client and a server can each include multiple systems, subsystems, or components.

[0162] Aspects of embodiments can be implemented in the form of control logic using hardware circuitry (e.g. an APSIC or FPGA) and / or using computer software with a generally programmable processor in a modular or integrated manner. As used herein, a processor can include a single-core processor, multi-core processor on a same integrated device, 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 disclosure using hardware and a combination of hardware and software.

[0163] Any of the software components or functions described in this application can be implemented as software code to be executed by a processor using any suitable computer language such as, for example, Java, C, C++, C#, Objective-C, Swift, or scripting language such as Perl or Python using, for example, conventional or object-oriented techniques. The software code can be stored as a series of instructions or commands on a computer readable medium for storage and / or transmission. A suitable non-transitory computer readable medium can include random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard-drive or a floppy disk, or an optical medium such as a compact disk (CD) or DVD (digital versatile disk), flash memory, and the like. The computer readable medium can be any combination of such storage or transmission devices.

[0164] Such programs can also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and / or wireless networks conforming to a variety of protocols, including the Internet. As such, a computer readable medium can be created using a data signal encoded with such programs. Computer readable media encoded with the program code can be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer readable medium can reside on or within a single computer product (e.g. a hard drive, a CD, or an entire computer system), and can be present on or within different computer products within a system or network. A computer system can include a monitor, printer, or other suitable display for providing any of the results mentioned herein to a user.

[0165] Any of the methods described herein may be totally or partially performed with a computer system including one or more processors, which can be configured to perform the steps. Thus, embodiments can be directed to computer systems configured to perform the steps of any of the methods described herein, potentially with different components performing a respective step or a respective group of steps. Although presented as numbered steps, steps of methods herein can be performed at the same time or at different times or in a different order.Additionally, portions of these steps can be used with portions of other steps from other methods. Also, all or portions of a step can be optional. Additionally, any of the steps of any of the methods can be performed with modules, units, circuits, or other means of a system for performing these steps.

[0166] G. Device Cleaning

[0167] The nanopore sequencing device described above can be subjected to a cleaning protocol to prepare the cells for the process illustrated in FIG.4 and treat the device post-sequencing. The purpose of the cleaning protocols is to (a) remove any surface contaminants from the device surface, resulting, e.g., from manufacturing or if the device has been used, elements of the compositions used on the device surface, such as remaining lipid, (b) recover reduced pore lifetime and pore insertions in the lipid bilayer, (c) in the final cleaning step remove air bubbles from the surface and subject the surface to a final cleaning step, and (d) conditioning the electrode surface to appropriate electrochemical state needed for sequencing .

[0168] In one embodiment, the initial cleaning protocol comprises sequentially perfusing the device with the following solutions (i) one or more volumes of detergent solution; (ii) one or more volumes of a cleaning solution, an acidic solution and / or a basic solution; (iii) one or more volumes of system fluid; and drying the surface of the device with air between an application of one or more of (i)-(iii). The steps listed above may be repeated one or more times to effectively clean the surface of the device for subsequent use. For example, it may be preferable to apply several cycles of cleaning solution, acidic and / or basic washes sequentially in step (ii), or several cycles of only cleaning solution in step (ii), which can be comprised of the same or different cleaning solutions, several cycles of only an acidic wash in step (ii), which can be comprised of either the same or different acids, or several cycles of only a basic wash in step (ii), which likewise, can be comprised of either the same or different bases.

[0169] A specific embodiment of the initial cleaning protocol is shown in FIG. 10A. A volume of detergent solution is applied to the device, followed by drying the device surface with a volume of air. A subsequent volume of detergent solution isapplied one or more times, followed by one or more volumes of acidic and / or basic solution. In the embodiment shown in FIG.10A, a volume of acid solution is applied, followed by system fluid, and two volumes of basic solution, but the skilled artisan will appreciate that the order of operations and / or number of individual wash steps may be adjusted without departing from the spirit or scope of the invention. For example, one or more volumes of acid solution can be applied, followed by one or more volumes of basic solution; or one or more volumes of acidic solution can be applied followed by one or more volumes of system fluid, and one or more volumes of basic solution. After acid and / or basic solutions are applied, system fluid is added to the system, the device is perfused once more with one or more volumes of detergent. Optionally, the device is finally perfused with system fluid for subsequent treatment according to the protocol described in reference to FIG.4.

[0170] After the device is used in a first sequencing protocol, following the initial cleaning protocol illustrated in FIG.10A, the device is optionally subjected to a subsequent run cleaning protocol as illustrated in FIG.10B, including sequentially perfusing the device a volume of solution selected from: (i) detergent solution; (ii) basic solution; (iii) system fluid; and combinations thereof, and drying the surface of the device with air between an application of one or more of the solutions. The steps listed above may be repeated one or more times to effectively clean the surface of the device for subsequent use.

[0171] A specific embodiment of the subsequent cleaning protocol is shown in FIG.10B. A volume of detergent solution is applied to the device, followed by drying the surface of the device with a volume of air. A subsequent volume of detergent solution is applied one or more times, followed by one or more volumes of a basic solution. In the embodiment shown in FIG.10B, a volume of basic solution is applied, followed by system fluid, but the skilled artisan will appreciate that the order of operations and / or number of individual wash steps of the subsequent cleaning protocol may be adjusted without departing from the spirit or scope of the invention. For example, one or more volumes of a basic solution can be applied, with a volume ofacidic solution between the basic solutions. After basic solutions are applied, system fluid is added to the system, the device is perfused once more with one or more volumes of detergent, and the device is finally perfused with system fluid for subsequent treatment according to the protocol described in reference to FIG.4.

[0172] An alternative pre-sequencing cleaning protocol is shown in FIG.14D. One or more volumes of detergent solution is applied to the device, followed by drying the surface of the device with a volume of air. A subsequent volume of cleaning solution is added, one or more times, followed by one or more volumes of detergent solution, with air introduced between each volume of reagent. The device is finally perfused with system fluid for subsequent use in the sequencing system.

[0173] A suitable detergent solution comprises 0.5-6% of a nonionic surfactant suspended in a buffered solution, such as Lutensol(R) (BASF, Ludwigshafen, Germany) C12-C14 alcohol ethoxylate, C9-C11 alcohol ethoxylate, C10-C18 alcohol ethoxylate, C12-C15 pareth-7 (polyethylene glycol ether of C12-C15 alcohol), Dowsil DS-1000, BYK-3450, and lauryl myristyl alcohol ethoxylate (C12-C14). In a specific embodiment, an ionic or non-ionic detergent, e.g., a Lutensol class of detergent, such as Lutensol M or Lutensol ON is the detergent component. The buffer can include 25- 250mM HEPES, Tris, TrisHCl, Phosphate, MOPS, MES, and PIPES, pH 6-9. For example, the detergent includes 0.5-1.5% Lutensol, 25-75mM HEPES, pH 6-8. The following is a list of suitable detergent solutions: (a) Lutensol M (C12-C14 alcohol ethoxylate) at 3%, HEPES 100 mM, pH 7.5; (b) C12-C15 Pareth-7 (polyethylene glycol ether) at 5%, Tris, 50mM, pH 7.4; (c) Lutensol ON (C10-C18 alcohol ethoxylate) at 1.5%, MES 25 mM, pH 6.0; (d) Dowsil DS-1000 at 0.8%, Tris-HCl, 200mM, pH 8.0; (e) BYK-3450 at 6%, PIPES 150 mM, pH 7.0; (f) Lauryl myristyl alcohol ethoxylate (C12-C14) at 2.5%, Phosphate 50mM, pH 7.2; (g) C12-C15 Pareth-7 (polyethylene glycol ether) at 4%, MOPS 100 mM, pH 6.8; (h) Lutensol M (C12-C14 alcohol ethoxylate) at 2%, Tris 250 mM, pH 7.4; (i) Lutensol ON (C9-C11 alcohol ethoxylate) at 0.5%, MES 150 mM, pH 6.5; (j) Dowsil DS- 1000 at 5%, HEPES 75mM, pH 7.3; (k) 1-5% Lutensol and 100 mM Tris base.

[0174] The acidic solution can include any suitable acidic component, includingbut not limited to, acetic acid, hydrochloric acid, citric acid, nitric acid, lactic acid, salicylic acid, tartaric acid, gluconic acid, oxalic acid, malonic acid, medronic acid, malic acid, etidronic acid, succinic acid, and mixtures thereof. In one embodiment, the acidic solution includes 0.5-1.5M acetic acid, 0.1-0.5M citric acid, or mixtures thereof, e.g., 1.0M acetic acid and 0.25M citric acid. Alternatively, the acidic solution comprises 100-300mM medronic acid, and in a specific embodiment, the acidic solution includes 200mM medronic acid. In one embodiment, the acidic component further comprises a chelating agent, including but not limited to, EDTA, EGTA, etc. Examples of suitable acidic solutions include but are not limited to: (a) 1.2 M acetic acid, 0.3 M citric acid, 10mM EDTA; (b) 0.9 M lactic acid, 0.4 M malic acid, 5mM EGTA; (c) 1.5 M acetic acid, 0.1 M gluconic acid, 0.1 M oxalic acid, EDTA 15 mM; (d) 1.0 M tartaric acid, 0.3 M succinic acid, 8mM EGTA; (e) 1.1 M malonic acid, 0.2 M medronic acid, 0.15 M citric acid, 5mM EDTA.

[0175] The basic solution can include any suitable basic component, including but not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, ammonium hydroxide, lithium hydroxide, tris, bis tris propane, triethanolamine, and combinations thereof. In one embodiment, the basic solution comprises 0.05-0.2M ammonium hydroxide, and more specifically, 0.10M ammonium hydroxide. Suitable basic solutions include: (a) 0.1M potassium hydroxide; (b) 0.08M ammonium hydroxide; (c) 0.15M sodium hydroxide; (d) 0.2M ammonium hydroxide; (e) 0.1M triethanolamine.

[0176] The system fluid can include, but is not limited to, water, including deionized water, ethanol, isopropyl alcohol, acetone, and mixtures thereof, alone or in combination with a water soluble biocide, including but not limited to ProClin, phenol, benzalkonium chloride, parabens, such as methylparaben and propylparaben, sodium benzoate, chlorhexidine, Kathon, isothiazolinones, and mixtures thereof. In a specific embodiment, the system fluid includes an aqueous mixture of isopropyl alcohol, e.g., between 2-30%, more specifically, 5-20%, e.g., 5-15%, and 0.025-1.25% Proclin, more specifically, 0.05-1.0%, e.g., 0.08-1.0%.

[0177] The cleaning solution includes up to 95% alcohol, e.g., 50-95% or 65- 85% of a low molecular weight alcohol, including but not limited to methanol, ethanol, propanol, isopropanol, sec-butanol, tert-butanol, n-butanol, octanol, hexanol, and mixtures thereof. For example, the cleaning solution can include the following compositions suspended in water: (a) 80% tert-butanol, 10% isopropanol; (b) 70% methanol, 20% ethanol; (c) 85% sec-butanol, 10% n-butanol; (d) 60% hexanol, 30% ethanol; (e) 95% tert-butanol; (f) 75% isopropanol, 15% octanol; (g) 80% n-butanol, 15% propanol; (h) 65% propanol, 25% tert-butanol; (i) 75% tert-butanol; (j) 90% ethanol, 5% methanol; (k) 50% hexanol, 35% octanol.

[0178] The protective solution includes (a) a low molecular weight alcohol, (b) a mildly acidic detergent, and (c) a biocide, e.g., (a) methanol, ethanol, propanol, isopropanol, sec-butanol, n-butanol, and / or hexanol, and combinations thereof, (b) CIP77, alone or in combination with CIP95, pH 2-3.5, and (c) Proclin 300. In a specific embodiment, the alcohol is isopropanol, e.g., 5-15%, e.g., 10%. COSA CIP77 is an example of a mildly acidic, inorganic surfactant free detergent including 50-100% phosphoric acid and 1-2.5% gluconic acid, and the pH of CIP77 is 0.8-1.2. Alternatives to CIP77 include but are not limited to aqueous phosphoric acid, COSA acid cleaners formulated for removing acid-soluble residues, and alkaline cleaners for CIP applications, e.g., CIP100 / ProKlenz, and any of the foregoing CIP alternatives can be used in place of or in combination with CIP77, alone or in combination with a suitable acidic component to adjust the pH accordingly. The following is a list of protective solutions that may be used in the methods described herein: (a) 20% ethanol, 2% COSA CIP77, 0.04% Proclin 300, pH 3.0; (b) 10% methanol, 10% propanol, 1.5% COSA CIP77, 0.5% CIP95, 0.03% Proclin 300, pH 2.5; (c) 15% isopropanol, 3% COSA CIP77, 0.05% Proclin 300, pH 3.2; (d) 8% n-butanol, 5% hexanol, 2.5% COSA CIP77, 0.04% Proclin 300, pH 3.0; (e) 12% isopropanol, 3% sec-butanol, 3% CIP95, 0.02% Proclin 300, pH 3.4; (f) 25% ethanol, 1% COSA CIP77, 0.04% Proclin 300, pH 3.0; (g) 15% methanol, 2.5% COSA CIP77, 0.5% CIP95, 0.05% Proclin 300, pH 2.8; (h) 20% propanol, 2% CIP95, 0.03% Proclin 300, pH 3.5; (i) 10% hexanol, 10% sec-butanol, 3% COSACIP77, 0.04% Proclin 300, pH 2.7; (j) 18% isopropanol, 1% COSA CIP77, 1% CIP95, 0.04% Proclin 300, pH 3.3.

[0179] In a specific embodiment, a standard pre-sequencing cleaning protocol comprises the following steps: (a) the device is suspended in system fluid and then perfused with 200-600uL of detergent for 10-60 seconds at a temperature of 15-50C; (b) a volume of air is introduced to the device, up to 100uL for up to 30 seconds at 15- 50C; (c) a second volume of detergent is introduced, 200-600uL of detergent for 10- 60 seconds at a temperature of 15-50C; (d) the device is perfused with a basic solution,400-800uL for 60-120 seconds at a temperature of 15-50C; (e) the device is flushed with excess system fluid, between 500-1500uL for 60-120 seconds at a temperature of 15-50C; (f) the device is subjected to an additional cycle of detergent washes with a volume of air between each detergent wash; and (g) the device is flushed with excess system fluid, 500-1500uL for 60-120 seconds at a temperature of 15-50C, at which time it was used in a sequencing run, as described herein.

[0180] In a more specific embodiment of the standard pre- sequencing cleaning protocol shown in FIG.10A, the volume of detergent is between 300-400uL and the volume of detergent is applied to the device during the pre-treatment protocol for between 20-50 seconds; the volume of basic solution is between 500- 700uL and the volume of basic solution is applied to the device during the pre- treatment protocol for between 60-100 seconds; and the volume of system fluid is between 750-1250uL and the volume of system fluid is applied to the device during the pre-treatment protocol for between 60-100 seconds.

[0181] In an alternative embodiment, a pre-sequencing cleaning protocol comprises the following steps: (a) a volume of detergent is introduced to the device, up to 200uL at 100 uL / s at a temperature of 15-50C; (b) a volume of air (20-40uL at 100 uL / s) is introduced at a temperature of 15-50C; (c) the device is perfused with cleaning solution, up to 200uL at 25uL / s at a temperature of 15-50C; (d) a volume of air (20-40uL at 100 uL / s) is introduced at a temperature of 15-50C; (e) the device is flushed with detergent, up to 200uL at 100 uL / s at a temperature of 15-50C; (f) one ormore of the foregoing steps are repeated, followed by the introduction of system fluid, 500-1500uL at 1000uL / s at a temperature of 15-50C, at which time it was used in a sequencing run, as described herein.

[0182] In a more specific embodiment, the protocol illustrated in FIG.14D is provided. Briefly, the device is washed with a volume of detergent, up to 200uL, e.g., 100uL, at 100 uL / s. A volume of air (20-40uL, e.g., 30uL, at 100 uL / s) is introduced, followed by the addition of a cleaning solution, up to 200uL, e.g., 100uL, at 25uL / s. An additional volume of air (20-40uL, e.g., 30uL, at 100 uL / s) is introduced, and the device is flushed with detergent, up to 200uL, e.g., 100uL, at 100 uL / s. An additional volume each of detergent and cleaning solution are introduced, with air introduced between each reagent, followed by the introduction of system fluid, 500-1500uL, e.g., 1000uL, at 1000uL / s. The process illustrated in FIG.14D is conducted at a temperature of 15- 50C, e.g., 20-40C, or 35C, at which time it can be used in a sequencing run, as described herein.

[0183] Following the standard pre-sequencing cleaning protocol and a sequencing run, the device is subjected to a standard post-sequencing (or end of run) cleaning protocol (FIG.11B) that includes: (a) perfusing the device with 20-60uL of system fluid for up to 30 seconds at 10-50C; (b) perfusing the device with 200-600uL of detergent for up to 60 seconds at 10-50C; and (c) flushing the device with 500- 1500uL of system fluid for 60-120 seconds at 10-50C. One or more of the steps listed above are followed by perfusing the device with a volume of air, e.g., up to 100uL for up to 30 seconds at 10-50C. In a specific embodiment, up to 50uL and more specifically, up to 30uL of air is added to the device for up to 15 seconds, and more specifically, up to 10 seconds, at 30-40C.

[0184] More specifically, the end of run cleaning protocol includes perfusing the device with one or more volumes of fluid in the following order:

[0185] 30-50uL of system fluid for up to 20 seconds at 10-30C;

[0186] 30uL of air is added to the device for up to 15 seconds at 30-40C;

[0187] 300-500uL of detergent for up to 60 seconds at 25-50C; and

[0188] 750-1250uL of system fluid for 60-100 seconds at 25-50C.

[0189] In a specific embodiment, the end of run cleaning protocol also includes perfusing the device with a basic solution, 400-800uL for 60-120 seconds at 25-50C. In a specific embodiment, 500-700uL of basic solution is applied for 60-100 seconds at 30-40C.

[0190] An alternative post-sequencing protocol is illustrated in FIG.14G. The process is conducted at 20-40C, e.g., 35C. Initially, the fluidic interface is used to draw air (20uL), detergent (approximately 200uL), and an additional volume of air (500uL). An initial volume of air is introduced to the device (30uL at 1000uL / s), followed by: (a) perfusing the device with 2 x 50-150uL of detergent at up to 100uL / s, with a brief interval, e.g., up to 10s, between detergent volumes; (b) perfusing the device with 2 x 50-150uL of cleaning solution at 20-30uL / s, with a brief interval, e.g., up to 10 s, between cleaning solution volumes; (c) flushing the device with 2 x 50- 150uL of detergent at up to 100uL / s, with a brief interval, e.g., up to 10s, between detergent volumes; and (d) introducing one or more volumes of protective solution, up to 1500uL at 100uL / s. One or more of the steps listed above are followed by perfusing the device with a volume of air, e.g., up to 100uL for up to 30 seconds at 10- 50C. In a specific embodiment, up to 50uL and more specifically, up to 30uL of air is added to the device for up to 15 seconds, and more specifically, up to 10 seconds, at 30-40C.

[0191] In a specific embodiment, the alternative post-sequencing protocol includes the following steps after an initial volume of air is introduced to the device: (a) perfusing the device with 2 x 100uL of detergent at up to 100uL / s, with a brief interval, e.g., 5s, between detergent volumes; (b) perfusing the device with 2 x 100uL of cleaning solution at 25uL / s, with a brief interval, e.g., 5s, between cleaning solution volumes; (c) flushing the device with 2 x 100uL of detergent at up to 100uL / s, with a brief interval, e.g., 5s, between detergent volumes; and (d) introducing one or more volumes of protective solution, 1000uL at 100uL / s. One or more of the steps listed above are followed by perfusing the device with a volume of air, e.g., up to 100uL forup to 30 seconds at 10-50C. In a specific embodiment, up to 50uL and more specifically, up to 30uL of air is added to the device for up to 15 seconds, and more specifically, up to 10 seconds, at 30-40C.

[0192] The pre- and post-sequencing cleaning protocols may be adjusted based on the conditions of the device. For example, the conditions may be adjusted to condition or activate the electrodes in the device and / or to treat an unused device that was not previously suspended in system fluid or other wetting reagent during storage. Under such conditions, the nature of the reagents used in the cleaning protocol and / or duration of each step or combination of steps and reagents may be altered without departing from the spirit or scope of the invention.

[0193] For example, alternative pre- and post-sequencing cleaning workflows (referred to herein as “alternative protocols”) are illustrated in FIGS.13A-13B. Before sequencing, the device is subjected to an initial alternative pre-sequencing cleaning protocol as illustrated in FIG.13A. The protocol includes the following step:

[0194] the device is perfused with 200-600uL of detergent solution for 10-60 seconds at 25-50C;

[0195] a volume of air is introduced to the device surface, and a second volume of detergent is introduced (200-600uL of detergent solution for 10-60 seconds at 25- 50C);

[0196] the device is treated with an acidic solution, e.g., including 100-300mM medronic acid, 500-1500uL for 60-120 seconds at a temperature of 25-50C;

[0197] a volume of excess system fluid is added, up to 1500uL for up to 120 seconds at a temperature of 25-50C;

[0198] the device is treated with a basic solution, e.g., including 200-400mM LiOH, up to 200uL for up to 30 seconds at 25-50C;

[0199] a volume of air is added and an additional volume of basic solution, 400- 800uL for 60-120 seconds at 25-50C;

[0200] the device is perfused with system fluid, up to 1500uL for 60-120 seconds at 25-50C;

[0201] the device is perfused with an additional cycle of detergent washes (one or more volumes of 200-600uL for 10-60 seconds at a temperature of 25-50C) with a volume of air between each detergent wash; and

[0202] finally, the device is washed with excess system fluid, up to1500uL for 60-120 seconds at a temperature of 25-50C, at which time it can be used in a sequencing run, as described herein.

[0203] The alternative pre-sequencing protocol is paired with an alternative end of run protocol as shown in FIG.13B. The device is subjected to:

[0204] one or more cycles of detergent washes (200-600uL of detergent solution for 10-60 seconds at 25-50C), with a volume of air between each detergent wash;

[0205] a volume of basic solution was introduced to the device, e.g., 50-150mM Tris, 400-800uL for 60-120 seconds at 25-50C;

[0206] the device is flushed with excess system fluid, up to 1500uL for 60-120 seconds at 25-50C;

[0207] the device is subjected to repeated detergent washes (200-400uL for 10-60 seconds at 25-50C), each separated by the introduction of a volume of air; and

[0208] the device was then washed in excess system fluid (up to 1500uL for 60- 120 seconds at 25-50C).

[0209] The complete alternative pre- and post-sequencing protocols are illustrated in FIG.11. As shown in FIG.11, the alternative protocols include an initial pre-treatment with medronic acid / lithium hydroxide, as described above, and then subjected to a sequencing run. Then the device is subjected to additional sequencing runs using the standard pre- and post-sequencing protocols described above and illustrated in FIGS.11A-11B. Alternatively, the device may be stored for later use by suspending the device in excess system fluid, e.g., up to 1500uL.

[0210] The cleaning protocols may be performed at a range of temperatures, e.g., 25-50C. In one embodiment, each step of the protocol is performed at a single temperature in the range of 25-50C. Alternatively, one or more steps may beperformed at a higher temperature, e.g., about 50C and the final wash with system fluid is performed at a lower temperature, e.g., 35C. Performing the wash steps, including one or more of the detergent, acid and / or basic wash steps, at a higher temperature may be useful in revitalizing aged electrodes. The duration of each wash step may also be extended to compensate for differences in devices, e.g., from lot to lot.

[0211] Moreover, in a specific embodiment of either the standard cleaning protocol or the alternative cleaning protocol, the volume of fluids introduced at each step of the protocols can be adjusted within the following parameters: (a) the volume of detergent introduced to the device is no more than 50% of the overall volume of the flow cell and the duration of the detergent wash step is up to 60 seconds; (b) the volume of acidic and / or basic solution is up to twice the volume of detergent used in the detergent wash step and the duration of the basic solution wash is up to twice as long as the detergent wash step; and (c) the device is flushed with excess system fluid for at least as long as the acid and / or basic solution wash steps.

[0212] In addition, the volumes, times, and temperatures of each step of the cleaning protocols described above may be adjusted based on the configuration of the nanopore sequencing device and the type of sequencing chemistry used in the device. In the specific embodiment, the conditions are suitable for SBX sequencing performed on a nanopore device comprising an array of up to 8M electrochemical cells.

[0213] The specific details of particular embodiments can be combined in any suitable manner without departing from the spirit and scope of embodiments of the disclosure. However, other embodiments of the disclosure can be directed to specific embodiments relating to each individual aspect, or specific combinations of these individual aspects.

[0214] Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, the disclosure is not limited to the details provided. There are many alternative ways of implementing the disclosure.The disclosed embodiments are illustrative and not restrictive. The above description of example embodiments of the disclosure has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form described, and many modifications and variations are possible in light of the teaching above.

[0215] EXAMPLES

[0216] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.

[0217] Example 1

[0218] The workflow for the sequencing run described in this Example, including pre- and post-sequencing protocols, in a fully automated nanopore sequencer is illustrated in FIG.12A-12D. FIG.12A illustrates the steps of the overall workflow including pre- and post-sequencing workflows; FIG.12B shows the complete sequencing workflow and timing of each step; FIG.12C shows the sequencing waveform used in a sequencing run; and FIG.12D shows the reuse of a nanopore sequencing device in an automated sequencing system.

[0219] The nanopore sequencing device used in the automatic sequencer included a nanopore array comprising a CMOS microchip with an array of 8 x 2(20) (“8M”) TiN electrodes within shallow wells (chip fabricated by Roche Sequencing Solutions, Santa Clara, Calif., USA). Methods for fabricating and using such nanopore array microchips can also be found in U.S. Patent Application Publication Nos. 2013 / 0244340 A1, US 2013 / 0264207 A1, and US2014 / 0134616 A1 each of which is hereby incorporated by reference herein. Each well in the array was manufactured using a standard CMOS process with surface modifications that allowed for constantcontact with biological reagents and conductive salts. Each well supported a phospholipid bilayer membrane with a nanopore embedded therein. The electrode at each well was individually addressable by a computer interface. All reagents used were introduced into a simple flow cell above the array microchip using a computer- controlled syringe pump. The chip supported analog to digital conversion and reported electrical measurements from all electrodes independently at a rate of over 1000 points per second. Changes in the electrical signal resulting from the passage of the XP molecules through the pore over time were made asynchronously at each of 8M addressable nanopore-containing membranes in the array at least once every millisecond (msec) and recorded on the interfaced computer. The reagents used in this Example are listed in Table 3A-3B.

[0220] Table 3A. Sequencing Reagent Compositions Reagent Composition Run buffer (R) 2M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM HEPES pH 7.4 Pore dilution (P) 50pM P-445 (wildtype, heptameric hemolysin), 2M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 0.001% tween20, 100mM HEPES pH 7.4 Osmo buffer (O) 1.6M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM MES (pH 5.0); final pH adjusted to 5.8 Sequencing buffer 1.25M NH4Cl, 1000mM Urea, 187.5mM K3(Fe(CN)6), 187.5mM K4(Fe(CN)6), 6.25% w / v PEG8000, 125mM MES (pH5), final pH adjusted 5.95 XP buffer (XP) 1M NH4Cl, 800mM Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 5% w / v PEG8000, 100mM MES (pH5), final pH adjusted 5.95, 2.8% ACN, 0.2% trehalose, 2mM HEPES, 1:5 dilution of XP sample Lipid (L) 8.1mg / ml Span 80 (0.82%), 1.9mg / ml DPhPE (8:1 mol ratio) 6.5% hexane 83.4% PDM20, 9.3% hexadecane (9:1 % ratio) System fluid (SF) 10% isopropyl alcohol, 0.09% ProclinDetergent (D) 1% Lutensol ON 70, 50mM HEPES pH 7 Base (B) 100mM tris base System waste 2M tris pH 8.8 (diluted to a minimum of 100mM with waste added) Table 3B. Reagents / Suppliers Reagents Supplier Cat # MES Sigma 69892-500G HEPES Sigma 54457-250G-F Tween20 Sigma P9416-100ML PEG8k Promega V3011 NH4Cl TCI A2037 Urea Sigma U1250-1KG K3[Fe(CN)6] Sigma 60299-100GF K4[Fe(CN)6] Sigma 60279-250G Mol Biology Grade H2O Corning 46-000-CM 30% NH4OH Sigma 221228-100ML-A Tris base Roche 10708976001 Lithium hydroxide monohydrate Sigma 62528-50G Medronic acid Combi-blocks QB-4738

[0221] As shown in FIG.12A, a device (“CD”) was placed in the automated sequencer and perfused with system fluid to wet the surface of the array. The device was subsequently subjected to a pre-sequencing cleaning protocol (“PSP”), as described in more detail below. The device was subjected to an initial set of electrical measurements to confirm the integrity of the electrode array (“short check”). For example, as described e.g., in U.S. Patent No.10,816,537, which is incorporated herein by reference in its entirety, physical checks of the cell's circuitry were performed, including but not limited to dry and / or wet checks performed before and after, respectively, any buffer or lipid solution was applied. For example, each cell of the sequencing chip was checked for an open-circuit or short-circuit state. Pore dilution buffer was applied to the device, followed by the lipid composition under conditionssufficient to form the lipid bilayer between the cis and trans chambers.

[0222] As described, e.g., in U.S. Patent No.10,816,537, the thickness of the lipid layer was monitored during the bilayer formation process and various feedback processes were used to ensure that the eventual state of the lipid layer was that of a lipid bilayer. For example, if, after a first application of a lipid solution to a cell, it was determined that the lipid layer was too thick and not a bilayer, a thinning procedure was initiated that included flowing multiple cycles of run buffer, e.g., up to 8 cycles, with each cycle comprising perfusing the device with about 125uL of run buffer at a flow rate of about 30uL / second, and the feedback processes described, e.g., U.S. Patent No.10,816,537 were used, e.g., after each cycle or multiple cycles, to ensure that a lipid bilayer was formed having uniform and consistent composition. Without wishing to be bound by any particular theory, the thinning process reduces multi- lamellar lipid membranes to a single bilayer. Pores were inserted into the lipid bilayer by electroporation, and the osmolarity of the buffer solution was adjusted by applying the osmolarity buffer.

[0223] A more detailed illustration is provided in FIG.12B. First, the sequencer was subjected to several system checks, as described above, followed by the addition of system fluid to wet the surface of the device. The device was then subjected to a series of pre-sequencing treatments, which consisted of several applications of detergent, with a volume of air between applications or several applications, followed by multiple volumes of base, system fluid, and a penultimate cycle of detergent washes with an intermittent application of air. The device was finally washed with system fluid before an additional short check was performed. Pore molecules were introduced, followed by the lipid composition and run buffer. The bilayer was formed in the presence of an additional volume of run buffer and electroporation proceeded. Osmolarity buffer was introduced and finally an XP buffer was also introduced. After a sequencing run was performed, the device was subjected to a post-sequencing cleaning protocol that included the application of several volumes of system fluid and detergent.

[0224] XP molecules were introduced in a stepwise manner as shown in FIG.7B. The first aliquot of XP molecules, about of the XP prepared, was added to the device and after 2 minutes, a sequencing run is initiated. About 2 minutes aftersequencing was initiated, an additional aliquot of XP was added ( ), and this processwas repeated until all of the remaining aliquots of XP were added. It was found that with the introduction of XP molecules in multiple aliquots over the span of a sequencing run, the overall throughput of the system was maintained over the duration of the sequencing run (as shown in the graph in FIG.7B).

[0225] The device was used in a sequencing run in which the sequencing waveform and conditions shown in FIG.12C were used.

[0226] After the sequencing run, the device was to an end of run cleaning protocol as shown in the steps shown in FIG.12B (“Post run clean”) . The device was either used in a subsequent sequencing run or stored for later use in the system fluid. If it was used in a subsequent sequencing run, the process was repeated as described above starting from the step of subjecting the device to a pre-sequencing cleaning protocol. As shown in FIG.12D, it was found that a device could be reused repeatedly in this workflow for more than 200 sequencing hours.

[0227] A. Standard Pre- and Post-Cleaning Protocols

[0228] Pre- and post-sequencing cleaning protocols (referred to herein as the “standard protocols”) used in the workflow described above are illustrated in FIGS. 10A-10B. As described above in reference to FIG.12A, a device was suspended in system fluid (not shown in FIG.10A-10B) and then subjected to an initial pre- sequencing cleaning protocol as illustrated in FIG.10A. First, the device was perfused with 375uL of detergent for 40 seconds at a temperature of 35C. A volume of air was introduced to the device surface, 30uL for 6 seconds at 35C, and a second volume of detergent was introduced (375uL for 40 seconds at a temperature of 35C). The device was perfused with a base solution, 600uL for 82 seconds at a temperature of 35C, followed by the addition of excess system fluid, 1000uL for 83 seconds at a temperature of 35C. The device was subjected to an additional cycle of detergentwashes (two volumes of 375L for 40 seconds at a temperature of 35C) with a volume of air between each detergent wash (30uL for 6 seconds at a temperature of 35C). Finally, the device was washed with excess system fluid, 1000uL for 83 seconds at a temperature of 35C, at which time it was used in a sequencing run, as described herein.

[0229] After a device was used in a sequencing run it was subjected to a post- cycle or end of run cleaning protocol as illustrated in FIG.10B. Sequencing was performed at 20C and once a run was completed, the device was perfused with system fluid, 42uL for 14 seconds at a temperature of 20C. A volume of detergent was introduced to the device surface, 375uL for 40 seconds at a temperature of 35C, followed by a volume of system fluid, 1001uL for 85 seconds at a temperature of 35C. The device was then suspended in system fluid and rewetted to maintain the integrity of the device surface (40uL every hour at a temperature of 20C).

[0230] B. Alternative Pre- and Post-Cleaning Protocols

[0231] Alternative pre- and post-sequencing cleaning workflows (referred to herein as “alternative protocols”) are illustrated in FIGS.13A-13B. The alternative protocols were found to be particularly suitable for unused devices that were not suspended in system fluid or other wetting reagent during storage or immediately prior to use. Alternatively, the alternative protocols were used to revitalize aged electrodes in a device. Before sequencing, the device was subjected to an initial alternative pre-sequencing cleaning protocol as illustrated in FIG.13A. First, the device was placed in the automated sequencer and perfused with 375uL of detergent for 40 seconds at a temperature of 35C. A volume of air was introduced to the device surface, 30uL for 6 seconds at 35C, and a second volume of detergent was introduced (375uL for 40 seconds at a temperature of 35C). The device was perfused with an acidic solution including 200mM medronic acid, 950uL for 79 seconds at a temperature of 35C, followed by the addition of excess system fluid, 1000uL for 83 seconds at a temperature of 35C. The device was perfused with a basic solution including 300mM LiOH, 100uL for 12 seconds at 35C, followed by a volume of air, 30uLfor 6 seconds at 35C, and an additional volume of basic solution, 600uL for 82 seconds at 35C. The device was perfused with system fluid, 1000uL for 83 seconds at 35C, followed by an additional cycle of detergent washes (two volumes of 375L for 40 seconds at a temperature of 35C) with a volume of air between each detergent wash (30uL for 6 seconds at a temperature of 35C). Finally, the device was washed with excess system fluid, 1000uL for 83 seconds at a temperature of 35C, at which time it was used in a sequencing run, as described herein.

[0232] After a device was used in a sequencing run it was subjected to a post- cycle alternative cleaning protocol as illustrated in FIG.13B. The device was subjected to two cycles of detergent washes (375uL for 40 seconds at 35C), with a volume of air (30uL for 6 seconds at 35C) between each detergent wash, and then a volume of basic solution was introduced to the device, 600uL for 82 seconds at 35C. The device was flushed with system fluid, 1000uL for 83 seconds at 35C, followed by repeated detergent washes (375uL for 40 seconds at 35C), each separated by the introduction of a volume of air. The device was then suspended in system fluid (1000uL for 83 seconds at 35C), for subsequent use.

[0233] Various conditions were tested in the standard and alternative protocols illustrated in FIGS.10A-10B and 13A-13B, including varying reagent alternatives, temperatures, and flow rates for one or more steps of the protocol. For example, various acids were evaluated as alternatives to the acid and / or base solutions applied in the standard and alternative protocols, such as replacing the acid and / or base with acetic acid, citric acid, or an initial treatment with citric acid followed by ammonium hydroxide. It was found that citric acid treatment recovered pore lifetime, but yields of effective nanopores in the lipid bilayer were lower than expected. Adding an ammonium hydroxide treatment after citric acid recovered the loss of pore counts observed with citric acid treatment alone. The standard end of run protocol was also adjusted to evaluate the addition of ammonium hydroxide after the detergent washes and it was found that this additional step improved pore insertions.

[0234] As shown in FIG.13C, the use of ammonium medronate was also evaluated instead of the basic solution used in the alternative protocol. Two separate volumes of ammonium medronate were introduced to the device in the middle of the cleaning protocol and it was found that this change was able to recover pore lifetimes.

[0235] Various temperatures were evaluated for the standard and alternative protocols for one or more steps of the protocols. In particular, it was found that a temperature range of 35-50C could be used for the detergent wash steps in the standard and alternative protocols and increasing the temperature within this range increased pore recovery. The volume and duration of each wash step was also evaluated in the standard and alternative protocols, as well as the duration of delays between individual wash steps.

[0236] Example 2

[0237] The alternative workflow for the sequencing run described in this Example, including pre- and post-sequencing protocols, in a fully automated nanopore sequencer is illustrated in FIG.14A-14D. FIG.14A illustrates the steps of the overall alternative workflow including pre- and post-sequencing workflows; FIG. 14B shows the waveform conditions used during sequencing; FIG 14C illustrates reagent stacking, as used in the pre-sequencing protocol shown in FIG.14D; and FIGs. 14E, and 14F(i)-14F(ii) show reagent stacking as used for post-sequencing cleaning, as shown in FIG.14G.

[0238] The nanopore sequencing device and array used in this Example were the same as that described in Example 1. The reagents used in this Example are listed in Table 4.

[0239] Table 4. Sequencing Reagent Compositions Reagent Composition Run buffer (T1R) 2M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM HEPES pH 8.2Pore dilution (P) 150pM P-445 (wildtype, heptameric hemolysin), 2M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 0.001% tween20, 100mM HEPES pH 8.2 Osmo buffer (O) 1.6M NH4Cl, 0.8M Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 100mM MES (pH 5.0); final pH adjusted to 5.8 Sequencing buffer 1.25M NH4Cl, 1000mM Urea, 187.5mM K3(Fe(CN)6), 187.5mM K4(Fe(CN)6), 6.25% w / v PEG8000, 125mM MES (pH5), final pH adjusted 5.95 XP buffer (XP) 1M NH4Cl, 800mM Urea, 150mM K3(Fe(CN)6), 150mM K4(Fe(CN)6), 5% w / v PEG8000, 10mM MES (pH5), final pH adjusted 5.95, 2.2-3.2% ACN, 0.2% trehalose, 2mM HEPES, 1:5 dilution of XP sample Lipid (L) 5.31mg / ml span80 (0.54%), 1.25mg / ml DPhPE (8:1 molar ratio between lipid components), 4.26% hexane, 4%tert-butanol, 82.08% PDM20, 4.26%hexadecane, 0.51% pentadecane, 0.51% tetradecane, 0.51% tridecane, 0.51% dodecane, 0.51% undecane, 0.51% decanted (9:1% ratio between PDM20 and alkanes) System fluid (SF) 10% isopropyl alcohol, 0.09% Proclin300 Basic Detergent 1-5% Lutensol ON 70, 100mM tris base (Tr2) System waste 2M tris pH 8.8 (diluted to a minimum of 100mM with waste added) SM Protective 10% isopropyl alcohol, 0.9% Proclin300, 0.5% CIP77 solution (Tr3) Cleaning Solvent 75% tert-butanol (C) Xp Storage 11-16% acetonitrile, 1% trehalose, 10mM HEPES Solution (X) Table 4B. Reagents / Suppliers Reagent Vendor Catalog # Tween-20 Sigma P9416-100MLMBG water Corning 46-000-CM HEPES Sigma 54457-250G-F 221228- 30% NH4OH Sigma 100ML-A NH4Cl TCI A2037 Urea Thermoscientific J75826-A1 K3[Fe(CN)6] Sigma 60299-100GF K4[Fe(CN)6] Sigma 60279-250G MES Sigma 69892-500G 471712- tert-Butanol Sigma 100ML Isopropyl alcohol Supelco PX1838-1 ProClin 300 Sigma 48912-U PEG8000 Promega V3011 Lutensol ON70 BASF 50070761 Tris base Roche 10708976001 P-445 PNZ N / A DPyPE Corden LP-R4-147 Span 80 Sigma S6760-250ML PDM20 Roche SC N / A Decane (C10) Sigma 457116 Undecane (C11) Sigma 94000 Dodecane (C12) Sigma 297879 Tridecane (C13) Sigma 91490 Tetradecane (C14) Sigma 87139Pentadecane (C15) Sigma 76509 Hexadecane (C16) Sigma H6703-100ML Hexane Thermo fisher 043263.AK CIP77 Roche 3504913001

[0240] The complete workflow is shown in FIG.14A. A device (“CD” or “SM”) was placed in the automated sequencer and perfused with system fluid to wet the surface of the array. The device was subjected to a pre-sequencing cleaning protocol (“PSP”) that included the introduction of the basic detergent solution (Tr2), followed by cleaning solvent (C), and system fluid (SF). After PSP, as described above in Example 1, the device was subjected to an initial set of electrical measurements to confirm the integrity of the electrode array (“short check”) and hemoflow was initiated by introducing pore dilution buffer (P) (100 uL P at 4uL / s). The lipid composition was applied (L) (up to 150 uL L at a rate of up to 15 uL / s, at once or in one or more small volumes of L with up to 3 minutes between each volume added. Run buffer (Tr1) was introduced without applying a waveform, 8 x 125 uL Tr1 at 30uL / s with a 15 second pause between volumes of Tr1, under conditions sufficient to form the lipid bilayer between the cis and trans chambers. As described in Example 1, the thickness of the lipid layer was monitored during the bilayer formation process and various feedback processes were used to ensure that the eventual state of the lipid layer was suitable for sequencing. Pores were inserted into the lipid bilayer by adaptive poration, and the osmolarity of the buffer solution was adjusted by applying the osmolarity buffer (O) at a rate of 45 uL at 5 uL / s. After osmoflow, the system was briefly paused prior to introducing XP to the device. As described hereinabove in reference to FIGs.7B, XP was introduced in multiple steps, as shown in Table 5.

[0241] Table 5. Sequencing Load Conditions Run Total Initial load Pre-seq. Secondary load type sample incubation loaded Volume Speed Flow time (m) Volume Speed Flow (uL) (uL) (uL / s) time (m) (uL) (uL / s) time (m) 55uL 4hr 255 60 0.1 10 2 195 0.014 232 55uL 1hr 255 60 0.1 10 2 195 0.056 58 Fast: 255 60 1.0 1 1 195 0.056 58 55uL 1hr Fast: 255 80 1.78 0.75 1 175 0.09 32 55uL 35m Fast: 255 80 1.78 0.75 1 175 0.09 58 55uL 1hr

[0242] The device was used in a sequencing run in which the sequencing waveform and conditions shown in FIG.14B were used. After the sequencing run, the device was subjected to an end of run cleaning protocol as described in more detail below. The device was either used in a subsequent sequencing run or stored for later use in the system fluid. If it was used in a subsequent sequencing run, the process was repeated as described above starting from the step of subjecting the device to a pre- sequencing cleaning protocol.

[0243] A more detailed illustration of the PSP used in this Example is provided in FIGs.14C-14D. As shown in FIG.14C, the various reagents used in the PSP were sequentially drawn into a fluidic introduction port in the sequencing system as follows: (i) system fluid; (ii) a volume of air, (iii) cleaning solution, (iv) a volume of air, (v) basic detergent solution, (vi) a volume of air, (vii) cleaning solution, (viii) a volume of air, (ix) basic detergent solution, and (x) a volume of air, such that as the solution in the port was added to the device, each of these reagents (i)-(x) were introduced in reverse order (see, e.g., FIG.14D). Reagent volumes, flow rates, and temperatures for the PSP are shown in FIG.14D, and the device was incubated for 5 seconds after the introduction of each reagent in the process. Once the process was completed, theadapter on the fluidic introduction port was cleaned and the tip was removed and cleaned.

[0244] The post-sequencing cleaning protocol used is shown in FIGS.14E-14G. Reagents were introduced to the device by stacking, as described above in relation to FIG.14C and described above. The first set of stacked reagents are shown in FIG.14E (including basic detergent solution and cleaning solvent with volumes of air separating each bolus of reagent in the fluid introduction port) and each successive reagent was added to the device as shown in FIG.14G. After the last volume of air, protective solution was introduced in a reagent stack as shown in FIGs.14F-14G. Reagent volumes, flow rates, and temperatures for the post-sequencing protocol are shown in FIG.14G, and the device was incubated for 5 seconds after the introduction of each reagent in the process. Once the process was completed, the adapter on the fluidic introduction port was cleaned and the tip was removed and cleaned.

[0245] Using the protocols described in this Example, the total sequencing run time was reduced from over 100 minutes to 64.8 minutes, the pre-sequencing time was reduced from 85.3 minutes to 56 minutes, post-sequencing time was reduced from 19.2 minutes to 8.8 minutes, total non-sequencing time was reduced from 105 minutes to less than 60 minutes, and pore count, lifetime, reuse performance, multi- cycle consistency, and accuracy were improved.

Claims

WHAT IS CLAIMED IS:

1. A method of sequencing a target nucleic acid comprising: (a) providing a nanopore sequencing device comprising a plurality of electrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution; (b) preparing the device for sequencing by forming a lipid bilayer on the plurality of electrochemical cells having a nanopore molecule inserted in the lipid bilayer, thereby operably connecting the cis and trans chambers by the nanopore, wherein the lipid composition comprises: (a) a phospholipid; (b) a bilayer additive; (c) an annular solvent; (d) silicone oil; and (e) a cosolvent; wherein (x) a molar ratio of phospholipid to bilayer additive in the composition is between 1:6 to 1:10; and / or (y) a molar ratio of silicone oil to cosolvent is between 6:1 to 12:1.; (c) perfusing the cis chambers with an XP buffer comprising XP molecules; Applying an electrical stimulus to the plurality of electrochemical cells that causes at least a portion of the XP molecules to translocate through the nanopore; and (d) detecting changes in an electrical signal in the cell as the XP molecules translocate through the nanopore, wherein the changes in the electrical signal are indicative of a sequence of a target nucleic acid.

2. The method of claim 1 wherein the nanopore is up to 2nM heptameric hemolysin suspended in a pore dilution buffer.

3. The method of any one of the preceding claims, wherein the lipid composition comprises at least 1.5 mg / ml phospholipid, at least 6 mg / ml bilayer additive, between 5-10% annular solvent, between 8-12% cosolvent, and between 75-85% silicone oil.

4. The method of any one of the preceding claims, further comprising, prior to step (b), subjecting the device to a pre-sequencing cleaning protocol.

5. The method of any one of the preceding claims further comprising, subjecting the device to an end of run cleaning protocol after the detecting step (d).

6. A lipid bilayer composition comprising: a phospholipid;a bilayer additive; an annular solvent; silicone oil; and a cosolvent, wherein (i) a molar ratio of phospholipid to bilayer additive in the composition is between 1:6 to 1:10; and / or (ii) a molar ratio of silicone oil to cosolvent is between 6:1 to 12:

1.

7. The composition of claim 6 wherein the composition comprises one or more of the following: (a) the phospholipid is selected from the group consisting of DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, and mixtures thereof; (b) the bilayer additive comprises single-tail lipids, lauryl dimethylamine-N- oxide, octyl beta-D-glucopyranoside, sodium dodecyl sulfate, Triton X-100, CHAPS, Span20, Span60, Span80, lysophospholipids, sphingosine, monoglycerides, cationic lipids, nonionic surfactants, and combinations thereof; (c) the annular solvent comprises hexane, chloroform, dichloromethane, toluene, cyclohexane, benzene, diethyl ether, petroleum ether, isooctane, carbon tetrachloride, and mixtures thereof; (d) the silicone oil comprises PDM20, mineral oil, squalene, polyalphaolefins, medium-chain triglycerides, isopropyl myristate, caprylic / capric triglycerides, perfluoropolyether, hydrogenated polyisobutene, and mixtures thereof; and (e) the cosolvent comprises decane, tridecane, octadecane, squalene, cyclohexane, heptane, hexane, hexadecane, toluene, xylene, and mixtures thereof.

8. The composition of claim 7, wherein the lipid composition comprises at least 1.5 mg / ml phospholipid, at least 6 mg / ml bilayer additive, between 5-10% annular solvent, between 8-12% cosolvent, and between 75-85% silicone oil.

9. A nanopore sequencing device comprising a plurality of electrochemical cells, wherein each electrochemical cell comprises (a) a nanopore embedded in a lipid bilayer comprising the composition of any one of claims 6-8; and (b) cis and transchambers separated by the lipid bilayer and operably connected by the nanopore, wherein the cis and trans chambers each contain an electrode, and an electrolyte solution.

10. A method of preparing a nanopore sequencing device for use in a sequencing assay: providing the nanopore sequencing device comprising a plurality of electrochemical cells, wherein each electrochemical cell comprises cis and trans chambers each containing an electrode and an electrolyte solution, and introducing a nanopore composition into the device using an iterative pulsed flow to increase uniform delivery of nanopore molecules across the fluidic path.

11. The method of claim 10 wherein the iterative pulsed flow comprises two or more applications of a portion of the nanopore composition at a flow rate of between 1-500 uL / sec.

12. The method of any one of claims 10-11 wherein the iterative pulsed flow comprises two or more applications of a portion of the nanopore composition at a flow rate of between 5-100 uL / sec.

13. The method of any one of claims 10-12 wherein the iterative pulsed flow comprises two or more applications of a portion of the nanopore composition at a flow rate of between 20-50 uL / sec.

14. The method of any one of claims 10-13 wherein each application of the portion of nanopore composition is followed by for a waiting period of up to 10 minutes before an additional application is introduced.

15. The method of any one of claims 10-14 wherein each application of the portion of nanopore composition is followed by for a waiting period of up to 5 minutes before an additional application is introduced.

16. The method of any one of claims 10-15 wherein each application of the portion of nanopore composition is followed by for a waiting period of up to 2 minutes before an additional application is introduced.

17. The method of any one of claims 10-16 wherein the iterative pulsed flow comprises 2 to 20 applications of the portion of nanopore composition.

18. The method of any one of claims 10-17 wherein the pulsed flow rate comprises between 3 to 5 applications of the portion of nanopore composition.

19. The method of any one of claims 10-18 wherein the flow rate of the two or more applications of the portion of the nanopore composition is 25 uL / sec.

20. The method of any one of claims 10-19 wherein the waiting period is about 60 seconds.

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