Calibration of nanopore array devices

Abstract

A method of generating a polymer sequence using a nanopore sequencing device, the nanopore array device comprising nanopore channels (35) formed in a membrane (32) separating two ionic solutions (33, 36), the nanopore channels connecting the ionic solutions, the method comprising: translocating a series of polymers through the nanopore channels; generating a measurement signal during translocation of each polymer through the nanopore channel; analyzing a set of measurement signals acquired from each polymer during translocation of the polymer through the nanopore channel to determine a scale factor for each polymer, applying the scale factor to the measurement signals generated during translocation of the polymer through the nanopore channel to determine a normalized measurement signal value, and generating a polymer sequence for each polymer in the series from the corresponding normalized measurement signal value for each polymer.

Description

[0001] The present invention relates to methods for calibrating nanopore devices. More specifically, the present invention relates to methods for calibrating nanopore array devices. Most specifically, the present invention relates to methods for calibrating nanopore array devices for sensing molecular entities of analytes.

[0002] The use of nanopores to sense interactions with molecular entities (e.g., polynucleotides) is a powerful technology that has recently undergone significant development. Nanopore devices comprising arrays of nanopore sensing elements have been developed to enhance data collection by allowing multiple nanopores to sense interactions in parallel, typically from the same sample.

[0003] Nanopore devices can generally use electrical signals across the nanopore channel to generate measurement signals that are interpreted to sense and / or characterize molecular entities as they interact with the nanopore. Typically, the electrical signal is applied as a potential difference or current across the array of nanopore channels, which provides a meaningful measurement signal to be interpreted. The measurement can include, for example, one of ionic current, resistance, or voltage.

[0004] Typically, an electrical signal from an array device is applied to the system at one or more predetermined values. Changes in the measured signal over time can then be interpreted to determine the molecular entities present in the analyte. However, in array devices, variations in extrinsic factors (i.e., standard conditions) or intrinsic factors (i.e., the "health" of the nanopore channel or membrane) across the array can cause variations in the measured signal across the array and contaminate or distort the measured signal from the nanopore channel.

[0005] In a first embodiment of the present invention, a method for generating a polymer sequence using a nanopore sequencing device is provided. The nanopore array device includes a nanopore channel formed in a membrane separating two ionic solutions, and the nanopore channel connects the ionic solutions. The method includes:

[0006] causing a series of polymers to translocate through a nanopore channel; generating a measurement signal during the translocation of each polymer through the nanopore channel; analyzing a set of measurement signals from each polymer acquired during the translocation of the polymer through the nanopore channel to determine a scaling factor for each polymer; applying the scaling factor to the measurement signals generated during the translocation of the polymer through the nanopore channel to determine a normalized measurement signal value; and generating a polymer sequence for each polymer in the series from the corresponding normalized measurement signal value for each polymer.

[0007] By determining a scaling factor for each polymer that translocates through the pore, a more accurate signal estimate for each polymer can be achieved. The scaling factor is determined from a set of measurements from the region of the polymer to ensure consistent signal levels as much as possible, substantially or completely independent of the channel, pore, flow cell, or other experimental or standard conditions. Using such a scaling factor will then improve the quality and robustness of the normalized signal, enabling it to be used to generate polymer sequences from, for example, base callers or other computational sequencing techniques.

[0008] Additionally, improved read scaling methods, based on carefully selected quantiles of the full read distribution, have been found to reduce genomic bias in low-entropy (i.e., low-variation) polymers. Furthermore, when combined with scaling factors on the measured signals of multiple polymers, read-to-read noise and drift from standard conditions between reads can be reduced using the methods of the present invention. This is not an easy task, as there are many variables that affect each channel, such as temperature variations, electrolyte mediator concentrations at the electrodes, and the health of the nanopore and membrane. Therefore, it is important to calculate rolling statistics (i.e., scaling factors determined for each polymer in the series) rather than global statistics (i.e., scaling factors applied to the entire series of polymers), as the scaling properties of the nanopore channel can vary over longer timescales.

[0009] The set of measurement signals obtained from each polymer during the translocation of the polymer through the nanopore channel for determining the scale factor of each polymer can be a measurement of the adapter region or a portion of the adapter region of each polymer. The adapter used for nanopore sequencing of, for example, a polynucleotide can include at least one single-stranded polynucleotide or a non-polynucleotide region. For example, Y-shaped adapters used in nanopore sequencing are known in the art, such as those disclosed in WO22243691 and WO21255476, which are disclosed herein in their entirety by reference.

[0010] In this regard, the scaling factor determined from the adapter region of each polymer in a series of polymers also provides an estimate of the changes within the system. This is more accurate than relying on measurement signals obtained from local non-adapter regions of the polymers because the sequence / content of the adapter region is known for each polymer in the series and has a known length. Therefore, using the adapter region to determine the scaling factor ensures that known regions of the polymer are used. This in turn means that the user does not rely on an estimate of the sequence in the reads or the polymer itself to determine the scaling factor, as both of these measurements require the user to know which portion of the polymer will be used for scaling and / or can rely on post-analysis of the sequenced content before scaling is applied. The adapters of all polymers in the series of polymers can be identical. This means that in a perfect system, there should be no variation in the scaling factor determined from measurements generated by the nanopore channel. This can be used to assess intrinsic / extrinsic changes to the nanopore channel during use.

[0011] Additionally, the adapter region is typically the first part of the polymer to be sequenced and is typically of known length. Therefore, the scaling factor can be accurately determined from analysis of known adapters. This adapter region-based scaling can be further refined by determining the median signal and excluding any signal measurements above / below a specific quantile, typically 10 (i.e., lower bottom 5 quantile and upper top 95 quantile), and even more typically 20 (i.e., lower bottom 10 quantile and upper top 90 quantile). This ensures that irrelevant or erroneous data points are excluded from the scaling applied to the measured signal.

[0012] The length of at least one polymer in the series of polymers may be shorter than the adapter region of the polymer. In this case, the polymer may be a short chain or fragment of a larger polymer. This may mean that the broken portion is part of the target polymer that is broken, and the portion can be reconstructed to determine the entire target polymer sequence. The method of the present invention is particularly suitable for generating proportional factors for polymers of this length because the adapter region provides a consistent and reliable set of measurements that can be used to scale each set of measurements that is much shorter. In addition, the polymer to be analyzed may not be long enough to provide enough measurements for an accurate proportional factor, and for example, signal noise may have a greater weight in determining the proportional factor, and therefore adversely affect the accuracy of the proportional factor. It can be considered that the reconstructed sequence is constructed from more accurate sequencing estimates based on the proportional factors applied to each fragment.

[0013] The scale factors from a range of polymers can be analyzed to determine the condition of the nanopore channel nanopore device. As mentioned above, the scaled properties of the nanopore channel can change over long timescales. By analyzing the changes in the scale factors from each of the polymers, especially when using a consistent adapter region for each polymer, the user can be informed if there are unusual or unexpected drifts or changes, which may indicate that the nanopore channel is not operating as expected due to, for example, temperature changes, damage to the nanopore or membrane, and depletion of the electrolyte medium in the system.

[0014] A nanopore sequencing device may include an array of nanopore channels. In this case, a user may monitor changes in the scale factor of each channel. In this case, the nanopore sequencing device may select a nanopore channel from the array based on the scale factor or changes in the scale factor associated with the nanopore channel. The methods of the present invention may be used so that the device may be configured to select a functional nanopore channel from the array of nanopore channels based on examining the scale factor or changes in the scale factor of each channel.

[0015] The nanopore in the nanopore channel can be a biological pore supported on a membrane. The nanopore can be a solid-state pore or a biological pore supported on a solid-state membrane. The nanopore device can include a sensor electrode, and the measurement can include an electrical measurement. Furthermore, the nanopore device can include a sensor electrode, and the measurement obtained by the sensor can indicate ion flow through the nanopore.

[0016] The polymer may comprise a series of polymer units to be recognized by the nanopore device. The polymer is a polynucleotide, and the polymer units are nucleotides. In a specific embodiment, the polymer or polynucleotide is genomic DNA. The use of the present invention is particularly beneficial for polymers such as genomic DNA and RNA, where it can be assumed that there is low entropy in the molecular portion to be sensed. In other words, the improved scaling based on the region of the polymer translocated through the nanopore channel provides better fidelity or resolution for samples with highly repetitive patterns of specific molecular portions. For example, it can be assumed that genomic DNA has regions of G and C base repeats that may be incorrectly counted or misinterpreted by prior art methods.

[0017] In one example, a median value for the measured signal values can be determined and used to determine the normalized measured signal value. This can be improved by determining the median signal and excluding any signal measurements above or below a certain quantile, typically 10 (i.e., the lower 5th quantile and the upper 95th quantile). This ensures that irrelevant or erroneous data points are excluded from the scaling applied to the measured signal.

[0018] In order to achieve a better understanding, embodiments of the invention will now be described, by way of non-limiting examples, with reference to the accompanying drawings, in which:

[0019] Figure 1 is a diagram of a nanopore array device;

[0020] Figure 2 is a schematic cross-sectional view of a portion of a nanopore array device;

[0021] Figure 3 shows three example traces from a nanopore channel as a polymer translocates through the nanopore channel, where the x-axis indicates time and the y-axis indicates ionic current; and

[0022] Figure 4 is a graph showing polymers of various GC contents versus quantile of the included data along the x-axis and median ion current value plotted along the y-axis.

[0023] Nanopore array device 1 for sensing the interaction of molecular entities Figure 1 The nanopore array device 1 comprises a sensing apparatus 2 , which comprises a sensor device 3 and a detection circuit 4 connected to the sensor device 3 .

[0024] The sensor device 3 comprises an array of sensing elements 30, each supporting a respective nanopore capable of interacting with a molecular entity. The sensing elements 30 comprise respective electrodes 31. In use, each sensing element 30 outputs an electrical measurement at its electrode 31 that is dependent on the interaction of the molecular entity with the nanopore. Figure 1 It is shown schematically in FIG, but can have a variety of configurations, some non-limiting examples of which are as follows.

[0025] In one example, the sensor device 3 may have Figure 2 The sensor device 2 is shown in the form shown. Here, the sensor device 2 includes an array of sensor elements 30, each of which includes a membrane 32 supported across a well 33 in a substrate 34, wherein a nanopore 35 is inserted into the membrane 32. The membrane 31 can be made of amphiphilic molecules (such as lipids), as described below. Each membrane 32 seals the corresponding well 33 from a sample chamber 36, which extends through the array of sensor elements 30 and is in fluid communication with each nanopore 35. Each well 33 has a sensor electrode 32 arranged therein. A common electrode 37 is provided in the sample chamber 36 for providing a common reference signal (typically an electric potential or voltage) to each sensor element 30. In use, the sample chamber 36 receives a sample containing molecular entities that interact with the nanopore 35 of the sensor element 30.

[0026] For clarity, Figure 2Two sensing elements 30 are shown in FIG, but generally any number of sensing elements 30 may be provided. Generally, a large number of sensing elements 30 may be provided to optimize the data collection rate, such as 256, 1024, 4096 or more sensing elements 30.

[0027] The sensor device 3 may have a detailed construction as disclosed in WO 2009 / 077734 or WO 2014 / 064443, which are incorporated herein by reference in their entirety.

[0028] The associated elements of the nanopore and the sensing element 30 may be as follows, but are not limited to: Figure 2 Example shown.

[0029] A nanopore is a pore, typically having a size on the order of nanometers. In embodiments where the molecular entity is a polymer that interacts with the nanopore while translocating through the nanopore, in such cases the nanopore is of a suitable size to allow the polymer to pass therethrough.

[0030] The nanopore can be a protein pore or a solid-state pore. The size of the pore can be such that only one polymer can translocate through the pore at a time.

[0031] When the nanopore is a protein pore, it may have the following properties.

[0032] The nanopore may be a transmembrane protein pore. Transmembrane protein pores used in accordance with the present invention include, but are not limited to, beta-toxins such as α-hemolysin, anthrax toxin, and leukocidin; and bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp) (e.g., MspA), lysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter lipoprotein (NalP). α-helical bundle pores include a barrel or channel formed by α-helices. Suitable α-helical bundle pores include, but are not limited to, inner membrane proteins and α outer membrane proteins such as WZA and ClyA toxins. The transmembrane pore may be derived from lysine. The pore may be derived from CsgG, such as disclosed in WO-2016 / 034591, which is incorporated herein by reference in its entirety. The pore may be a DNA origami pore.

[0033] The protein pore may be a naturally occurring pore or may be a mutant pore. The pore may be entirely synthetic.

[0034] When the nanopore is a protein pore, it can be inserted into a membrane supported in the sensor element 30. Such a membrane can be an amphiphilic layer, such as a lipid bilayer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer can be a coblock polymer, such as disclosed in WO 2014 / 064444. Alternatively, the protein pore can be inserted into an orifice provided in a solid-state layer, such as disclosed in WO 2012 / 005857.

[0035] The nanopore may include an aperture formed in the solid-state layer, which may be referred to as a solid-state pore. The aperture may be a well, gap, channel, groove, or slit disposed in the solid-state layer, along which an analyte may pass or enter. The solid-state layer may be formed of both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials (such as Si3N4, Al2O3, and SiO), organic and inorganic polymers (such as polyamides), plastics (such as Teflon®), or elastomers (such as two-component addition-cured silicone rubber), and glass. The solid-state layer may be formed of graphene.

[0036] The molecular entity interacts with the nanopore in the sensing element 30 , resulting in the output of an electrical signal at the electrode 31 that is dependent on the interaction.

[0037] In one type of sensor device 3, the electrical signal may be an ionic current flowing through the nanopore. Similarly, electrical properties other than ionic current may be measured. Some examples of alternative types of properties include, but are not limited to, ionic current, impedance, tunneling properties, such as tunneling current (e.g., as disclosed in Ivanov AP et al., Nano Lett. 2011 Jan 12; 11(1): 279-85, which is incorporated herein by reference in its entirety), and FET (field effect transistor) voltage (e.g., as disclosed in WO2005 / 124888, which is incorporated herein by reference in its entirety). One or more optical properties may be used, optionally in combination with electrical properties (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1): 014301, which is incorporated herein by reference in its entirety). The property may be a transmembrane current, such as an ionic current flowing through the nanopore. The ionic current may typically be a DC ionic current, although in principle an alternative is to use an AC current (i.e., the amplitude of an AC current flowing under an applied AC voltage).

[0038] The interaction may occur during translocation of the molecular entity relative to, eg, through, the nanopore.

[0039] The electrical signal provides a series of measurements of properties associated with the interaction between the molecular entity and the nanopore. This interaction can occur at the constriction region of the nanopore. For example, where the molecular entity is a polymer comprising a series of polymer units that translocate relative to the nanopore, the measurement can be of a property that depends on the translocation of the successive polymer units relative to the pore.

[0040] The ionic solution can be provided on either side of the nanopore. The sample containing the molecular entity of interest as a polymer can be added to one side of the nanopore, e.g. Figure 2 The polymer is placed in a sample chamber 36 in a sensor device of a membrane and allowed to translocate relative to the nanopore, for example, under a potential difference or chemical gradient. An electrical signal can be obtained during translocation of the polymer relative to the pore, for example, during translocation of the polymer through the nanopore. The polymer can be partially translocated relative to the nanopore.

[0041] In order to allow measurement when polymer translocation passes through nanopore, the translocation rate can be controlled by a binding moiety that is bound to the polymer. Typically, the binding moiety can move the polymer through the nanopore under the action of an applied field or against the applied field. The binding moiety can be a molecular motor, for example, used when the binding moiety is an enzyme, enzymatic activity, or used as a molecular brake. When the polymer is a polynucleotide, a variety of methods for controlling the translocation rate have been proposed, including the use of polynucleotide-bound enzymes. Suitable enzymes for controlling the translocation rate of polynucleotides include but are not limited to polymerases, helicases, exonucleases, single-stranded and double-stranded binding proteins and topoisomerases, such as gyrase. For other polymer types, a binding moiety that interacts with this polymer type can be used. The binding moiety may be any of the binding moieties disclosed in WO-2010 / 086603, WO-2012 / 107778 and Lieberman KR et al., J Am Chem Soc. 2010;132(50):17961-72), and the voltage-gated scheme (Luan B et al., Phys Rev Lett. 2010;104(23):238103), all of which are incorporated herein by reference in their entirety.

[0042] Binding moieties can be used in a variety of ways to control polymer motion. The binding moiety can move the polymer through the nanopore under the influence of an applied field or against the applied field. The binding moiety can be used as a molecular motor, for example, where the binding moiety is an enzyme, enzymatic activity, or as a molecular brake. The translocation of the polymer can be controlled by a molecular ratchet that controls the movement of the polymer through the pore. The molecular ratchet can be a polymer-binding protein.

[0043] The polynucleotide handling enzyme may be one of the types described in, for example, WO 2015 / 140535 or WO-2010 / 086603.

[0044] Translocation of the polymer through the nanopore can occur, cis to trans or trans to cis, either with or against an applied potential. Translocation can occur under an applied potential that can control the translocation.

[0045] Exonucleases that act gradually or continuously on double-stranded DNA can be used on the cis side under an applied potential to force the remaining single strand through the pore, or on the trans side under a reverse potential. Similarly, helicases that unwind double-stranded DNA can be used in a similar manner. Sequencing applications are also possible, which require strand translocation against the applied potential, but the DNA must first be "caught" by the enzyme under a reverse potential or no potential. If the potential is switched back after binding, the strand will pass through the pore from cis to trans and be held in an extended conformation by the current. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can act as molecular motors, pulling the recently translocated single strand back through the pore in a controlled, stepwise manner (trans to cis) against the applied potential. Alternatively, single-stranded DNA-dependent polymerases can act as molecular brakes to slow the movement of the polynucleotide through the pore. Any of the moieties, techniques or enzymes described in WO-2012 / 107778 or WO-2012 / 033524, both of which are incorporated herein by reference in their entirety, may be used to control polymer motion.

[0046] The sensing element 30 and / or the molecular entity can be adapted to capture the molecular entity in the vicinity of the corresponding nanopore. For example, the sensing element 30 can further include a capture moiety configured to capture the molecular entity in the vicinity of the corresponding nanopore. The capture moiety can be any of the binding moieties or exonucleases described above, also for the purpose of controlling translocation, or can be provided separately.

[0047] The capture moiety may be attached to the nanopore of the sensing element.At least one capture moiety may be attached to the nanopore of each sensor element.

[0048] The capture moiety can be a tag or tether that binds to a molecular entity. In this case, the molecular entity can be tailored to achieve this binding.

[0049] Such tags or tethers can be attached to the nanopore, for example as disclosed in WO 2018 / 100370, which is incorporated herein by reference in its entirety, and as further described below.

[0050] Alternatively, where the nanopore is inserted into a membrane, such tags or tethers may be attached to the membrane, for example as disclosed in WO 2012 / 164270, which is incorporated herein by reference in its entirety.

[0051] The methods described herein may include the use of an adapter that adapts the polymer to optimize its translocation through the nanopore. The adapter is typically coordinated to one or both ends of the polymer and may include one or more spacers for preventing the motor protein loaded onto the adapter. For example, polynucleotide adapters suitable for nanopore sequencing of polynucleotides are known in the art. The adapter for nanopore sequencing of polynucleotides may include at least one single-stranded polynucleotide or non-polynucleotide region. For example, Y-type adapters for nanopore sequencing are known in the art, such as disclosed in WO22243691 and WO21255476, the entire text of which is disclosed herein by reference. The Y adapter typically includes (a) a double-stranded region and (b) a single-stranded region or a non-complementary region at the other end. If the Y adapter includes a single-stranded region, it can be described as having an overhang. The presence of the non-complementary region in the Y adapter gives the adapter its Y shape because, unlike the double-stranded portion, the two chains do not typically hybridize to each other. The Y adapter may include one or more anchors.

[0052] The Y adapter preferably includes a leader sequence that preferentially penetrates the pore. The leader sequence typically includes a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader preferably includes a polynucleotide, and more preferably includes a single-stranded polynucleotide. The adapter can be connected to the DNA molecule using any method known in the art. Each polymer will typically include the same adapter molecule, and therefore measurements of the adapter region or a portion of the adapter region can be advantageously used to provide a reliable scaling factor.

[0053] The polynucleotide adapter can include a membrane anchor or transmembrane pore anchor attached to the adapter. For example, the membrane anchor or transmembrane pore anchor can promote the positioning of the adapter and the coupled polynucleotide near the nanopore. The anchor can be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. In one embodiment, the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein, or amino acid, such as cholesterol, palmitate, or tocopherol.

[0054] The anchor may comprise a linker, or 2, 3, 4 or more linkers. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers may be linear, branched, or cyclic. Suitable linkers are described in WO 2010 / 086602. Examples of suitable anchors and methods of attaching anchors to adapters are disclosed in WO 2012 / 164270 and WO 2015 / 150786, both of which are incorporated herein by reference in their entirety.

[0055] Examples of tags and tethers attached to nanopores are as follows.

[0056] Nanopores used in the methods described herein can be modified to include one or more binding sites for binding to one or more analytes (e.g., molecular entities) and thereby serve as capture moieties. In some embodiments, the nanopore can be modified to include one or more binding sites for binding to an adaptor attached to the analyte. For example, in some embodiments, the nanopore can bind to a leader sequence of an adaptor attached to the analyte. In some embodiments, the nanopore can bind to a single-stranded sequence in an adaptor attached to the analyte.

[0057] In some embodiments, the nanopore is modified to include one or more tags or tethers, each tag or tether including a binding site for an analyte. In some embodiments, the nanopore is modified to include one tag or tether per nanopore, each tag or tether including a binding site for an analyte.

[0058] In some embodiments, the tag or tether may comprise or be an oligonucleotide.

[0059] Other examples of tags or tethers include, but are not limited to, a His tag, biotin or streptavidin, an antibody that binds to the analyte, an aptamer that binds to the analyte, an analyte binding domain such as a DNA binding domain (including, for example, a peptide zipper such as a leucine zipper, a single-stranded DNA binding protein (SSB)), and any combination thereof.

[0060] Any method known in the art can be used to attach a tag or tether to the outer surface of the nanopore, such as the cis side of the membrane. For example, one or more tags or tethers can be attached to the nanopore via one or more cysteines (cysteine bonds), one or more primary amines (such as lysine), one or more non-natural amino acids, one or more histidines (His tags), one or more biotin or streptavidin, one or more antibody-based tags, one or more enzyme modifications of epitopes (including, for example, acetyltransferases), and any combination thereof. Suitable methods for performing such modifications are well known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz) and Liu CC and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444. Figure 1 Any of the amino acids numbered 1-71 in SEQ ID NO: 1, which is incorporated herein by reference in its entirety.

[0061] In some embodiments where one or more tags or tethers are attached to the nanopore via a cysteine bond, one or more cysteines may be introduced by substitution into one or more monomers forming the nanopore.

[0062] The transmembrane pore can be modified to enhance capture of polynucleotides. For example, the pore can be modified to increase the positive charge within the pore entrance and / or within the barrel of the pore. Such modifications are known in the art. For example, WO 2010 / 055307 discloses mutations in α-hemolysin that increase the positive charge within the barrel of the pore.

[0063] Modified MspA, lysine, and CsgG pores comprising mutations that enhance polynucleotide capture are disclosed in WO 2012 / 107778, WO 2013 / 153359, and WO 2016 / 034591, respectively, all of which are incorporated herein by reference in their entireties. Any of the modified pores disclosed in these publications may be used herein.

[0064] The arrangement of the detection circuit 4 will now be discussed. The detection circuit 4 is connected to the electrodes 31 of each sensor element 30 and has the primary function of processing the electrical signals output therefrom. The detection circuit 4 also has the function of controlling the application of a bias signal to each sensor element 30.

[0065] The detection circuit 4 includes a plurality of detection channels 40. Each detection channel 40 receives an electrical signal from a single sensor electrode 31 and is arranged to amplify the electrical signal. Therefore, the detection channel 40 is designed to amplify very small currents with sufficient resolution to detect characteristic changes caused by the interaction of interest. The detection channel 40 is also designed with a sufficiently high bandwidth to provide the time resolution required to detect each such interaction. These limitations require sensitive and therefore expensive components. Each detection channel 40 can be similar to a standard single-channel recording device as described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19): 7702-7, Lieberman KR et al., J Am Chem Soc. 2010; 132(50): 17961-72 and WO-2000 / 28312. Alternatively, each detection channel 40 may be arranged as described in detail in WO 2010 / 122293, WO 2011 / 067559 or WO 2016 / 181118.

[0066] The analyte of interest to be detected by the nanopore can be a polynucleotide, such as DNA or RNA. The nucleotides can be naturally occurring or non-naturally occurring. The nucleotides can be modified. Specifically, the polynucleotide can be genomic DNA or genomic RNA, and the methods of the present invention can be used to more accurately determine the GC bias in the genome. The genome can be from any source and can be eukaryotic or prokaryotic. The genome can be bacteria, viruses, plants, animals, algae, protozoa, or archaea. The analyte can be a polypeptide or a polysaccharide.

[0067] The number of sensing elements 30 in the array is greater than the number of detection channels 40, and the nanopore array device is operable to obtain polymer measurements from selected sensing elements 30 in a multiplexed manner, particularly an electrically multiplexed manner. This is achieved by providing a switch arrangement 42 between the sensor electrodes 31 of the sensing elements 30 and the detection channels 40. For clarity, Figure 1 A simplified example with four sensing elements 30 and two detection channels 40 is shown, but the number of sensor elements 30 and detection channels 40 is typically much larger. For example, for some applications, the sensor device 2 may include a total of 4096 sensing elements 30 and 1024 detection channels 40.

[0068] The switch arrangement 42 may be arranged as described in detail in WO 2010 / 122293. For example, the switch arrangement 42 may include a plurality of 1 to N multiplexers, each multiplexer connected to a set of N sensing elements 30 from a detection channel 40, and may include appropriate hardware (such as latches) to select the state of the switches.

[0069] The nanopore array device 1 can be operated to amplify electrical signals from the sensing elements 30 selected in an electrically multiplexed manner by switching the switch arrangement 42. The detection circuit 4 includes a data processor 5 that receives output signals from the detection channels 40. The data processor 5 acts as a controller that controls the switch arrangement 42 to connect the detection channels 40 to the corresponding sensing elements 30, as further described below.

[0070] In addition, the detection circuit 4 includes a bias control circuit 41 to perform the function of controlling the application of a bias signal to each sensor element 30. The bias control circuit 41 is connected to the common electrode 37 and the sensor electrode 31 of each sensing device 30. The bias signal is selected to bias the sensor electrode 31 relative to the common electrode 37, thereby controlling the translocation of the molecular entity relative to the nanopore. Generally, the bias signal provided to a given sensor element 30 can be a driving bias signal that causes translocation to occur at the sensor element 30, or an inhibiting bias signal that inhibits translocation to occur at the sensor element 30.

[0071] The bias control circuit 41 is controlled by the data processor 5. The data processor has an operating mode for the bias control circuit 41. That is, three independent test bias signals are provided to all sensor elements 30, thereby inducing an ion current with respect to the nanopore of each sensor element 30. The current corresponding to each test signal is recorded in the data processor 5 as an amplified electrical signal.

[0072] The data processor 5 is arranged as follows. It is connected to the output of the detection channel 40 and is supplied with the amplified electrical signal therefrom. The data processor 5 stores and analyzes the amplified electrical signal from the test bias signal to generate a calibrated signal. The data processor 5 also controls other elements of the detection circuit, including the bias voltage circuit 41 described above and the switch arrangement 42 described below. The data processor 5 forms part of the detection circuit 2 and may be provided in a common package with the detection circuit, possibly on a common circuit board. The data processor 5 may be implemented in any suitable form, such as as a processor running an appropriate computer program or as an ASIC (Application Specific Integrated Circuit).

[0073] The data processor 5 of the nanopore array device 1 is connected to an analysis system 6. The data processor 5 also provides the amplified output signal to the analysis system 6. The analysis system 6 performs further analysis on the amplified electrical signal, which is the raw signal representing the measurement result of the property measured at the nanopore. Such an analysis system 6 can, for example, estimate the overall identity of a molecular entity or, in the case where the molecular entity is a polymer, the identity of its polymer units. Thus, the analysis system can be configured as a computer device running an appropriate program. Such a computer device can be connected to the data processor 5 of the nanopore array device 1 directly or via a network, for example, within a cloud-based system.

[0074] Figure 3 Three example traces are provided, which show the measured signals of three different polymers translocating through the nanopore channel. These polymers are different DNA chains or DNA fragments with various sequences and lengths.

[0075] The shaded area starting at time 0 represents the region in which a set of measurements were taken to normalize the signal across the entire set of measurements generated from the polymer. In these specific examples, for scaling purposes, the region of interest is the adapter region of the polymer. As shown in the example traces, the adapter region is not always the same length or sequence.

[0076] The trajectory of the change in amplitude along the x-axis is the overall trajectory of the ionic current through the nanopore channel as the polymer translocates through the nanopore channel. Changes in the ionic current indicate the fraction of molecules within the nanopore channel, and computational methods (such as base calling programs) can be used to interpret such changes in the measured signal.

[0077] In this example, these traces of measured signal are normalized relative to a scaling factor generated when comparing the traces in the shaded region to the expected value for that region or to a previous read from the same region in another polymer in the series of polymers being analyzed. These normalized measured signals are then more easily interpreted by, for example, a base caller or another computational program that can interpret complex signals.

[0078] Additionally, a set of lines parallel to the x-axis show the quantiles of the signal measurements, which are calculated from the median value of the signal generated when the polymer translocates through the nanopore.

[0079] These quantiles can be used to exclude extreme values measured in the scaled region of interest, thereby improving the overall accuracy of the scale factor to be applied.

[0080] The problem with using full-strand-based scaling methods is that the calculated scaling factors will depend on the genomic content of the reads, not just on the scaling properties of the nanopore channel. When using scaling factors generated from data obtained from full-strand translocation through the nanopore channel, the goal would be to generate more precise scaling parameters that programs like base-calling models would learn to rely on during training.

[0081] This in turn can exacerbate the problem of bias (ie, the amount of G or C homogeneity within a resolved strand region, which is particularly seen in genomic polymers such as DNA and RNA).

[0082] To illustrate the problem, Figure 4 A curve graph of the median signal quantiles of different genomes is provided. In order to highlight the main influence, GC content is calculated from the main genome in the data set. The curve graph shows the signal quantiles of different genomes, which is shaded according to GC content. The median (q50) and the quantile near it have a large numerical range, which depends largely on the GC content of the genome. On the other hand, the 20th quantile and the 90th quantile are closely gathered in the genome. This method excludes the extreme value of the measurement signal from things such as noise when generating scale factors and normalized data. This improves the overall accuracy and fidelity of the data because the data are input into the base calling program, for example, polymer sequences are generated from the normalized measurement signal.

Claims

1. A method for generating polymer sequences using a nanopore sequencing device, The nanopore array device includes a nanopore channel formed in a membrane separating two ionic solutions, and the nanopore channel connects the ionic solutions. The method comprises: translocating a series of polymers through the nanopore channel; generating a measurement signal during translocation of each polymer through the nanopore channel; analyzing a set of measurement signals from each polymer acquired during translocation of the polymer through the nanopore channel to determine a scaling factor for each polymer; applying the scaling factor to the measurement signal generated during translocation of the polymer through the nanopore channel to determine a normalized measurement signal value; as well as The series of polymer sequences for each polymer is generated from the corresponding normalized measured signal values for each polymer.

2. The method of claim 1, wherein the set of measurement signals from each polymer acquired during translocation of the polymer through the nanopore channel for determining the scaling factor for each polymer are measurements of the adaptor region of each polymer.

3. The method of claim 2, wherein at least one polymer in the series of polymers is shorter in length than the adapter region of the polymer.

4. A method according to any one of the preceding claims, wherein the scaling factor is analysed across the range of polymers to determine the condition of the nanopore channel nanopore device.

5. The method according to any one of the preceding claims, wherein the nanopore sequencing device comprises an array of nanopore channels, and wherein a measurement signal corresponding to the polymer is generated during translocation through a respective nanopore channel. 6 . The method of claim 5 , wherein the nanopore sequencing device selects the nanopore channel from the array based on the scale factor or a scale factor associated with a nanopore channel.

7. A method according to any one of the preceding claims, wherein the nanopore is a biological pore.

8. The method of any preceding claim, wherein the polymer comprises a series of polymer units to be recognized by the nanopore device.

9. The method of claim 8, wherein the polymer is a polynucleotide and the polymer units are nucleotides.

10. The method according to claim 8 or 9, wherein the polymer or the polynucleotide is genomic DNA. 11 . The method according to claim 1 , wherein a median value of the measurement signal values is determined and used to determine the normalized measurement signal value.

12. The method of any preceding claim, wherein the nanopore device comprises a sensor electrode and the measuring comprises an electrical measurement.

13. A method according to any preceding claim, wherein the nanopore device comprises a sensor electrode and the measurements made by the sensor are indicative of ion flow through the nanopore.

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