Formation and calibration of nanopore sequencing cells

• US 10,317,392 B2
• Filed: 06/23/2017
• Issued: 06/11/2019
• Est. Priority Date: 06/23/2016
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Mager, Morgan, Mannion, John
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/632,190
• Publication Number:
  US 20170370903A1
• Time in Patent Office: 718 Days
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/4163 checking the operation of, or calibrating, the measuring apparatus G01N27/3274, G01N27/4175 and G01N33/0006 take precedence
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Improved multi-cell nanopore-based sequencing chips and methods can employ formation, characterization, calibration, and/or normalization techniques. For example, various methods may include one or more steps of performing physical checks of cell circuitry, forming and characterizing a lipid layer on the cells, performing a zero point calibration of the cells, forming and characterizing nanopores on the lipid layers of each cell, performing a sequencing operation to accumulate sequencing signals from the cells, normalizing those sequencing signals, and determining bases based on the normalized sequencing signals.

Claims

1. A method of using a sequencing cell, the method comprising:

obtaining a plurality of measured signal values for the sequencing cell having a voltage applied across the sequencing cell, the sequencing cell including a nucleic acid, wherein the applied voltage includes an alternating signal having a first portion and a second portion relative to a reference voltage;

determining a first set of one or more signal values measured during the first portion of the alternating signal;

determining a second set of one or more signal values measured during the second portion of the alternating signal, wherein the first set of one or more signal values and second set of one or more signal values are determined with no molecule in a nanopore of the sequencing cell;

determining a normalization factor based on the second set of one or more signal values;

determining a third set of one or more signal values measured during the first portion of the alternating signal when a tag molecule is threaded in the nanopore of the sequencing cell, the tag molecule corresponding to a particular nucleotide; and

normalizing the third set of one or more signal values using the normalization factor to obtain one or more normalized signal values.

2. The method of claim 1, wherein each of the plurality of measured signal values corresponds to a voltage or a current of the sequencing cell as measured at a particular time.

3. The method of claim 1, wherein the normalization factor accounts for gain drift resulting from a capacitance of a circuit of the sequencing cell changing over time.

4. The method of claim 1, wherein the normalization factor accounts for a baseline shift resulting from an accumulation of charge of a circuit of the sequencing cell over time.

5. The method of claim 1, wherein an absolute value of a difference of one of the second set of one or more signal values and the reference voltage is used to determine the normalization factor.

6. The method of claim 1, further comprising:

filtering the second set of one or more signal values to obtain one or more filtered signal values; and

using the one or more filtered signal values to determine the normalization factor.

7. The method of claim 1, wherein filtering the second set of one or more signal values uses a Kalman filter.

8. The method of claim 1, wherein obtaining the plurality of measured signal values for the sequencing cell includes:

charging a capacitive element of a circuit of the sequencing cell, wherein the nanopore comprises a resistive element of the circuit;

partially discharging the capacitive element for a specified amount of time or to a specified voltage level; and

measuring a voltage at a point in the circuit after the specified amount of time or measuring a duration of time required for the point on the circuit to reach the specified voltage level.

9. The method of claim 1, wherein obtaining the plurality of measured signal values for the sequencing cell includes:

charging a capacitive element of a circuit of the sequencing cell, wherein the nanopore comprises a resistive element of the circuit;

partially discharging the capacitive element for a specific amount of time or to a specified current level; and

measuring a current in the circuit after the specified amount of time or measuring a duration of time required for a current at a point on the circuit to reach the specified current level.

10. The method of claim 8, wherein the first portion of the alternating signal includes a plurality of sub-periods of cycles during which a voltage between a first electrode and a second electrode of the sequencing cell is such that the tag molecule may be forced into the nanopore.

11. The method of claim 10, wherein the alternating signal is applied to the first electrode, wherein the second electrode is at the reference voltage, and wherein the capacitive element is charged to be at the reference voltage.

12. The method of claim 1, further comprising:

determining the normalization factor also based on the first set of one or more signal values.

13. The method of claim 1, wherein determining the normalization factor based on the second set of one or more signal values includes:

determining a second voltage value that approximates a first open-channel voltage value that would occur during the first portion of the alternating signal, the determining of the second voltage value using a baseline shift computed using the second set of one or more signal values; and

using the second voltage value as the normalization factor or using the second voltage value and other voltage values to determine an average voltage value that is used as the normalization factor.

14. The method of claim 1, further comprising:

determining a sequence of the nucleic acid using the one or more normalized signal values.

15. A method of using a sequencing cell, the method comprising:

obtaining a plurality of measured signal values for the sequencing cell having a voltage applied across the sequencing cell, the sequencing cell including a nucleic acid, wherein the applied voltage includes an alternating signal having a first portion and a second portion relative to a reference voltage;

determining an initial signal value measured during the first portion of the alternating signal;

determining subsequent signal values measured at various times;

using the subsequent signal values to solve a set of equations for a gain drift m and a baseline shift b, the gain drift m resulting from a capacitance of a circuit of the sequencing cell changing over time, and the baseline shift b resulting from an accumulation of charge of the circuit of the sequencing cell over time, wherein each value of the subsequent signal values is defined as including the gain drift m and the baseline shift b;

using the gain drift m, the baseline shift b, and the initial signal value to determine a normalization factor;

determining one or more signal values measured during the first portion of the alternating signal when a tag molecule is threaded in a nanopore of the sequencing cell, the tag molecule corresponding to a particular nucleotide; and

normalizing the one or more signal values using the normalization factor.

16. The method of claim 15, wherein the set of equations comprises a model that relates one or more first measured signal values measured during the first portion of the alternating signal to one or more second measured signal values measured during the second portion the alternating signal.

17. The method of claim 15, further comprising:

determining an initial signal value measured during the second portion of the alternating signal; and

using the gain drift m, the baseline shift b, the initial signal value measured during the second portion of the alternating signal, and the initial signal value measured during the first portion of the alternating signal to determine a normalization factor.

18. The method of claim 16, wherein the model comprises a linear system of equations comprising the following:

P(t)=m*(1+b*c)*P0

N(t)=m*(1−

b)*N0

where m is the gain drift, b is the baseline shift, and c is a baseline shift ratio between signal values measured the first portion and the second portion,

wherein P0 is the initial signal value measured during the first portion of the alternating signal and N0 is an initial signal value measured during the second portion of the alternating signal, and

wherein P(t) is a subsequent signal value measured during the first portion and N(t) is a subsequent signal value measured during the second portion.

19. The method of claim 16, wherein, during a time when a tag molecule is threaded in the nanopore of the sequencing cell, the normalization factor is computed to be an estimated value of a signal value that would have occurred in absence of the tag molecule being threaded in the nanopore.

20. The method of claim 15, wherein each of the plurality of measured signal values corresponds to a voltage or a current of the sequencing cell as measured at a particular time.

21. A system comprising:

a sequencing cell that includes a nanopore, the nanopore configured to receive a tag that is connected to a nucleotide, thereby creating a threading event;

a signal generator that applies an alternating signal across the nanopore of the sequencing cell, each cycle of the alternating signal comprising a first portion and a second portion, wherein voltage levels of the second portion are opposite of a reference voltage than voltage levels of the first portion;

an analog-to-digital converter that acquires a plurality of measured signal values from the sequencing cell; and

a digital processor configured to;

determine a first set of one or more signal values measured during the first portion of the alternating signal;

determine a second set of one or more signal values measured during the second portion of the alternating signal, wherein the first set of one or more signal values and second set of one or more signal values are determined with no molecule in the nanopore of the sequencing cell;

determine a normalization factor based on the second set of one or more signal values;

determine a third set of one or more signal values measured during the first portion of the alternating signal when a tag molecule is threaded in the nanopore of the sequencing cell, the tag molecule corresponding to a particular nucleotide; and

normalize the third set of one or more signal values using the normalization factor to obtain one or more normalized signal values.