IP Library › Granted Patent US 12,321,837
Granted Patent B2
US 12,321,837 · App. 17/430,695 · Granted Jun 3, 2025

Systems and methods for improved nanopore-based analysis of nucleic acids

Inventors: Jens H. Gundlach (Seattle, WA); Matthew Noakes (Seattle, WA); Henry D. Brinkerhoff (Seattle, WA)
Assignee: UNIVERSITY OF WASHINGTON
G06N20/10C12Q1/6869G01N33/48721G06N20/20
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Quick Facts
Patent No.
US 12,321,837
App. No.
17/430,695
Granted
Jun 3, 2025
Kind
B2
Abstract

In some embodiments, a computer-implemented method of determining an identity of one or more monomer subunit residues of a polymer analyte is provided, In some embodiments, a raw current signal generated by using a variable voltage to translocate the polymer analyte through a nanopore. In some embodiments, change points are detected in the raw current signal to determine a series of states, In some embodiments, capacitance compensation is performed on the raw current signal for each state to create an ionic current-vs-voltage curve for each state. In some embodiments, the ionic current-vs-voltage curves is converted to conductance-vs-voltage curves. In some embodiments, filtering is performed for the series of states to create a series of filtered states. In some embodiments, the identity of one or more monomer subunit residues of the polymer analyte is determined based on the series of filtered states.

Claims (65)

1. A computer-implemented method of determining an identity of one or more monomer subunit residues of a polymer analyte, the method comprising:

transmitting, by a computing system, an instruction to a nanopore system that causes a voltage source of the nanopore system to generate a variable voltage between a first conductive liquid medium and a second conductive liquid medium;

receiving, by the computing system, a raw current signal generated as a polymer analyte translocates through a nanopore of the nanopore system in response to the variable voltage;

detecting, by the computing system, change points in the raw current signal to determine a series of states, wherein detecting change points in the raw current signal includes determining basis functions derived from a dimensionality-reduced version of the raw current signal;

performing, by the computing system, capacitance compensation on the raw current signal for each state to create an ionic current-vs-voltage curve for each state;

converting, by the computing system, the ionic current-vs-voltage curves to conductance-vs-voltage curves;

performing, by the computing system, filtering for the series of states to create a series of filtered states; and

determining, by the computing system, the identity of one or more monomer subunit residues of the polymer analyte based on the series of filtered states.

2. The computer-implemented method of claim 1 , wherein detecting change points in the raw current signal includes:

removing flicker from the raw current signal to create a flicker-free current signal; and

using the flicker-free current signal to detect the change points.

3. The computer-implemented method of claim 1 , wherein the dimensionality-reduced version of the raw current signal is generated via principal component analysis.

4. The computer-implemented method of claim 1 , wherein detecting change points in the raw current signal includes using an over-fitting bias removal technique to remove over-fitting bias from a likelihood-maximization process for determining the change points, wherein the overfitting bias removal technique is at least one of a Change Point Information Criterion or an Aikaike Information Criterion.

5. The computer-implemented method of claim 1 , wherein performing capacitance compensation on the raw current signal for each state to create an ionic current-vs-voltage curve for each state includes:

determining a phase of the raw current signal based on an applied voltage signal;

binning data points in the raw current signal based on a location of each data point in the phase of the raw current signal;

averaging data points in each bin to determine an average current-voltage characteristic for up portions of the phase and for down portions of the phase;

determining a difference between the average current-voltage characteristic for up portions of the phase and the average current-voltage characteristic for down portions of the phase to find an asymmetry between the up portions and the down portions;

fitting a parabola to the asymmetry between the up portions and the down portions over a second quartile and a third quartile of the voltage to determine a residual function;

determining an up correction function and a down correction function based on the residual function;

applying the up correction function to the average current-voltage characteristic for up portions of the phase and applying the down correction function to the average current-voltage characteristic for down portions of the phase; and

applying a correction based on the average current-voltage characteristics to each data point of the raw current signal to create the ionic current-vs-voltage curves.

6. The computer-implemented method of claim 1 , further comprising determining one or more features for each state based on the conductance-vs-voltage curves.

7. The computer-implemented method of claim 6 , wherein the conductance-vs-voltage curves include a full feature vector, and wherein determining the one or more features for each state includes, for each state, performing dimensionality reduction on the full feature vector, wherein performing dimensionality reduction includes performing a principal component analysis to determine a reduced set of descriptive features of the full feature vector.

8. The computer-implemented method of claim 1 , wherein converting the ionic current-vs-voltage curves to the conductance-vs-voltage curves includes performing conductance normalization to remove any portion of the current signal not associated with a monomer subunit identity of the polymer analyte translocating through the nanopore.

9. The computer-implemented method of claim 8 , wherein performing conductance normalization includes:

determining a mean conductance at each voltage over each state; and

for each state:

subtracting the mean conductance from the measured conductance of the state to obtain a reduced conductance curve;

fitting a linear model to the reduced conductance curve to obtain a slope;

linearly fitting the slope to a mean voltage of the reduced conductance curves to obtain a bias; and

subtracting the bias from the reduced conductance curve to remove the portion of the conductance that is not dependent upon monomer subunit position.

10. The computer-implemented method of claim 1 , wherein performing filtering for the series of states to create a series of filtered states includes:

applying a removal filter to remove states that are not informative of the monomer subunit residues;

applying a recombination filter to remove states that represent repeated measurements of the same monomer subunit position; and

applying a reordering filter to identify and correct out-of-order states.

11. The computer-implemented method of claim 10 , wherein applying the removal filter includes determining a probability that a state should be removed using a machine learning classification algorithm, wherein the machine learning classification algorithm is a support vector machine.

12. The computer-implemented method of claim 11 , wherein determining the probability that the state should be removed using the machine learning classification algorithm includes determining the probability based on one or more features of the state, one or more features of a preceding state, and one or more features of a subsequent state.

13. The computer-implemented method of claim 12 , wherein determining the probability that the state should be removed using the machine learning classification algorithm includes determining the probability based further on at least one of:

a value of a single conductance measurement in the state that most deviates from an overall mean conductance;

an average mean square difference between a full feature vector of the state and a reduced-dimensionality feature vector of the state; and

a score of a best match for the state against a k-mer model.

14. The computer-implemented method of claim 10 , wherein applying the recombination filter to remove states that represent repeated measurements of the same monomer subunit position includes performing a Needleman-Wunsch-style alignment of each state against itself.

15. The computer-implemented method of claim 14 , wherein performing the Needleman-Wunsch-style alignment of each state against itself includes:

determining step-type probabilities at each transition based on conductance curve overlap information using a machine learning classification algorithm; and

applying a self-alignment penalty to newly created states;

wherein the machine learning classification algorithm is an ensemble of support vector machines.

16. The computer-implemented method of claim 10 , wherein applying the reordering filter to identify and correct out-of-order states includes:

using a machine learning classification technique to assign a probability that each transition was a step, a skip, or a backstep; and

using a dynamic programming technique to reorder the states in a most likely true order;

wherein the machine learning classification algorithm is an ensemble of support vector machines with associated logit functions.

17. The computer-implemented method of claim 1 , wherein determining the identity of one or more monomer subunit residues of the polymer analyte based on the series of filtered states includes performing sequencing using a hidden Markov model (HMM) solver.

18. The computer-implemented method of claim 17 , wherein performing sequencing using the HMM solver includes:

using information regarding a likelihood of a sequencing enzyme used to translocate the polymer analyte through the nanopore in a given state to help determine state probabilities for a score matrix used with the HMM solver.

19. The computer-implemented method of claim 17 , wherein performing sequencing using the HMM solver includes:

using probabilities of steps, skips, and bad levels determined while performing filtering on the series of states to help determine state probabilities for a score matrix used with the HMM solver.

20. A non-transitory computer-readable medium having computer-executable instructions stored thereon that, in response to execution by one or more processors of a computing system, cause the computing system to perform actions for determining an identity of one or more monomer subunit residues of a polymer analyte, the actions comprising:

transmitting, by the computing system, an instruction to a nanopore system that causes a voltage source of the nanopore system to generate a variable voltage between a first conductive liquid medium and a second conductive liquid medium;

receiving, by the computing system, a raw current signal generated as a polymer analyte translocates through a nanopore of the nanopore system in response to the variable voltage;

detecting, by the computing system, change points in the raw current signal to determine a series of states;

performing, by the computing system, capacitance compensation on the raw current signal for each state to create an ionic current-vs-voltage curve for each state;

converting, by the computing system, the ionic current-vs-voltage curves to conductance-vs-voltage curves;

determining, by the computing system, one or more features for each state based on the conductance-vs-voltage curves, wherein the conductance-vs-voltage curves include a full feature vector, and wherein determining the one or more features for each state includes, for each state, performing dimensionality reduction on the full feature vector to determine a reduced set of descriptive features of the full feature vector;

performing, by the computing system, filtering for the series of states to create a series of filtered states; and

determining, by the computing system, the identity of one or more monomer subunit residues of the polymer analyte based on the series of filtered states.

Assignments (2)
CONFIRMATORY LICENSE Recorded Dec 12, 2023
From: UNIVERSITY OF WASHINGTON
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065967/0376 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 21, 2021
From: GUNDLACH, JENS H.; NOAKES, MATTHEW; BRINKERHOFF, HENRY D.
To: UNIVERSITY OF WASHINGTON
Reel/Frame 057549/0854 →
Continuity (2)
Provisional Application 62805870 · Feb 14, 2019
Related Publication 20220366313A1 · Nov 17, 2022
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