Granted Patent B2

US 12,216,110 · App. 15/487,329 · Granted Feb 4, 2025

Inventors: Stuart William Reid (Oxford, GB); Gavin Harper (Sonning, GB); Clive Gavin Brown (Cambridge, GB); James Anthony Clarke (Kidlington, GB); Andrew John Heron (Oxford, GB)

Assignee: Oxford Nanopore Technologies PLC

G01N33/48721C12Q1/6869G01N27/44791G01N33/483G06F17/18G06N7/01G16B30/00G16B30/10B82Y15/00

Abstract

Claims (45)

1. A computer-implemented method of estimating a sequence of polymer units in a polymer, the method comprising:

controlling translocation of the polymer through a nanopore using an enzyme;

detecting, using a measurement circuit coupled to electrodes arranged on each side of the nanopore, a sequence of measurements of the polymer, each measurement being indicative of an ion current flowing through the nanopore during said translocation of the polymer through the nanopore;

using at least one processor, coupled to a memory, to perform:

accessing, in the memory, a probabilistic model comprising, for a set of possible k-mers, wherein a k-mer is a group of k polymer units where k is a positive integer:

transition weightings each representing a probability of transition from an origin k-mer to a destination k-mer immediately subsequent in a sequence representing the sequence of polymer units, and wherein, when k is greater than one, transitions to destination k-mers that have a sequence in which the first k-1 polymer units are the same as the last k-1 polymer units of associated origin k-mers have greater probabilities than other transitions; and

emission weightings each representing a probability of obtaining given values of ion current measurements for a respective k-mer of the set of possible k-mers;

determining likelihoods, for each ion current measurement in the detected sequence of measurements of the polymer, that the ion current measurement was produced by respective k-mers of the set of possible k-mers, by optimizing a sequence of states of the probabilistic model based on the detected sequence of ion current measurements, the transition weightings, and the emission weightings; and

determining an estimated sequence of polymer units in the polymer based on the likelihoods determined for each ion current measurement.

2. The method of claim 1 , wherein optimizing the sequence of states of the probabilistic model comprises optimization via the Viterbi algorithm.

3. The method of claim 1 , wherein the probabilistic model comprises a Hidden Markov Model (HMM).

4. The method of claim 1 , wherein the polymer is a polynucleotide.

5. The method of claim 4 , wherein translocation of the polynucleotide through the nanopore is assisted by an enzyme.

6. The method of claim 1 , wherein the sequence of measurements comprises plural series of measurements each related to said polymer, wherein the value of each measurement is dependent on a k-mer; and

wherein determining at least one estimated sequence of polymer units involves treating the plural series of measurements as arranged in plural, respective dimensions.

7. The method of claim 6 , wherein each series of measurements are measurements of a same region of the same polymer.

8. The method of claim 6 , wherein the plural series of measurements comprise a first series of measurements and a second series of measurements, wherein the first series of measurements are measurements of a first region of a polymer and the second series of measurements are measurements of a second region of a polymer that is related to said first region.

9. The method of claim 8 , wherein the first region and the second region are related regions of the same polymer.

10. The method of claim 8 , wherein the related regions are complementary.

11. The method of claim 1 , wherein providing the probabilistic model comprises accessing, using the at least one processor, the probabilistic model stored in a computer-readable storage medium.

12. The method of claim 1 , wherein the nanopore is inserted into a biological membrane, and wherein detecting the sequence of measurements of the polymer comprises measuring a current across the membrane.

13. The method of claim 1 , further comprising operating a control circuit to supply a voltage to the electrodes during said translocation of the polymer through the nanopore.

14. The method of claim 1 , wherein the enzyme controls a rate of translocation of the polymer through the nanopore throughout said translocation.

15. The method of claim 1 , wherein the transition weightings comprise k preferred transitions.

16. The method of claim 1 , wherein determining the estimated sequence of polymer units comprises determining an estimated sequence of k-mers and determining the estimated sequence of polymer units based on the estimated sequence of k-mers.

17. At least one non-transitory computer-readable storage medium having instructions encoded thereon that, when executed by at least one processor, cause the at least one processor to perform a method of estimating a sequence of polymer units in a polymer, the method comprising:

controlling translocation of the polymer through a nanopore using an enzyme;

detecting, using a measurement circuit coupled to electrodes arranged on each side of the nanopore, a sequence of measurements of the polymer, each measurement being indicative of an ion current flowing through the nanopore during said translocation of the polymer through the nanopore;

using at least one processor, coupled to a memory, to perform:

accessing, in the memory, a probabilistic model comprising, for a set of possible k-mers, wherein a k-mer is a group of k polymer units where k is a positive integer:

transition weightings each representing a probability of transition from an origin k-mer to a destination k-mer immediately subsequent in a sequence representing the sequence of polymer units, and wherein, when k is greater than one, transitions to destination k-mers that have a sequence in which the first k-1 polymer units are the same as the last k-1 polymer units of associated origin k-mers have greater probabilities than other transitions; and

emission weightings each representing a probability of obtaining given values of ion current measurements for a respective k-mer of the set of possible k-mers;

determining likelihoods, for each ion current measurement in the detected sequence of measurements of the polymer, that the ion current measurement was produced by respective k-mers of the set of possible k-mers, by optimizing a sequence of states of the probabilistic model based on the detected sequence of ion current measurements, the transition weightings, and the emission weightings; and

determining an estimated sequence of polymer units in the polymer based on the likelihoods determined for each ion current measurement.

18. A system comprising:

at least one processor; and

at least one non-transitory computer-readable storage medium having instructions encoded thereon that, when executed by the at least one processor, cause the at least one processor to perform a method of estimating a sequence of polymer units in a polymer, the method comprising:

controlling translocation of the polymer through a nanopore using an enzyme;

detecting, using a measurement circuit coupled to electrodes arranged on each side of the nanopore, a sequence of measurements of the polymer, each measurement being indicative of an ion current flowing through the nanopore during said translocation of the polymer through the nanopore;

using at least one processor, coupled to a memory, to perform:

accessing, in the memory, a probabilistic model comprising, for a set of possible k-mers, wherein a k-mer is a group of k polymer units where k is a positive integer:

transition weightings each representing a probability of transition from an origin k-mer to a destination k-mer immediately subsequent in a sequence representing the sequence of polymer units, and wherein, when k is greater than one, transitions to destination k-mers that have a sequence in which the first k-1 polymer units are the same as the last k-1 polymer units of associated origin k-mers have greater probabilities than other transitions; and

emission weightings each representing a probability of obtaining given values of ion current measurements for a respective k-mer of the set of possible k-mers;

determining likelihoods, for each ion current measurement in the detected sequence of measurements of the polymer, that the ion current measurement was produced by respective k-mers of the set of possible k-mers, by optimizing a sequence of states of the probabilistic model based on the detected sequence of ion current measurements, the transition weightings, and the emission weightings; and

determining an estimated sequence of polymer units in the polymer based on the likelihoods determined for each ion current measurement.

Assignments (2)

Continuity (4)

References Cited (198)

Cited By (3)

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