Granted Patent B2

US 12,630,866 · App. 18/166,986 · Granted May 19, 2026

Inventors: Shankar Balasubramanian (Cambridge, GB); Jens Fullgrabe (Great Chesterford, GB); Walraj Singh Gosal (Cambridge, GB); Joanna Dawn Holbrook (London, GB); Sidong Liu (Saffron Walden, GB); David Morley (St. Albans, GB); Oliver Nentwich (Cambridge, GB); Tobias Ost (Ely, GB); Michael Steward (Royston, GB); Albert Vilella (Cambridge, GB); Nicolas James Walker (Cambridge, GB); Shirong Yu (Cambridge, GB); Helen Rachel Bignell (Cambridge, GB); Rita Santo San-Bento (Lyons, FR)

Assignee: Biomodal Limited

C12Q1/6827C12N15/1058C12N15/52C12Q1/6806C12Q1/6811C12Q1/686C12Q1/6869

Abstract

Claims (30)

1 . A method, comprising:

(a) providing a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide are linked, wherein the second polynucleotide is at least partially complementary to the first polynucleotide, and wherein the first polynucleotide comprises at least a portion of a genomic polynucleotide;

(b) contacting the first polynucleotide and the second polynucleotide with an agent that specifically glycosylates 5-hydroxymethylcytosine (5-hmC);

(c) determining sequences of the first polynucleotide and the second polynucleotide to determine a first identity of a first base at a locus of the first polynucleotide and a second identity of a second base at a corresponding locus of the second polynucleotide using sequencing; and

(d) using a computer comprising a processor, a memory, and instructions stored thereupon that, when executed, determine an identity of a true base of the genomic polynucleotide corresponding to the locus of the first polynucleotide based at least in part on the identity of the first base and the identity of the second base.

2 . The method of claim 1 , wherein the second polynucleotide does not comprise a portion of the genomic polynucleotide.

3 . The method of claim 1 , wherein the second polynucleotide is generated from the first polynucleotide.

4 . The method of claim 1 , wherein, prior to or during (c), the first polynucleotide and the second polynucleotide are linked covalently via a hairpin.

5 . The method of claim 1 , further comprising, prior to (a), generating the second polynucleotide by conducting one or more chemical reactions or enzymatic reactions on the genomic polynucleotide.

6 . The method of claim 1 , further comprising, prior to (a), generating the second polynucleotide via a nucleic acid extension reaction.

7 . The method of claim 1 , wherein the identity of the true base is determined before aligning data generated during the sequencing to a reference nucleic acid sequence.

8 . The method of claim 1 , further comprising contacting the first polynucleotide and the second polynucleotide with an entity having DNA methyltransferase activity.

9 . The method of claim 8 , wherein the entity having DNA methyltransferase activity comprises DNA (cytosine-5)-methyltransferase 1 (DNMT1) or DNA (cytosine-5)-methyltransferase 5 (DNMT5).

10 . The method of claim 8 , wherein contacting the first polynucleotide and the second polynucleotide with the entity having DNA methyltransferase activity is performed after (b).

11 . The method of claim 1 , wherein the agent that specifically glycosylates 5hmC comprises β-glucosyltransferase.

12 . The method of claim 1 , further comprising contacting the first polynucleotide and the second polynucleotide with an oxidizing agent.

13 . The method of claim 12 , wherein the oxidizing agent comprises a methylcytosine dioxygenase.

14 . The method of claim 13 , wherein the methylcytosine dioxygenase comprises a ten-eleven translocation (TET) enzyme or derivative thereof.

15 . The method of claim 12 , wherein contacting the first polynucleotide and the second polynucleotide with the agent that specifically glycosylates 5hmC is performed after contacting the first polynucleotide and the second polynucleotide with the oxidizing agent.

16 . The method of claim 12 , further comprising, prior to contacting the first polynucleotide and the second polynucleotide with the oxidizing agent, contacting the first polynucleotide and the second polynucleotide with an entity having DNA methyltransferase activity.

17 . The method of claim 1 , further comprising contacting the first polynucleotide and the second polynucleotide with a deamination agent after (b) and prior to (c).

18 . The method of claim 17 , wherein the deamination agent comprises a deaminase.

19 . The method of claim 18 , wherein the deaminase comprises an apolipoprotein B mRNA editing enzyme (APOBEC) or a functional fragment thereof.

20 . The method of claim 18 , wherein the deaminase comprises a single-stranded DNA deaminase or a functional fragment thereof.

21 . The method of claim 17 , prior to the contacting the first polynucleotide and the second polynucleotide with the deamination agent, separating a first portion of the first polynucleotide from a second portion of the second polynucleotide.

22 . The method of claim 17 , further comprising contacting the first polynucleotide and the second polynucleotide with the deamination agent in a presence of a helicase.

23 . The method of claim 22 , wherein the helicase comprises an amino acid sequence that has at least 90% identity to UvrD helicase, Geobacillus sterothermophilus Bad protein, a PcrA helicase, or functional fragment thereof.

24 . The method of claim 23 , wherein the helicase comprises a UvrD helicase, Geobacillus sterothermophilus Bad protein, a PcrA helicase, or fragment thereof.

25 . The method of claim 1 , wherein a false positive rate for determining the identity of the true base at the original locus of the original polynucleotide is no higher than 1 in 100,000.

26 . The method of claim 25 , wherein the false positive rate for determining the identity of the true base of the genomic polynucleotide is no higher than 1 in 1,000,000.

Assignments (2)

Continuity (13)

References Cited (400)