CONCURRENT SEQUENCING OF FORWARD AND REVERSE COMPLEMENT STRANDS ON CONCATENATED POLYNUCLEOTIDES FOR METHYLATION DETECTION

1 . A method of preparing at least one polynucleotide sequence for detection of modified cytosines, comprising:

synthesising at least one polynucleotide sequence comprising a first portion and a second portion,

wherein the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template, and the first portion comprises a forward strand of the template, and the second portion comprises a reverse complement strand of the template; or wherein the first portion comprises a reverse strand of the template, and the second portion comprises a forward complement strand of the template,

wherein the template is generated from a target polynucleotide to be sequenced via complementary base pairing, and wherein the target polynucleotide has been pre-treated using a conversion reagent,

wherein the conversion reagent is configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or wherein the conversion reagent is configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil.

2 . The method according to claim 1 , wherein the target polynucleotide has been pre-treated using a conversion reagent configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil.

3 . The method according to claim 1 , wherein the target polynucleotide has been pre-treated using a conversion reagent configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil.

4 . The method according to claim 1 , wherein the conversion agent comprises a chemical agent and/or an enzyme.

5 . The method according to claim 4 , wherein the chemical agent comprises a boron-based reducing agent.

6 . The method according to claim 5 , wherein the boron-based reducing agent is an amine-borane compound or an azine-borane compound.

7 . The method according to claim 5 , wherein the boron-based reducing agent is selected from the group consisting of pyridine borane, 2-picoline borane, t-butylamine borane, ammonia borane, ethylenediamine borane and dimethylamine borane.

8 . The method according to claim 4 , wherein the chemical agent comprises sulfite; preferably bisulfite; more preferably sodium bisulfite.

9 . The method according to claim 4 , wherein the enzyme comprises a cytidine deaminase.

10 . The method according to claim 9 , wherein the cytidine deaminase is a wild-type cytidine deaminase or a mutant cytidine deaminase; preferably a mutant cytidine deaminase.

11 - 18 . (canceled)

19 . The method according to claim 1 , wherein the target polynucleotide is treated with a further agent prior to treatment with the conversion reagent.

20 .- 28 . (canceled)

29 . The method according to claim 1 , wherein the modified cytosine is selected from the group consisting of: 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

30 . The method according to claim 1 , wherein the forward strand of the template is not identical to the reverse complement strand of the template.

31 . (canceled)

32 . The method according to claim 1 , wherein the method further comprises a step of preparing the first portion and the second portion for concurrent sequencing.

33 . (canceled)

34 . The method according to claim 1 , wherein a proportion of first portions is capable of generating a first signal and a proportion of second portions is capable of generating a second signal, wherein an intensity of the first signal is substantially the same as an intensity of the second signal.

35 . The method according to claim 1 , wherein the method further comprises a step of selectively processing the at least one polynucleotide sequence comprising the first portion and the second portion, such that a proportion of first portions are capable of generating a first signal and a proportion of second portions are capable of generating a second signal, wherein the selective processing causes an intensity of the first signal to be greater than an intensity of the second signal.

36 .- 43 . (canceled)

44 . The method according to claim 1 , wherein the at least one polynucleotide sequence comprising the first portion and the second portion is/are attached to a solid support, preferably wherein the solid support is a flow cell.

45 .- 51 . (canceled)

52 . The method according to claim 1 , wherein the step of synthesising the at least one polynucleotide sequence comprising a first portion and a second portion comprises:

synthesising a first precursor polynucleotide fragment comprising a complement of the first portion and a hybridisation complement sequence,

synthesising a second precursor polynucleotide fragment comprising a second portion and a hybridisation sequence,

annealing the hybridisation complement sequence of the first precursor polynucleotide fragment with the hybridisation sequence on the second precursor polynucleotide fragment to form a hybridised adduct,

synthesising a first precursor polynucleotide sequence by extending the first precursor polynucleotide fragment to form a complement of the second portion, and

synthesising the at least one polynucleotide sequence by forming a complement of the first precursor polynucleotide sequence.

53 .- 61 . (canceled)

62 . The method according to claim 1 , wherein the method further comprises concurrently sequencing nucleobases in the first portion and the second portion.

63 . A method of sequencing at least one polynucleotide sequence to detect modified cytosines, comprising:

preparing at least one polynucleotide sequence for detection of modified cytosines using a method according to claim 1 ;

concurrently sequencing nucleobases in the first portion and the second portion; and

identifying modified cytosines by detecting differences when comparing a sequence output from the first portion with a sequence output from the second portion.

64 . (canceled)

65 . (canceled)

66 . (canceled)

67 . (canceled)

68 . (canceled)

69 . (canceled)

70 . A computer program product comprising instructions which, when the program is executed by a processor, cause the processor to carry out a method according to claim 1 .

71 .- 73 . (canceled)