METHODS FOR PREPARING SIGNALS FOR CONCURRENT SEQUENCING

1 . A method of preparing at least one polynucleotide sequence for identification, comprising:

selectively processing at least one polynucleotide sequence comprising a first portion and a second portion, or at least one first polynucleotide sequence comprising a first portion and at least one second polynucleotide sequence comprising a 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.

2 . A method according to any one of claim 1 , wherein a concentration of the first portions capable of generating the first signal is greater than a concentration of the second portions capable of generating the second signal.

3 . A method according to claim 1 or 2 , wherein a ratio between the concentration of the first portions capable of generating the first signal and the concentration of the second portions capable of generating the second signal is between 1.25:1 to 5:1, preferably between 1.5:1 to 3:1, more preferably about 2:1.

4 . A method according to any preceding claim , comprising selectively processing at least one polynucleotide sequence comprising a first portion and a second portion.

5 . A method according to any preceding claim , comprising selectively processing at least one first polynucleotide sequence comprising a first portion and at least one second polynucleotide sequence comprising a second portion.

6 . A method according to any preceding claim , wherein the first signal and the second signal are spatially unresolved.

7 . A method according to any preceding claim , wherein the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template, and each of the first portions comprise a forward strand of the template, and each of the second portions comprise a reverse strand of the template or a forward complement strand of the template; or wherein each of the first portions comprise a reverse strand of the template, and each of the second portions comprise a forward strand of the template or a reverse complement strand of the template.

8 . A method according to any preceding claim , wherein the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template, and each of the first portions comprises a forward strand of a template, and each of the second portions comprises a reverse complement strand of the template; or wherein each of the first portions comprises a reverse strand of a template, and each of the second portions comprises a forward complement strand of the template.

9 . A method according to any preceding claim , wherein the at least one polynucleotide sequence comprising the first portion and the second portion, or the at least one first polynucleotide sequence comprising the first portion and the at least one second polynucleotide sequence comprising the second portion, is/are attached to a solid support, preferably wherein the solid support is a flow cell.

10 . A method according to any preceding claim , wherein the at least one polynucleotide sequence comprising the first portion and the second portion, or the at least one first polynucleotide sequence comprising the first portion and the at least one second polynucleotide sequence comprising the second portion, form a cluster on the solid support.

11 . A method according to any preceding claim , wherein the cluster is formed by bridge amplification.

12 . A method according to any preceding claim , wherein the at least one polynucleotide sequence comprising the first portion and the second portion forms a monoclonal cluster.

13 . A method according to any preceding claim , wherein the at least one first polynucleotide sequence comprising the first portion and the at least one second polynucleotide sequence comprising the second portion form a duoclonal cluster.

14 . A method according to any preceding claim , wherein a first region occupied by the at least one first polynucleotide sequence comprising the first portion within the duoclonal cluster is the same as, or substantially overlapping with, a second region occupied by the at least one second polynucleotide sequence comprising the second portion within the duoclonal cluster.

15 . A method according to any preceding claim , wherein the solid support comprises at least one first immobilised primer and at least one second immobilised primer.

16 . A method according to any preceding claim , wherein the first immobilised primer comprises a sequence as defined in SEQ ID NO:1 or 5, or a variant or fragment thereof; and the second immobilised primer comprises a sequence as defined in SEQ ID NO: 2, or a variant or fragment thereof.

17 . A method according to any preceding claim , comprising selectively processing at least one polynucleotide sequence comprising a first portion and a second portion, and wherein each polynucleotide sequence is attached to a first immobilised primer.

18 . A method according to claim 17 , wherein each polynucleotide sequence comprising the first portion and the second portion further comprises a second adaptor sequence, wherein the second adaptor sequence is substantially complementary to the second immobilised primer.

19 . A method according to any preceding claim , comprising selectively processing at least one first polynucleotide sequence comprising a first portion and at least one second polynucleotide sequence comprising a second portion, wherein each first polynucleotide sequence is attached to a first immobilised primer, and wherein each second polynucleotide sequence is attached a second immobilised primer.

20 . A method according to claim 19 , wherein each first polynucleotide sequence comprises a second adaptor sequence and wherein each second polynucleotide sequence comprises a first adaptor sequence, wherein the second adaptor sequence is substantially complementary to the second immobilised primer and wherein the first adaptor sequence is substantially complementary to the first immobilised primer.

21 . A method according to any preceding claim , wherein selectively processing comprises preparing for selective sequencing or conducting selective sequencing.

22 . A method according to any preceding claim , wherein selectively processing comprises conducting selective amplification.

23 . A method according to any preceding claim , wherein selectively processing comprises contacting first sequencing primer binding sites located after a 3′-end of the first portions with first primers and contacting second sequencing primer binding sites located after a 3′-end of the second portions with second primers, wherein the second primers comprises a mixture of blocked second primers and unblocked second primers.

24 . A method according to claim 23 , wherein the blocked second primer comprises a blocking group at a 3′ end of the blocked second primer.

25 . A method according to claim 23 or 24 , wherein the blocking group is selected from the group consisting of: a hairpin loop, a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3′-OH group, a phosphate group, a phosphorothioate group, a propyl spacer, a modification blocking the 3′-hydroxyl group, or an inverted nucleobase.

26 . A method according to any one of claims 23 to 25 , wherein the blocked second primer comprises a sequence as defined in SEQ ID NO: 11 to 16 or a variant or fragment thereof and/or the unblocked second primer comprises a sequence as defined in SEQ ID NO: 11 to 14 or a variant or fragment thereof.

27 . A method according to any one of claims 1 to 22 , wherein the selective processing comprises selectively removing some or substantially all of second immobilised primers that are not yet extended, and conducting a further amplification cycle in order to selectively amplify the first polynucleotide sequence(s) relative to the second polynucleotide sequence(s).

28 . A method according to any one of claims 1 to 22 , wherein selectively processing comprises selectively blocking some or substantially all of second immobilised primers that are not yet extended using a primer blocking agent, wherein the primer blocking agent is configured to limit or prevent synthesis of a strand extending from the second immobilised primer, and conducting a further amplification cycle in order to selectively amplify the first polynucleotide sequence(s) relative to the second polynucleotide sequence(s).

29 . A method according to claim 28 , wherein the primer blocking agent is added whilst first polynucleotide sequence(s) are hybridised to the second immobilised primers.

30 . A method according to claim 28 , wherein the method comprises contacting some or substantially all of the second immobilised primers with an extended primer sequence, wherein the extended primer sequence is substantially complementary to the second immobilised primer and further comprises a 5′ additional nucleotide; and adding the primer blocking agent, wherein the primer blocking agent is complementary to the 5′ additional nucleotide.

31 . A method according to claim 30 , wherein the primer blocking agent is a blocked nucleotide.

32 . A method according to claim 31 , wherein the blocked nucleotide comprises a blocking group at a 3′ end of the blocked nucleotide.

33 . A method according to claim 32 , wherein the blocking group is selected from the group consisting of: a hairpin loop, a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3′-OH group, a phosphate group, a phosphorothioate group, a propyl spacer, a modification blocking the 3′-hydroxyl group, or an inverted nucleobase.

34 . A method according to any one of claims 32 to 33 , wherein the blocked nucleotide is A or G.

35 . A method of sequencing at least one polynucleotide sequence, comprising:

preparing at least one polynucleotide sequence for identification using a method according to any one of claims 1 to 34 ; and

concurrently sequencing nucleobases in the first portion and the second portion based on the intensity of the first signal and the intensity of the second signal.

36 . A method according to claim 35 , wherein the step of concurrently sequencing nucleobases comprises performing sequencing-by-synthesis or sequencing-by-ligation.

37 . A method according to any one of claims 35 to 36 , wherein the method further comprises a step of conducting paired-end reads.

38 . A primer, wherein the primer comprises a sequence as defined in SEQ ID NO: 11 to 16, or a variant or fragment thereof.

39 . A primer according to claim 38 , wherein a 3′ end of the primer comprises a 3′-OH group.

40 . A primer according to any one of claims 38 to 39 , wherein a 3′ end of the primer comprises a blocking group at a 3′ end of the primer.

41 . A primer according to any one of claims 38 to 40 , wherein the blocking group is selected from the group consisting of: a hairpin loop, a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3′-OH group, a phosphate group, a phosphorothioate group, a propyl spacer, a modification blocking the 3′-hydroxyl group, or an inverted nucleobase.

42 . Use of a primer according to any one of claims 38 to 41 in preparing at least one polynucleotide sequence for identification according to any one of claims 1 to 34 .

43 . A kit comprising instructions for preparing at least one polynucleotide sequence for identification according to any one of claims 1 to 34 ; and/or sequencing at least one polynucleotide sequence according to any one of claims 35 to 37 .

44 . A kit according to claim 43 , further comprising a primer according to any one of claims 38 to 41 .

45 . A kit according to any one of claims 43 to 44 , further comprising an amplification mixture comprising a recombinase, a DNA polymerase, a single-stranded DNA binding protein (SSB) and a glycosylase, wherein the glycosylase is either FPG glycosylase or uracil glycosylase or the USER enzyme mix.

46 . A kit according to any one of claims 43 to 45 , further comprising a primer-blocking agent(s), wherein the primer-blocking agent is preferably a blocked nucleotide, more preferably a blocked A or G.

47 . A kit according to any one of claims 43 to 46 , further comprising at least one extended primer sequence(s), wherein the extended primer sequence is selected from SEQ ID NO: 25 to 36, and wherein the extended primer sequence further comprises a 5′ additional nucleotide, wherein the 5′ additional nucleotide is complementary to the primer-blocking agent.

48 . An amplification composition comprising a recombinase, a DNA polymerase, a single-stranded DNA binding protein (SSB) and primer-blocking agent, wherein the primer-blocking agent is preferably a blocked nucleotide, more preferably a blocked A or G.

49 . An amplification composition according to claim 48 , wherein the composition further comprises at least one extended primer sequence(s), wherein the extended primer sequence is selected from SEQ ID NO: 25 or 36, and wherein the extended primer sequence comprises a 5′ additional nucleotide, wherein the 5′ additional nucleotide is complementary to the primer-blocking agent.

50 . A data processing device comprising means for carrying out a method according to any one of claims 1 to 34 or 35 to 37 .

51 . A data processing device according to claim 50 , wherein the data processing device is a polynucleotide sequencer.

52 . 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 any one of claims 1 to 34 or 35 to 37 .

53 . A computer-readable storage medium comprising instructions which, when executed by a processor, cause the processor to carry out a method according to any one of claims 1 to 34 or 35 to 37 .

54 . A computer-readable data carrier having stored thereon a computer program product according to claim 52 .

55 . A data carrier signal carrying a computer program product according to claim 52 .