Detection of methylcytosine and its derivatives using S-adenosyl-L-methionine analogues (XSAMS)

Abstract

Examples provided herein relate to the detection of methylcytosine and its derivatives using S-adenosyl-L-methionine analogs (xSAMs). Compositions and methods for performing such detection are disclosed. The target polynucleotide may include cytosine (C) and methylcytosine (mC). The method may include (a) protecting C in the target polynucleotide from deamination, and (b) after step (a), deaminating mC in the target polynucleotide to form thymine (T). Protecting C from deamination may include adding a protecting group to the 5-position of C, for example, using a methyltransferase enzyme from xSAM to add a first protecting group.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 161,330, entitled “DETECTING METHYLCYTOSINE AND ITS DERIVATIVES USING S-ADENOSYL-L-METHIONINE ANALOGS (xSAMS),” filed March 15, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to compositions and methods for detecting methylcytosine.

[0003] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 8549102516_SL.txt. The text file is 2.06KB, was created on Mar. 9, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0004] In organisms such as humans, selected cytosines (C) in the genome can be methylated. For example, S-adenosyl-L-methionine (SAM) is known to be a ubiquitous methyl donor for various biological methylation reactions catalyzed by enzymes called methyltransferases (MTases). The enzyme 5-MTase can add a methyl group to the 5-position of cytosine to form 5-methylcytosine (5mC) in a manner as described in Deen et al., "Methyltransferase-directed labeling of biomolecules and its applications," Angewandte Chemie International Edition 56:5182-5200 (2017), the entire contents of which are incorporated herein by reference. Other enzymes can oxidize the methyl group of cytosine to form the 5mC derivative 5-hydroxymethylcytosine (5hmC), which can be further oxidized to form the 5mC derivatives 5-formylcytosine (5fC) and 5-carboxycytosine (5caC).

[0005] 5mC and 5hmC are sometimes referred to as epigenetic markers, and it may be desirable to detect them in genomic sequences. The current gold standard method for detecting 5mC and 5hmC is bisulfite sequencing, which converts any unmethylated C in a sequence to uracil (U), but does not convert 5mC or 5hmC to the corresponding uracil derivative. When a sequence is amplified using polymerase chain reaction (PCR), uracil is amplified as thymidine (T), and therefore unmethylated C is sequenced as T. In comparison, 5mC and 5hmC are amplified as C, and therefore sequenced as C. Thus, any C in a sequence can be identified as corresponding to 5mC or 5hmC, since they were not converted to U. Such a scheme may be referred to as a "three-base" sequencing scheme, since any unmethylated C is converted to T. However, this type of scheme reduces sequence complexity and may result in reduced sequencing quality, lower mapping rates, and relatively uneven coverage of the sequence. Summary of the Invention

[0006] Examples provided herein relate to the detection of methylcytosine and its derivatives using S-adenosyl-L-methionine analogs (xSAMs). Compositions and methods for carrying out such detection are disclosed.

[0007] Some examples herein provide a method for modifying a target polynucleotide. The target polynucleotide may include cytosine (C) and methylcytosine (mC). The method may include (a) protecting C in the target polynucleotide from deamination. The method may include (b) after step (a), deaminating mC in the target polynucleotide to form thymine (T).

[0008] In some examples, protecting C from deamination includes adding a first protecting group to the 5-position of C. In some examples, the first methyltransferase enzyme adds a first protecting group to the 5-position of C. In some examples, the first methyltransferase enzyme has the structure: [ka] wherein X comprises a first protecting group and a methylene group through which the first protecting group is bonded to the sulfonium ion (S+); The first protecting group is added from an S-adenosyl-L-methionine analog (xSAM) having the structure:

[0009] In some examples, the first methyltransferase enzyme is selected from the group consisting of DNMT1, DNMT3A, DNMT3B, dam, and CpG(M.SssI).

[0010] In some examples, the first protecting group comprises an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye.

[0011] In some instances, the methyl group of mC inhibits the addition of X to position 5 of mC.

[0012] In some examples, the cytidine deaminase enzyme deaminates mC. In some examples, X fits within a first methyltransferase enzyme and inhibits the activity of the cytidine deaminase enzyme. In some examples, the cytidine deaminase enzyme comprises an APOBEC. In some examples, the APOBEC is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4.

[0013] In some examples, the target polynucleotide further comprises hydroxymethylcytosine (hmC), and step (b) comprises deaminating hmC in the target polynucleotide to form hydroxythymine (hT).

[0014] In some examples, the target polynucleotide further comprises hydroxymethylcytosine (hmC). The method may further comprise (c) protecting hmC in the target polynucleotide from deamination prior to step (b). In some embodiments, step (c) is performed after step (a). In some examples, protecting hmC from deamination comprises adding a second protecting group to the hydroxymethyl group of hmC. In some examples, the enzyme adds the second protecting group to the hydroxymethyl group of hmC. In some examples, the enzyme is selected from the group consisting of β-glucosyltransferase (βGT) and β-arabinosyltransferase (βAT). In some examples, the second protecting group comprises a sugar.

[0015] In some examples, the method includes performing steps (a) and (b) on a first sample containing the target polynucleotide, and performing steps (a), (b), and (c) on a second sample containing the target polynucleotide.

[0016] In some examples, the target polynucleotide further comprises a formylcytosine (fC), wherein the formyl group of fC inhibits deamination of fC during step (b).

[0017] In some examples, the target polynucleotide further comprises a formylcytosine (fC), and the method may further comprise (d) prior to step (b), converting fC to an unprotected C that is deaminated during step (b) to form uracil (U). In some examples, the thymine deglycosylase enzyme replaces the base of fC with C.

[0018] In some examples, the method includes performing steps (a) and (b) on a first sample containing the target polynucleotide, and performing steps (a), (b), and (d) on a third sample containing the target polynucleotide.

[0019] In some examples, the target polynucleotide further comprises a carboxyl cytosine (caC), wherein the carboxyl group of caC inhibits deamination of fC during step (b).

[0020] In some examples, the target polynucleotide further comprises a carboxyl cytosine (caC) and the method further comprises (e) prior to step (b), converting caC to an unprotected C that is deaminated during step (b) to form uracil (U). In some examples, the third methyltransferase enzyme removes the carboxyl group from caC. In some examples, the thymine deglycosylase enzyme replaces the base of caC with a C.

[0021] In some examples, the method includes performing steps (a) and (b) on a first sample comprising the target polynucleotide and performing steps (a), (b), and (e) on a fourth sample comprising the target polynucleotide. In some examples, the third sample is a fourth sample and the second methyltransferase is a third methyltransferase.

[0022] In some examples, the target polynucleotide comprises DNA.

[0023] In some examples, the target polynucleotide comprises a first and a second adaptor. In some examples, the first and second adaptors are added to the target polynucleotide prior to step (a). In some examples, the first and second adaptors are added to the target polynucleotide after step (b).

[0024] Some examples herein provide a method of sequencing a target polynucleotide. The method may include modifying the target polynucleotide according to any of the methods described above. The method may include generating a first amplicon of the modified target nucleotide. The first amplicon may include a first guanine (G) at a position complementary to the protected C and a first adenine (A) at a position complementary to the T. The method may include generating a second amplicon of the first amplicon, the second amplicon including a first unprotected C at a position complementary to the first G and a first thymine (T) at a position complementary to the first A. The method may include sequencing the first amplicon, the second amplicon, or both the first amplicon and the second amplicon. The method can include identifying mC based on a first A in the first amplicon, a first T in the second amplicon, or both a first A in the first amplicon and a first T in the second amplicon.

[0025] In some examples, the first amplicon includes a second A at a position complementary to the hT, and the second amplicon includes a second T at a position complementary to the second A. The method may further include identifying hmC based on the second A in the first amplicon, the second T in the second amplicon, or both the second A in the first amplicon and the second T in the second amplicon.

[0026] In some examples, the first amplicon includes a second G at a position complementary to hmC, and the second amplicon includes a second unprotected C at a position complementary to the second G. The method may further include identifying hmC based on the second G in the first amplicon, the second unprotected C in the second amplicon, or both the second G in the first amplicon and the second unprotected C in the second amplicon.

[0027] In some examples, the first amplicon includes a third G at a position complementary to fC, and the second amplicon includes a third unprotected C at a position complementary to the third G. The method may further include identifying fC based on the third G in the first amplicon, the third unprotected C in the second amplicon, or both the third G in the first amplicon and the third unprotected C in the second amplicon.

[0028] In some examples, the first amplicon includes a third A at a position complementary to the U, and the second amplicon includes a third T at a position complementary to the third A. The method may further include identifying fC based on the third A in the first amplicon, the third T in the second amplicon, or both the third A in the first amplicon and the third T in the second amplicon.

[0029] In some examples, the first amplicon includes a fourth G at a position complementary to caC, and the second amplicon includes a fourth unprotected C at a position complementary to the fourth G. The method may further include identifying caC based on the fourth G in the first amplicon, the fourth unprotected C in the second amplicon, or both the fourth G in the first amplicon and the fourth unprotected C in the second amplicon.

[0030] In some examples, the first amplicon includes a fourth A at a position complementary to the U, and the second amplicon includes a fourth T at a position complementary to the fourth A. The method may further include identifying caC based on the fourth A in the first amplicon, the fourth T in the second amplicon, or both the fourth A in the first amplicon and the fourth T in the second amplicon.

[0031] Some examples herein provide polynucleotides isolated from extracellular fluid samples. The polynucleotides may include cytosine (C) containing a blocking group at position 5; and thymine (T).

[0032] In some examples, the first protecting group comprises an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye.

[0033] In some examples, the polynucleotide comprises a hydroxymethylcytosine (hmC). In some examples, the hmC comprises a second protecting group. In some examples, the second protecting group comprises a sugar.

[0034] In some examples, the polynucleotide includes hydroxythymine (hT).

[0035] In some examples, the polynucleotide includes a formylcytosine (fC).

[0036] In some examples, the polynucleotide includes a carboxyl cytosine (caC).

[0037] In some instances, the polynucleotide includes uracil (U).

[0038] In some instances, the polynucleotide comprises DNA.

[0039] In some examples, the polynucleotide comprises a first and a second adaptor.

[0040] Some examples herein are of the following structure: [ka] wherein X comprises a first protecting group and a methylene group through which the protecting group is bonded to the sulfonium ion (S+); The present invention provides an S-adenosyl-L-methionine analog (xSAM) having the structure:

[0041] In some examples, the protecting group comprises an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye.

[0042] Some examples herein provide compositions that include a polynucleotide, any of the xSAMs described above, and a methyltransferase enzyme that adds a protecting group of the xSAM to a cytosine in the polynucleotide.

[0043] Some examples herein provide a composition comprising an isolated polynucleotide and a cytidine deaminase enzyme in an extracellular fluid. The polynucleotide may comprise (i) a cytosine (C) comprising a blocking group at position 5, and (ii) a methylcytosine (mC) or a hydroxymethylcytosine (hmC). The cytidine deaminase enzyme may deaminate mC to form thymine (T) or may deaminate hmC to form hydroxythymine (hT).

[0044] Some examples herein provide a composition comprising an isolated polynucleotide in an extracellular fluid and a methyltransferase enzyme. The polynucleotide may comprise (i) a cytosine (C) containing a blocking group at position 5, and (ii) a formylcytosine (fC) or a carboxylcytosine (caC). The composition may comprise an enzyme that converts fC or caC to C.

[0045] Some examples herein provide an isolated polynucleotide and a β-glucosyltransferase (βGT) or β-arabinosyltransferase (βAT) enzyme in an extracellular fluid. The polynucleotide may include (i) a cytosine (C) that includes a first protecting group at position 5, and (ii) a hydroxymethylcytosine (hmC). The βGT or βAT enzyme can add a second protecting group to the hmC.

[0046] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 shows a schematic of a series of reactions for detecting methylcytosine and its derivatives using S-adenosyl-L-methionine analogues (xSAMs).

[0048] [Diagram 2] FIG. 2 is a schematic representation of selected reactions of FIG. 1.

[0049] [Diagram 3] FIG. 13 shows a schematic series of further reaction schemes for detecting methylcytosine and its derivatives, and for distinguishing methylcytosine derivatives from one another, using xSAMs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Examples provided herein relate to the detection of methylcytosine and its derivatives using S-adenosyl-L-methionine analogs (xSAMs). Compositions and methods for carrying out such detection are disclosed.

[0051] As provided herein, a protecting group (X) is added to the 5-position of any unmethylated cytosine (C) in a polynucleotide sequence to generate XC, which is relatively stable to further reactions used to convert any methylcytosine (mC) to thymine (T) and any hydroxymethylcytosine (hmC) to hydroxythymine (hT). When the sequence is amplified using polymerase chain reaction (PCR), T and hT are amplified as thymine (T), and thus mC and its derivative hmC are sequenced as T. In comparison, unmethylated C is amplified and sequenced as C. Thus, any C in the sequence can be identified as corresponding to C, since it has not been converted to T. Such a scheme can be referred to as a "four-base" sequencing scheme, since any unmethylated C is sequenced as C. Compared to the "three-base" sequencing scheme, this scheme maintains the complexity of the sequence and can result in improved sequencing quality, higher mapping rates, and relatively uniform coverage of the sequence. Further reactions are provided to distinguish mC and its derivatives from one another, thus providing a further analytical tool to characterize any epigenetic marker in a genomic sequence.

[0052] We first provide a brief description of some of the terms used herein, followed by a description of some exemplary compositions and exemplary methods for detecting methylcytosine and its derivatives using xSAMS.

[0053] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The use of the term "having" and other forms such as "have", "has" and "had" is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "comprising at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0054] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and take into account small variations due to processing variations, etc. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0055] As used herein, "hybridize" refers to the non-covalent binding of a first polynucleotide to a second polynucleotide along the length of the polymer to form a double stranded "duplex". For example, two DNA polynucleotide strands can associate through complementary base pairing. The strength of association between a first and a second polynucleotide increases with the complementarity between the nucleotide sequences within the polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the duplex dissociates from each other.

[0056] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some instances also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (GT ... deoxyadenosine monophosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), and deoxythymidine monophosphate (DTMP).These include deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0057] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar and / or phosphate moiety as compared to naturally occurring nucleotides. Exemplary modified nucleobases include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil ... These include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, such as nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates.

[0058] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides that are linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs. A polynucleotide can be a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide can include non-naturally occurring DNA, such as enantiomeric DNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0059] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles a polynucleotide by polymerizing nucleotides into a polynucleotide. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by successively adding nucleotides to the primer to form a "complementary copy" polynucleotide having a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of the complementary copy polynucleotide. Any such copy may be referred to herein as an "amplicon." A DNA polymerase can bind to a target polynucleotide and then move downstream while growing the target polynucleotide by successively adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Polymerases may use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases may displace the strand upstream of the site where they add a base to the strand. Such polymerases may also be said to be strand displacing, that is, to have the activity of removing a complementary strand from the template strand that is read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, Bst (Bacillus stearothermophilus) polymerase, Exo-Klenow polymerase, or a large fragment of sequencing grade T7 exo-polymerase. Some polymerases cleave the strand in front of them and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0060] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added via a free 3'OH group. The length of the primer can be any suitable number of bases long and can include a suitable combination of natural and / or non-natural nucleotides. The target polynucleotide can include an "adapter" that hybridizes to the primer (having a sequence complementary to the primer) and can be amplified to generate a complementary copy polynucleotide by adding nucleotides to the free 3'OH group of the primer. The primer can be bound to a substrate.

[0061] As used herein, the term "substrate" refers to a material used as a support for the compositions described herein. Exemplary substrate materials can include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application includes a silica-based substrate, such as glass, fused silica, or other silica-containing materials. In some examples, the substrate can include silicon, silicon nitride, or hydrogenated silicone. In some examples, the substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In certain examples, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface that includes a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamides, enones, or acrylates can also be utilized as substrate materials or components. Other substrate materials can include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resin, polymer, quartz, resin, polymer, and copolymer.In some examples, the substrate and / or substrate surface may be or include quartz. In some other examples, the substrate and / or substrate surface may be or include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate may include a single material or multiple different materials. The substrate may be a composite or laminate. In some examples, the substrate includes an organosilicate material. The substrate may be flat, round, spherical, rod-like, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0062] In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions can be features in which one or more capture primers are present. The features can be separated by gap regions in which no capture primers are present. In some examples, the pattern can be an xy format of features in rows and columns. In some examples, the pattern can be a repeating arrangement of features and / or gap regions. In some examples, the pattern can be a random arrangement of features and / or gap regions. In some examples, the substrate includes an array of wells (recesses) in the surface. The wells can be provided with substantially vertical sidewalls. The wells can be manufactured as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As is understood in the art, the techniques used depend on the composition and shape of the array substrate.

[0063] The features within the patterned surface of the substrate may include wells in which a covalently bonded gel, such as patterned poly(N-(5-azidoacetamylpentyl)acrylamide-co-acrylamide) (PAZAM), is arrayed on glass, silicon, plastic, or other suitable material. This process creates a gel pad used for sequencing, which may be stable over many cycles of sequencing operations. Covalently bonding the polymer to the wells may be useful to maintain the gel in the structured features throughout the life of the structured substrate during various applications. However, in many instances, the gel does not need to be covalently bonded to the wells. For example, in some conditions, silane free acrylamide (SFA) that is not covalently bonded to any part of the structured substrate may be used as the gel material.

[0064] In a particular example, a structured substrate can be created by patterning a suitable material with wells (e.g., microwells or nanowells), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azido form of SFA (azido-SFA)), and polishing the gel-coated material, for example, by chemical or mechanical polishing, thereby retaining the gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions of the surface of the structured substrate between the wells. Primers can then be bound to the gel material. A solution containing a plurality of target polynucleotides (e.g., a fragmented human genome or a portion thereof) can then be contacted with the polished substrate such that individual target polynucleotides are seeded into individual wells through interaction with primers attached to the gel material, but the target polynucleotides do not occupy the interstitial regions due to the absence or inactivity of the gel material. Amplification of the target polynucleotides can be confined to the wells due to the absence or inactivity of the gel in the interstitial regions, preventing outward migration of clusters. The process is easily manufacturable, scalable, and utilizes conventional micro- or nano-fabrication methods.

[0065] A patterned substrate may include, for example, wells etched into a slide or chip. The pattern of wells etched and geometric shapes may be of a variety of different shapes and sizes, and such features may be physically or functionally separable from one another. Particularly useful substrates with such structural features include patterned substrates that allow selection of the size of solid particles, such as microspheres. An example of a patterned substrate with these characteristics is the etched substrate used in conjunction with BEAD ARRAY technology (Illumina, Inc., San Diego, CA).

[0066] In some examples, the substrates described herein form at least a part of a flow cell, are located within a flow cell, or are coupled to a flow cell. A flow cell may include a flow chamber that is divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that can be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).

[0067] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. A plurality can range in size from small, medium, large, to very large. A small size plurality can range, for example, from a few members to tens of members. A medium size plurality can range, for example, from tens of members to about 100 members or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, a plurality can range in size from 2 to well over 100 million members, as well as between all sizes measured by number of members and larger than the exemplary ranges listed above. Exemplary polynucleotides include, for example, about 1×10 5 That's it, 5 x 10 5 or more or 1×10 6 The term includes a population of more than one different polynucleotides. Thus, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the plurality of values ​​can be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.

[0068] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of an analysis or action. The analysis or action includes subjecting the polynucleotide to amplification, sequencing, and / or other procedures. A target polynucleotide may include additional nucleotide sequences to the target sequence being analyzed. For example, a target polynucleotide may include one or more adapters, including adapters that function as primer binding sites, that flank the target polynucleotide sequence being analyzed.

[0069] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terminology is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another species.

[0070] As used herein, the term "amplicon" when used in reference to a polynucleotide refers to a product of the replication of a polynucleotide, which product has a nucleotide sequence that is substantially the same as or complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" and "amplifying" refer to the process of creating an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Additional amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of the polynucleotide.

[0071] As used herein, the term "methylcytosine" or "mC" refers to a methyl group (-CH3 or -Me). The methyl group may be located at the 5-position of the cytosine, in which case mC may be referred to as 5mC.

[0072] As used herein, a "derivative" of methylcytosine refers to a methylcytosine having an oxidized methyl group. A non-limiting example of an oxidized methyl group is hydroxymethyl (-CH 2 An oxidized methyl group may be at the 5-position of cytosine, in which case hmC may be referred to as 5hmC, fC may be referred to as 5fC, or caC may be referred to as 5caC. Another non-limiting example of an oxidized methyl group is a formyl group (-CHO), in which case mC derivatives may be referred to as formylcytosine or fC. Another non-limiting example of an oxidized methyl group is a carboxyl (-COOH), in which case mC derivatives may be referred to as carboxylcytosine or caC. The oxidized methyl group may be located at the 5-position of cytosine, in which case hmC may be referred to as 5hmC, fC may be referred to as 5fC, or caC may be referred to as 5caC.

[0073] As used herein, a "derivative" of thymine (T) refers to a thymine having an oxidized methyl group. A non-limiting example of an oxidized methyl group is hydroxymethyl (-COH), in which case the T derivative may be referred to as hydroxythymine or hT. The oxidized methyl group may be located at the 5-position of thymine, in which case the hT may be referred to as 5hT.

[0074] As used herein, S-adenosyl-L-methionine (SAM) refers to the compound having the following structure: [ka] The methyl group attached to the sulfonium (S+) ion can be transferred to cytosine by a methyltransferase in a manner such as described in Deen et al., referenced above. A counterion such as chloride (Cl-) can be present or a proton can be removed from the COOH to provide a neutral atom. Additionally, amino acids in solution may be in zwitterionic isoforms (COO-, NH3+).

[0075] As used herein, the term S-adenosyl-L-methionine analog (xSAM) refers to a compound having the following structure: [ka] In the formula, X refers to a compound having a structure including a protecting group and a methylene group through which the protecting group is attached to S. X may be compatible with the activity of one or more enzymes or inhibit the activity of one or more enzymes. For example, as described in more detail herein, X may be compatible with the activity of a methyltransferase enzyme, such that the methyltransferase can act on xSAM to transfer X bound to the sulfonium ion of xSAM to cytosine to form XC, in a manner similar to that described in Deem et al., and the methyltransferase can act on SAM to transfer the sulfonium-bound methyl group to cytosine to form mC. Additionally, or alternatively, X may be incompatible with the activity of a cytidine deaminase enzyme, such that the cytidine deaminase enzyme cannot act on XC to deaminate XC in a manner similar to how cytidine deaminase otherwise acts on C to form U, mC to form T, or hmC to form hT. Non-limiting examples of X include methylene alkyne groups, [ka] Methylene carboxyl group [ka] Methyleneamino group [ka] Methylene hydroxymethyl group [ka] Methyleneisopropyl group [ka] or methylene dye group [ka] Examples include:

[0076] As used herein, "methyltransferase enzyme" or "MTase" refers to an enzyme that can add (or "methylate") or remove (or "demethylate") a methyl group from a substrate. Some methyltransferases can add a methyl group (Me) from SAM to a substrate (e.g., C), and can also, alternatively, add a protecting group (X) from XSAM to such a substrate (e.g., C). Non-limiting examples of methyltransferases suitable for adding a protecting group X from XSAM to C include mammalian methyltransferases such as DNMT1, DNMT3A, and DNMT3B, as described in Jin et al., "DNA methytransferases (DNMTs), DNA damage repair, and cancer," Adv Exp Med Biol. 754:3-29 (2013), the entire contents of which are incorporated herein by reference, and bacterial methyltransferases such as dam and CpG(M.SssI), which are commercially available from New England Biolabs (Ipswitch, MA). Some methyltransferases can remove oxidized methyl groups (such as formyl or carboxyl) from substrates such as caC. Non-limiting examples of methyltransferases that can decarboxylate caC in the absence of SAM include the bacterial C5-methyltransferases M.HhaI and M.SssI (the latter can also be used to add a protecting group X from XSAM to C in the manner described above). For further details on the use of methyltransferases to remove the carboxyl group from caC to form C, see Liutkeviciute et al., "Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases," J. Am. Chem. Soc. 136(16):5884-5887 (2014), the entire contents of which are incorporated herein by reference.

[0077] As used herein, "thymine deglycosidase" (TDG) refers to an enzyme that excises a base from fC or caC and replaces the excised base with a C, a reaction that may be referred to as base excision repair (BER). For further details regarding TDG and BER, see Kohli et al., "TET enzymes, TDG and the dynamics of DNA methylation," Nature 502(7472):472-479 (2013), the entire contents of which are incorporated herein by reference.

[0078] As used herein, "cytidine deaminase enzyme" refers to an enzyme that deaminates cytosine and / or one or more cytosine derivatives. Deamination can occur at the 6-position of cytosine or a cytosine derivative. For example, a cytidine deaminase enzyme can deaminate cytosine to form U, can deaminate mC to form T, and / or can deaminate hmC to form hT. A cytidine deaminase enzyme may not necessarily deaminate all possible cytosine derivatives. For example, a cytidine deaminase enzyme may not necessarily deaminate cytosine containing an X at position 5, may not necessarily deaminate fC to form formyluridine (fU), and / or may not necessarily deaminate caC to form carboxyuridine (caU). A non-limiting example of a cytidine deaminase enzyme that may deaminate cytosine to form U, mC to form T, and / or hmC to form hT, and may or may not deaminate fC to form fU, and / or caC to form caU, is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC). Non-limiting examples of such APOBECs include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4.

[0079] As used herein, "β-glucosyltransferase enzyme" or "βGT" refers to an enzyme that adds a glucose group (e.g., glucose or a glucose derivative) to hmC, e.g., to the hydroxymethyl group at the 5-position of hmC to form β-glucosyl-5-hydroxymethylcytosine (1,2). A non-limiting example of a βGT is T4 phage β-glucosyltransferase (T-4BGT), commercially available from New England Biolabs (Ipswitch, Mass.).

[0080] As used herein, "β-arabinosyltransferase enzyme" or "βAT" refers to an enzyme that adds an arabinose group to hmC, e.g., to the hydroxymethyl group at position 5 of hmC to form arabinosyl-hmC. A non-limiting example of a βAT is T4-like phage RB69 ORF003c, described in Thomas et al., "The odd 'RB' Phage-identification of arabinosylation as a new epigenetic modification of DNA in T4-like phage RB69," Viruses 10(6):313, 18 pages (2018), the entire contents of which are incorporated herein by reference.

[0081] As used herein, a "protecting group" is intended to mean a chemical group that inhibits the activity of an enzyme. For example, a protecting group attached to the 5-position of cytosine via a methylene group can inhibit the activity of a cytidine deaminase enzyme that would otherwise deaminate cytosine to form uracil. As another example, a protecting group (e.g., a sugar such as glucose or arabinose) at the hydroxymethyl group at the 5-position of hmC can inhibit the activity of a cytidine deaminase enzyme that would otherwise deaminate hmC to form hydroxythymine.

[0082] Compositions and methods for detecting methylcytosine and its derivatives using xSAMS Some examples provided herein relate to the detection of methylcytosine and its derivatives using xSAMs. Compositions and methods for carrying out such detection are disclosed.

[0083] For example, a target polynucleotide having a sequence that includes cytosine (C) and methylcytosine (mC), and may also include hydroxymethylcytosine (hmC), may be modified in such a manner that C is protected from deamination, and then mC is deaminated to form thymine (T), and hmC is deaminated to form hydroxythymine (hT). If the sequence is subsequently amplified using polymerase chain reaction (PCR) in a manner as described in more detail below, T and any hT will be amplified as thymidine (T), and thus mC and hmC may be sequenced as T. In comparison, unmethylated (and protected) C is amplified and sequenced as C. Thus, any C in the sequence may be identified as corresponding to C, since it has not been converted to T or a T derivative as mC and hmC have. Thus, the method provides a "four base" sequencing method in which unmethylated C may be sequenced as C, thus preserving the genomic information carried by that base. In a manner described in more detail below, mC and hmC can be distinguished from one another using additional reaction schemes.

[0084] 1 shows a schematic of a series of reactions for detecting methylcytosine (mC) and its derivatives using xSAMS. As shown in FIG. 1, protecting C from deamination can include adding a first protecting group to the 5-position of C. For example, a first methyltransferase enzyme (MTase) can add X to the 5-position of C to form XC in the manner shown in FIG. 1, where X comprises a protecting group and a methylene group, and the protecting group is attached to C via the methylene group. Illustratively, the first methyltransferase enzyme has the following structure: [ka] X may be added from xSAM having the structure: X includes a first protecting group and a methylene group through which the first protecting group is attached to the sulfonium ion. In a non-limiting example, the first protecting group may include an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye. The xSAM having the first protecting group and the methylene group attached to the sulfonium can act as a surrogate cofactor instead of the SAM having the methyl group attached to the sulfonium, and thus the methyltransferase can covalently attach a methylene group (having the first protecting group attached thereto) to the 5-position of any unmethylated C in the target polynucleotide to form 5XC. During the action of the methyltransferase, a composition can be formed that includes a polynucleotide, xSAM, and a methyltransferase enzyme that adds X from xSAM to C in the polynucleotide. It is understood that an appropriate amount of the methyltransferase and xSAM can be mixed with the polynucleotide in the extracellular fluid. For example, since xSAM is a stoichiometric reagent, at least the amount of xSAM may be added as C present in the genomic sample, or an excess of xSAM may be added.

[0085] As shown in FIG. 1, it should be noted that the methyltransferase may not be able to add X (and therefore may not be able to add the first protecting group) to any mC and / or any mC derivative in the target polynucleotide. For example, the methyl group of mC may inhibit the addition of X (and the first protecting group) to the 5-position of mC because a methyl group already occupies that position. Similarly, the hydroxymethyl group of any hmC may inhibit the addition of X (and the first protecting group) to the 5-position of hmC, the formyl group of any fC may inhibit the addition of X (and the first protecting group) to the 5-position of fC, and the carboxyl group of any caC may inhibit the addition of X (and the first protecting group) to the 5-position of caC.

[0086] Following protection of C in the target polynucleotide, mC and / or any of its derivatives can be deaminated, for example, using a cytidine deaminase enzyme. In this regard, the first protecting group may be selected to fit within the first methyltransferase enzyme, and thus may be compatible with the activity of the first methyltransferase enzyme, but the first protecting group may inhibit the activity of the cytidine deaminase enzyme. Furthermore, the formyl group of any fC may inhibit the activity of the cytidine deaminase enzyme, and the carboxyl group of any caC may inhibit the activity of the cytidine deaminase enzyme. In comparison, the methyl group of mC and the hydroxymethyl group of hmC may be compatible with the activity of the cytidine deaminase enzyme. Thus, as shown in FIG. 1, XC, any fC, and any caC cannot be deaminated by the cytidine deaminase enzyme, but any mC may be deaminated to form T, and any hmC may be deaminated to form hT. During the action of cytidine deaminase enzyme, a composition comprising polynucleotide and cytidine deaminase enzyme can be formed in extracellular fluid.Polynucleotide can comprise XC and mC and / or hmC.Cytidine deaminase enzyme can deaminate mC to form T or deaminate hmC to form hT.It is understood that an appropriate amount of cytidine deaminase enzyme can be mixed with polynucleotide in extracellular fluid.For example, cytidine deaminase can be added in catalytic amount, for example, less than the number of mC and hmC to be deaminated.

[0087] As shown in FIG. 1, PCR can then be performed to generate amplicons of the target polynucleotide. In the first set of amplicons, the unmethylated protected C is amplified as C, while T and hT are amplified as T, and fC and caC are amplified as C. It will be understood that a second set of complementary amplicons is also generated using PCR, in which the unmethylated protected C is amplified as G, T and hT are amplified as A, and fC and caC are amplified as G. The amplicons can then be sequenced using known techniques, such as sequencing by synthesis (SBS). The positions in the target polynucleotide where mC and hmC are located, where T and hT are generated using deamination, and where C is protected using the present xSAM can be determined by comparing the sequence of the resulting amplicon to the sequence of an amplicon where mC and hmC are not deaminated and therefore are amplified and sequenced as C (or as G in the complementary amplicon). Bases that are T (or A) in the deaminated amplicon and C (or G) in the non-deaminated amplicon can be identified as corresponding to hC and / or hmC.

[0088] For example, Figure 2 shows a schematic of selected reactions from Figure 1. Figure 2 shows an exemplary polynucleotide sequence CCGT(5hmC)GGAC(mC)GC (SEQ ID NO: 1). The other C is protected with a protecting group (X) transferred from xSAM by a methyltransferase enzyme. A cytidine deaminase enzyme (e.g., APOBEC) is then used to deaminate 5hmC and 5mC, resulting in the sequence CCGT(5hT)GGAC(T)GC (SEQ ID NO: 2), which is amplified by PCR and then [ka] where the bolded Ts correspond to 5hmC and mC in the original sequence. The presence and location of 5hmC and mC in the target polynucleotide is determined by the sequence [ka] (The bolded Cs correspond to 5hmC and mC in the original sequence) and comparing the sequences of these amplicons of the target polynucleotide with the sequences of the amplicons after protection and deamination. Through such a comparison, it can be seen that the bolded Cs have been "converted" from C to T, indicating that deamination has occurred and thus mC or hmC was originally present at those positions.

[0089] Additionally, as further discussed above, the present disclosure provides methods for distinguishing methylcytosine and certain of its derivatives from one another. For example, Figure 3 generally illustrates a further set of reaction schemes for detecting mC and its derivatives, and for distinguishing methylcytosine derivatives from one another, using xSAMS.

[0090] As shown in FIG. 3, mC and hmC can be distinguished from each other using an additional reaction after protecting C with xSAMs but before deamination. Such a reaction protects hmC in the target polynucleotide from deamination, so that hmC is not converted to hT during deamination (and thus is amplified and sequenced as C), while mC is converted to T (and thus is amplified and sequenced as T). Protecting hmC from deamination can include adding a second protecting group to the hydroxymethyl group of hmC to form gmC. Illustratively, a glycosyltransferase, such as a β-glucosyltransferase (βGT) or β-arabinosyltransferase (βAT) enzyme, can add a second protecting group to the hydroxymethyl group of hmC. The second protecting group may include a sugar transferred from a sugar donor, such as glucose or a glucose derivative (e.g., UDP-glucose, or UDP-6-azido-glucose) transferred from a glucosyl donor, or arabinose (e.g., UDP-arabinose) transferred from an arabinose donor, forming sugar-methylcytosine (sMC). During the action of the glycosyltransferase, a composition may be formed in the extracellular fluid that includes a polynucleotide and an enzyme. The polynucleotide may include XC and hmC, and the enzyme may add a second protecting group to the hmC. It is understood that an appropriate amount of the enzyme may be mixed with the polynucleotide in the extracellular fluid. For example, the enzyme may be added in a catalytic amount, and the sugar donor may be added in a stoichiometric amount or in excess.

[0091] Unprotected methylcytosines in the polynucleotide can then be deaminated to form Ts, for example, using a cytidine deaminase enzyme in a manner as described with reference to Figure 1, and the sequences can then be amplified and sequenced. Note that the use of glucose derivatives, such as 6-azido-glucose, can allow for further modifications to glucose, such as via click chemistry reactions of dyes with azides in a manner as described in Song et al., "Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation," PNAS 113(16):4338-4343 (2016), the contents of which are incorporated herein by reference in their entirety.

[0092] To distinguish hmC from mC, the C protection and deamination steps described with reference to FIG. 1 can be performed on a first sample containing a target polynucleotide, followed by amplification and sequencing, and the C protection, hmC protection, and deamination steps described with reference to FIG. 3 can be performed on a second sample containing a target polynucleotide, followed by amplification and sequencing. The sequence of the amplicon from the first sample can be compared to the sequence of the amplicon from the second sample and / or the amplicon of the original sequence. Through such comparison, it can be understood that a C that is "converted" from C to T in the first sample compared to the original sequence corresponds to mC or hmC, and that such a C that is not similarly "converted" from T to C in the second sample compared to the first sample corresponds to hmC.

[0093] Additionally or alternatively, as shown in FIG. 3, fC and caC can be distinguished from C using one or more additional reactions after protecting C using xSAMs but before deamination. More specifically, if the target polynucleotide contains fC and / or caC, the formyl group from any fC and / or the carboxyl group from any caC can be removed before deamination to form an unprotected C that can be deaminated to form a U. Removal of the carboxyl group can be performed using a methyltransferase enzyme as described elsewhere herein, or the base of fC or caC can be replaced with a C using thymine deglycosylase (TDG) in a manner as described elsewhere herein. The unprotected C in the polynucleotide can then be deaminated to form a U, for example, using a cytidine deaminase enzyme in a manner as described with reference to FIG. 1, and the sequence can then be amplified and sequenced. To distinguish fC and / or caC from C, the C protection and deamination steps described with reference to FIG. 1 may be performed on a first sample containing the target polynucleotide, followed by amplification and sequencing, and the C protection, fC and / or caC deprotection, and deamination steps described with reference to FIG. 3 may be performed on a second sample containing the target polynucleotide, followed by amplification and sequencing. The sequence of the amplicon from the first sample may be compared with the sequence of the amplicon from the second sample and / or with the amplicon of the original sequence. Through such comparison, it may be understood that a C that remains a C in the first sample compared with the original sequence corresponds to C, fC, or caC, and such a C that is "converted" from C to T in the second sample compared with the first sample corresponds to fC or caC. During the action of the methyltransferase or TDG enzyme, a composition may be formed in the extracellular fluid that includes a polynucleotide and an enzyme. The polynucleotide may include XC and fC and / or caC. The enzyme may convert fC and / or caC to C. It is understood that a suitable amount of a methyltransferase enzyme, for example, in a catalytic amount, can be mixed with the polynucleotide in the extracellular fluid.

[0094] In some examples provided herein, the target polynucleotide comprises DNA, however, it is understood that the methods and compositions of the present invention may be suitably modified to detect mC and / or its derivatives in any suitable type of polynucleotide, such as RNA. The polynucleotide may be isolated from an extracellular fluid sample and may comprise C comprising a first protecting group at the 5-position and T as provided using the reaction scheme described with reference to Figures 1-2. The first protecting group may be attached to C via a methylene group and may illustratively comprise an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye. The polynucleotide may further comprise hmC, which may comprise a second protecting group, such as a sugar (e.g., glucose or arabinose), as provided using the reaction scheme described with reference to Figure 3. Alternatively, the polynucleotide may further comprise hT as provided using the reaction scheme described with reference to Figures 1-2. The polynucleotide may further comprise a formylcytosine (fC) and / or may comprise a carboxylcytosine (caC), as provided using the reaction scheme described with reference to Figures 1-2. Alternatively, the polynucleotide may comprise a U, as provided using the reaction scheme described with reference to Figure 3.

[0095] To facilitate amplification and sequencing, the target polynucleotide may include, for example, a first and second adaptor flanking the sequence of interest. Such adaptors may be added to the target polynucleotide prior to protecting C using xSAMS, may be added to the target polynucleotide after the deamination step, or may be added at any other suitable time.

[0096] To provide some further details regarding the sequencing of a target polynucleotide modified in any suitable manner provided herein, first and second complementary amplicons of the modified target nucleotides can be generated. The first amplicon can include a first C at a position complementary to the protected C (XC) and a first adenine (A) at a position complementary to the T. The second amplicon can include a first unprotected C at a position complementary to the first G and a first thymine (T) at a position complementary to the first A. The first amplicon, the second amplicon, or both the first amplicon and the second amplicon can be sequenced. The mC can be identified based on the first A in the first amplicon, the first T in the second amplicon, or both the first A in the first amplicon and the first T in the second amplicon, for example, in a manner as described with reference to Figures 1 and 2.

[0097] In some examples, such as those described with reference to Figures 1 and 2, the first amplicon includes a second A at a position complementary to hT, and the second amplicon includes a second T at a position complementary to the second A. hmC can be identified based on the second A in the first amplicon, the second T in the second amplicon, or both the second A in the first amplicon and the second T in the second amplicon. In other examples, such as those using additional reactions described with reference to Figure 3, the first amplicon includes a second G at a position complementary to hmC, and the second amplicon includes a second unprotected C at a position complementary to the second G. hmC can be identified based on the second G in the first amplicon, the second unprotected C in the second amplicon, or both the second G in the first amplicon and the second unprotected C in the second amplicon.

[0098] In some examples, such as those described with reference to Figures 1 and 2, the first amplicon includes a third G at a position complementary to fC, and the second amplicon includes a third unprotected C at a position complementary to the third G. fC can be identified based on the third G in the first amplicon, the third unprotected C in the second amplicon, or both the third G in the first amplicon and the third unprotected C in the second amplicon. In other examples, such as those using the further reaction described with reference to Figure 3, the first amplicon includes a third A at a position complementary to U, and the second amplicon includes a third T at a position complementary to the third A. fC can be identified based on the third A in the first amplicon, the third T in the second amplicon, or both the third A in the first amplicon and the third T in the second amplicon.

[0099] In some examples, such as those described with reference to Figures 1 and 2, the first amplicon includes a fourth G at a position complementary to caC, and the second amplicon includes a fourth unprotected C at a position complementary to the fourth G. caC can be identified based on the fourth G in the first amplicon, the fourth unprotected C in the second amplicon, or both the fourth G in the first amplicon and the fourth unprotected C in the second amplicon. In other examples, such as those using the further reaction described with reference to Figure 3, the first amplicon includes a fourth A at a position complementary to U, and the second amplicon includes a fourth T at a position complementary to the fourth A. caC can be identified based on the fourth A in the first amplicon, the fourth T in the second amplicon, or both the fourth A in the first amplicon and the fourth T in the second amplicon.

[0100] Further comments While various illustrative examples have been described above, it will be apparent to one skilled in the art that various changes and modifications can be made herein without departing from the present invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the present invention.

[0101] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein.

Claims

1. 1. A method for modifying a target polynucleotide containing cytosine (C) and methylcytosine (mC), comprising the steps of: (a) protecting the C in the target polynucleotide from deamination; and (b) after step (a), deaminating the mC in the target polynucleotide to form thymine (T); The method comprising:

2. 2. The method of claim 1 , wherein protecting the C from deamination comprises adding a first protecting group to the 5-position of the C and / or a first methyltransferase enzyme adds the first protecting group to the 5-position of the C.

3. The first methyltransferase enzyme has the structure: 【Chemistry 1】 3. The method of claim 2, wherein the first protecting group is added from an S-adenosyl-L-methionine analog (xSAM) having the formula:

4. 4. The method of claim 2 or 3, wherein the first methyltransferase enzyme is selected from the group consisting of DNMT1, DNMT3A, DNMT3B, dam, and CpG (M.SssI), and / or the first protecting group comprises an alkyne group, a carboxyl group, an amino group, a hydroxymethyl group, an isopropyl group, or a dye.

5. The method of any one of claims 2 to 4, wherein the methyl group of mC inhibits addition of X to the 5-position of mC.

6. 6. The method of any one of claims 1 to 5, wherein a cytidine deaminase enzyme deaminates the mC.

7. 7. The method of claim 6, wherein X fits within the first methyltransferase enzyme and inhibits the activity of the cytidine deaminase enzyme.

8. 8. The method of claim 6 or 7, wherein the cytidine deaminase enzyme comprises APOBEC.

9. 9. The method of claim 8, wherein the APOBEC is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4.

10. 10. The method of any one of claims 1 to 9, wherein the target polynucleotide further comprises hydroxymethylcytosine (hmC), and step (b) comprises deaminating the hmC in the target polynucleotide to form hydroxythymine (hT).

11. The target polynucleotide further comprises hydroxymethylcytosine (hmC), and the method further comprises: (c) prior to step (b), protecting the hmC in the target polynucleotide from deamination. The method of any one of claims 1 to 9, further comprising:

12. The method of claim 11 , wherein step (c) is carried out after step (a).

13. 13. The method of claim 11 or 12, wherein protecting the hmC from deamination comprises adding a second protecting group to a hydroxymethyl group of the hmC.

14. 14. The method of claim 13, wherein an enzyme adds the second protecting group to the hydroxymethyl group of the hmC.

15. 15. The method of claim 14, wherein the enzyme is selected from the group consisting of β-glucosyltransferase (βGT) and β-arabinosyltransferase (βAT).

16. The method of any one of claims 13 to 15, wherein the second protecting group comprises a sugar.

17. 17. The method of any one of claims 11 to 16, comprising performing steps (a) and (b) on a first sample comprising the target polynucleotide, and performing steps (a), (b), and (c) on a second sample comprising the target polynucleotide.

18. 18. The method of any one of claims 1 to 17, wherein the target polynucleotide further comprises a formylcytosine (fC), the formyl group of which inhibits deamination of said fC during step (b).

19. The target polynucleotide further comprises a formylcytosine (fC), and the method further comprises: (d) prior to step (b), converting said fC to an unprotected C which is deaminated during step (b) to form uracil (U). The method of any one of claims 1 to 17, further comprising:

20. 20. The method of claim 19, wherein the thymine deglycosylase enzyme replaces the fC base with C.

21. 21. The method of claim 19 or 20, comprising performing steps (a) and (b) on a first sample containing the target polynucleotide, and performing steps (a), (b) and (d) on a third sample containing the target polynucleotide.

22. 22. The method of any one of claims 1 to 21, wherein the target polynucleotide further comprises a carboxyl cytosine (caC), the carboxyl group of which inhibits the deamination of fC during step (b).

23. The target polynucleotide further comprises a carboxyl cytosine (caC), and the method further comprises: (e) prior to step (b), converting said caC to an unprotected C that is deaminated during step (b) to form uracil (U). The method of any one of claims 1 to 22, further comprising:

24. a second methyltransferase enzyme removes the carboxyl group from the caC; and / or 24. The method of claim 23, wherein the thymine deglycosylase enzyme replaces the base of caC with C.

25. 25. The method of claim 23 or 24, comprising performing steps (a) and (b) on a first sample containing the target polynucleotide, and performing steps (a), (b), and (e) on a fourth sample containing the target polynucleotide.

26. 26. The method of any one of claims 1 to 25, wherein the target polynucleotide comprises (i) DNA and / or (ii) a first and a second adaptor.

27. 27. The method of claim 26, wherein the first and second adaptors are added to the target polynucleotide prior to step (a).

28. 27. The method of claim 26, wherein the first and second adaptors are added to the target polynucleotide after step (b).

29. 1. A method for sequencing a target polynucleotide, comprising: Modifying a target polynucleotide according to any one of claims 1 to 28; generating a first amplicon of the modified target nucleotide, the first amplicon comprising a first guanine (G) at a position complementary to the protected C and a first adenine (A) at a position complementary to the T; generating a second amplicon of the first amplicon, the second amplicon including a first unprotected C at a position complementary to the first G and a first thymine (T) at a position complementary to the first A; sequencing the first amplicon, the second amplicon, or both the first amplicon and the second amplicon; identifying the mC based on the first A in the first amplicon, the first T in the second amplicon, or both the first A in the first amplicon and the first T in the second amplicon; The method comprising:

30. the first amplicon comprises a second A at a position complementary to the hT, the second amplicon comprises a second T at a position complementary to the second A, and the method further comprises: identifying said hmC based on said second A in said first amplicon, said second T in said second amplicon, or both said second A in said first amplicon and said second T in said second amplicon.

30. The method of claim 29 when dependent on claim 10, further comprising:

31. the first amplicon comprises a second G at a position complementary to the hmC, and the second amplicon comprises a second unprotected C at a position complementary to the second G, and the method further comprises:

30. The method of claim 29 dependent on claim 11, further comprising identifying the hmC based on the second G in the first amplicon, the second unprotected C in the second amplicon, or both the second G in the first amplicon and the second unprotected C in the second amplicon.

32. the first amplicon comprises a third G at a position complementary to the fC, the second amplicon comprises a third unprotected C at a position complementary to the third G, and the method further comprises:

30. The method of claim 29 dependent on claim 18, further comprising identifying the fC based on the third G in the first amplicon, the third unprotected C in the second amplicon, or both the third G in the first amplicon and the third unprotected C in the second amplicon.

33. the first amplicon comprises a third A at a position complementary to the U, the second amplicon comprises a third T at a position complementary to the third A, and the method further comprises:

30. The method of claim 29 dependent on claim 19, further comprising identifying the fC based on the third A in the first amplicon, the third T in the second amplicon, or both the third A in the first amplicon and the third T in the second amplicon.

34. the first amplicon comprises a fourth G at a position complementary to the caC, the second amplicon comprises a fourth unprotected C at a position complementary to the fourth G, and the method further comprises:

30. The method of claim 29 dependent on claim 22, further comprising identifying the caC based on the fourth G in the first amplicon, the fourth unprotected C in the second amplicon, or both the fourth G in the first amplicon and the fourth unprotected C in the second amplicon.

35. the first amplicon comprises a fourth A at a position complementary to the U, the second amplicon comprises a fourth T at a position complementary to the fourth A, and the method further comprises:

30. The method of claim 29 dependent on claim 23, further comprising identifying the caC based on the fourth A in the first amplicon, the fourth T in the second amplicon, or both the fourth A in the first amplicon and the fourth T in the second amplicon.

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