Methods and adapters

Abstract

The present invention relates to methods for characterizing, e.g., sequencing, at least a portion of a telomere and adapters for use in such methods.

Description

Technical Field

[0001] The present invention relates to methods for characterizing at least a portion of a telomere, such as sequencing it, and adapters for use in such methods. Background Art

[0002] Telomeres (i.e., regions of repetitive DNA sequences located at the ends of eukaryotic chromosomes) are difficult to characterize, such as by sequencing them. This is due to a number of reasons, including, but not limited to, that telomeres are highly repetitive and nonlinear, looping on themselves to form displacement loops (D-loops), and that telomere-binding proteins bind along the D-loops. Methods for sequencing telomeres are known, but these methods generally involve restriction digestion and / or PCR and Southern blot analysis (Lai, TP., Zhang, N., Noh, J. et al. Nat Commun 8, 1356 (2017); Bendix L, Horn PB, Jensen UB, Rubelj I, Kolvraa S. Aging Cell. 2010 Jun; 9(3): 383-97; and Bennett HW, Liu N, Hu Y, King MC. FEBS Lett. 2016 Dec; 590(23): 4159-4170). In these methods, the sequence of the telomere must be reconstructed from a shorter sequence, and these methods focus on characterizing short telomeres. There are problems with using PCR because polymerases cannot efficiently replicate long stretches of repetitive sequences such as those found in telomeres.

[0003] Biopores (and other nanopores) have great potential as direct electrical biosensors for polymers and various small molecules. Specifically, nanopores have recently been of interest as potential DNA sequencing technologies. When a potential is applied across a nanopore, the current changes while an analyte (such as a nucleotide) resides briefly in the barrel for a period of time. Nanopore detection of nucleotides gives current changes of known characteristics and duration. In chain sequencing methods, a single polynucleotide chain is passed through a hole and the identity of the nucleotide is derived. Chain sequencing can involve the use of molecular brakes to control the movement of polynucleotides through the hole.

[0004] Nanopores have been used to characterize telomeres by extending their 3' overhangs with polyA tails (Sholes SL, Karimian K, Gershman A, Kelly TJ, Timp W, Greider CW. Genome Res. 2022 Apr; 32(4):616-628. doi: 10.1101 / gr.275868.121.), but this method is not specific to telomeres, and many other 3' overhangs in genomic DNA are extended. The method also uses a polymerase to fill in the overhangs before additional labeling. Improved methods for characterizing telomeres are needed. Summary of the invention

[0005] The inventors have determined a specific method for characterizing at least a portion of a telomere, such as sequencing it. The method involves connecting a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of the telomere. As described in more detail below, the 3' end of the adapter specifically hybridizes with the first portion of the protruding strand, and therefore only the end of the telomere will be adapted. The telomere adapter can then be used to characterize the connected non-protruding strand in the 5' to 3' direction from the end of the telomere of at least a portion of the telomere. The telomere adapter is not connected to any other part of the chromosome, and therefore the method specifically characterizes at least a portion of the telomere. In essence, the method is an enrichment strategy for specifically characterizing at least a portion of the telomere. A portion or the entire telomere may be specifically characterized, and the method may also involve characterizing one or more of: a portion or the entire subtelomere, a portion or the entire chromatin (i.e., genomic DNA), a portion or the entire relative subtelomere, and a portion or the entire relative telomere of the relative telomere. The method may be used in combination with nanopore sequencing, but not necessarily. The method of the present invention has several advantages, including but not limited to specific and efficient characterization of at least a portion of the telomere, high resolution of sequence and length, the ability to go beyond the telomere and further characterize the chromosome, including the possibility of characterizing the entire chromosome, no restriction digestion and fragment size control is required, no PCR is required, the ability to identify methylated nucleotides or other modifications in the telomere, the ability to identify the presence or absence of telomere-bound proteins and characterize such proteins.

[0006] In a preferred embodiment, the method further comprises generating a double strand break at an opposite end of the at least a portion of the telomere relative to the telomere end, and characterizing the unligated protruding strands of the at least a portion of the telomere in opposite directions (i.e., toward the end of the telomere). This allows characterization of both strands of the at least a portion of the telomere and provides increased resolution, particularly in sequencing repetitive telomere sequences and identifying modifications such as methylation.

[0007] The present invention provides a method for characterizing at least a portion of a telomere, the method comprising:

[0008] (a) ligating a polynucleotide telomere adaptor to the 5' end of a non-overhanging strand located at the end of the telomere, wherein the 3' end of the adaptor specifically hybridizes to a first portion of the overhanging strand and the 5' end of the adaptor does not hybridize to an opposing portion of the overhanging strand; and

[0009] (b) characterizing the ligated non-protruding strand of the at least a portion of the telomere in the 5' to 3' direction from the end of the telomere using the telomere adaptor.

[0010] The present invention also provides a method for characterizing at least a portion of a telomere, the method comprising (a) connecting a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of the telomere, wherein the 3' end specifically hybridizes with a first portion of the protruding strand and the 5' end of the adapter does not hybridize with the opposite portion of the protruding strand, (b) using a polymer-guided effector protein to generate a double-strand break at the opposite end of at least a portion of the telomere relative to the telomere end and connecting a sequencing adapter to the opposite end, and (c) using the telomere adapter to characterize the connected non-protruding strand of at least a portion of the telomere in the 5' to 3' direction from the end of the telomere, and using the sequencing adapter to characterize the unconnected protruding strand of at least a portion of the telomere in the 5' to 3' direction toward the end of the telomere.

[0011] The present invention also provides:

[0012] - A polynucleotide telomere adaptor, wherein the 3' end of the adaptor specifically hybridizes to a first portion of a protruding strand located at the end of a telomere, and the 5' end of the adaptor does not hybridize to an opposite portion of the protruding strand located at the end of the telomere;

[0013] - A population of six telomere adaptors, each of which has a 3' end that specifically hybridizes to one of six possible sequences of a first portion of a protruding strand located at the end of a telomere and a 5' end that does not hybridize to an opposite portion of the protruding strand located at the end of the telomere.

[0014] - A kit for characterizing at least a portion of a telomere, the kit comprising (a) one or more polynucleotide telomere adaptors of the invention or a population of six telomere adaptors of the invention, and (b) one or more splint polynucleotides or one or more polynucleotide extensions; and

[0015] - A system comprising (a) one or more polynucleotide telomere adaptors of the invention or a population of six telomere adaptors of the invention, and (b) a nanopore. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] It should be understood that the drawings are only for the purpose of illustrating specific embodiments of the invention and are not intended to be limiting.

[0017] Figure 1 Shown are T1 to T6 telomeric adaptors as used in Example 1. Telomeric adaptors are attached to the C-rich strand located at the chromosome end.

[0018] Figure 2 Shown is the bonding of a telomere splint according to Example 1. T1 = telomere adaptor; S1 = telomere splint variant; S2 = telomere splint variant.

[0019] Figure 3 Ligation of nanopore sequencing adapters (Oxford Nanopore Technologies) is shown. The sequencing adapter is ligated to the telomeric adapter T1. The bonded splint S1 facilitates the ligation reaction.

[0020] Figure 4 The ligation of exemplary "click" chemistry telomere adapters is shown. T1 to T6 represent different adapters. The "click" chemistry telomere adapters ligate to the C-rich strand located at the chromosome end.

[0021] Figure 5 Shown is the addition of "click" nanopore sequencing adaptors (Oxford Nanopore Technologies Ltd.) using "click" chemistry.

[0022] Figure 6 Binding of a biotinylated telomeric adaptor is shown. The biotinylated telomeric adaptor is attached to the C-rich strand located at the chromosome end.

[0023] Figure 7Ligation of nanopore sequencing adaptors (Oxford Nanopore Technologies Ltd.) is shown. Sequencing adaptors are added after dA tailing at both ends of double-stranded DNA molecules.

[0024] Figure 8 The mappable telomere read counts per chromosome for a given method are shown. 1x Teloseq started the protocol as described in the examples with 5 μg DNA input, while 3x Teloseq started the protocol with 15 μg input. As expected, 3x Teloseq had 3 times the read counts of 1x Teloseq.

[0025] Fig. 9 The read coverage for each chromosome under a given condition is shown. The reads are filtered and assigned to chromosomes. When a read has multiple mapping alignments, a single alignment is selected by selecting the longest read coverage, highest identity, and overlapping telomere and subtelomeric regions. The coverage is normalized by dividing the total number of telomere reads for a given chromosome by the total number of telomere reads for a given method. The coverage between WGS and the "Teloseq" method of the present invention is relatively the same, while the restriction digest method shows a bias towards certain chromosomes.

[0026] Fig.10 The total number of telomere reads for each best run of each method identified using the noise-removing repeat finder is shown (dark blue). The number of telomere reads that mapped to the ends of the chromosomes is shown (light blue). If a read did not map to the end, it was discarded from the analysis. WGS was a control. The 1x Teloseq protocol started with 5 μg of DNA input, where the 3x Teloseq protocol started with 15 μg of input.

[0027] Fig.11 The percentage of mappable and unmappable telomeric reads is shown. Teloseq has about 10% unmappable telomeric reads. In contrast, BamHI has about 40% unmappable telomeric reads.

[0028] Fig.12 The percentage of methylated CpG sites in a single flow pool of 3x Teloseq is shown. The blue bar is the percentage of methylated CpG sites, and the red line shows the total CpG sites along the aligned region. For example, chromosome 3Q has over 800 CpG sites, yet less than 40% are methylated, while chromosome 11Q has 200 CpG sites, and over 80% are methylated.

[0029] Fig.13 An example of a methylated CpG (dark grey) site on chromosome 11 in a single run of 3x telomere is shown. Telomere reads were re-base called with Remora to call methylation.

[0030] Fig.14 The "Teloseq" method of the invention enabling identification of the greatest number of telomere sequences is shown. Each triplet of bars shows (reading from left to right): telomere reads found; telomere reads classified; anchored reads.

[0031] Fig.15 Telomere bioinformatics workflow and telomere enrichment with HG002 are shown.

[0032] Fig.16 Haploidized HG002 telomeric reads and read counts for chromosome 18.q are shown.

[0033] Fig.17 Telomere length by chromosome and haplotype is shown.

[0034] Description of Sequence Listing

[0035] SEQ ID NO: 1-6 show the sequences of the telomere adapters used in Example 1 (Table 3 and Figure 1 ).

[0036] SEQ ID NO: 7 shows the preferred 5' end of the telomeric adaptor of the invention. This is present in all SEQ ID NOs: 1-6 and 14-19.

[0037] SEQ ID NO:8 shows the preferred sequence of the splint polynucleotide and is the reverse complement of SEQ ID NO:7.

[0038] SEQ ID NOs: 9-10 show the splint polynucleotides used in the Examples (Table 5).

[0039] SEQ ID NO: 11 shows the polynucleotide extension used in the Examples (Table 7).

[0040] SEQ ID NOs: 12-13 show the sequences of the biotinylated telomeric adaptors used in the Examples (Table 9).

[0041] SEQ ID NOs: 14-19 show the sequences of the telomeric adaptors used in Example 10 (Table 14).

[0042] SEQ ID NO: 20 shows the splint polynucleotide used in Example 10 (Table 15).

[0043] SEQ ID NO: 21 shows Figure 1-6 The sequence of the top (protruding) strand of an exemplary chromosome end in .

[0044] SEQ ID NO:22 shows Figure 1-6 The sequence of the bottom (non-protruding) strand of an exemplary chromosome end in (in the 5' to 3' direction).

[0045] SEQ ID NO: 23 shows Figure 1 The sequence formed by ligating the T1 telomeric adaptor to the non-protruding strand (in the 5' to 3' direction).

[0046] SEQ ID NO: 24 shows Figure 3 The sequence formed by ligating the sequencing adapter to the telomeric adapter (in the 5' to 3' direction) in the sequence (in the 5' to 3' direction). This sequence includes SEQ ID NO:23.

[0047] SEQ ID 25-30 shows Figure 4 The sequence of the extended telomeric adaptor in (in the 5' to 3' direction).

[0048] SEQ ID NO: 31 shows Figure 4 The sequence formed by ligating the extended T1 telomeric adaptor to the non-protruding strand (in the 5' to 3' direction).

[0049] SEQ ID NO:32 shows Figure 6 The sequence of the top chain.

[0050] SEQ ID NO: 33 shows Figure 6 The sequence of the bottom strand (in the 5' to 3' direction). DETAILED DESCRIPTION

[0051] It should be understood that different applications of the disclosed products and methods can be customized according to specific needs in the art.It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting.

[0052] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0053] definition

[0054] When an indefinite or definite article is used when referring to a singular noun (e.g., "a / an", "the"), this includes the plural form of the noun unless specifically stated otherwise. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequence or a chronological order. It should be understood that the terms used in this way are interchangeable where appropriate, and that the embodiments of the invention described herein can operate in other sequences than those described or illustrated herein. The following terms or definitions are provided only to assist in understanding the present invention. Unless specifically defined herein, all terms used herein have the same meaning as would be understood by a person skilled in the art to which the present invention belongs. For definitions and terms in the art, practitioners are particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Suppl. 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed as having a scope less than that understood by a person of ordinary skill in the art.

[0055] As used herein, the term "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass deviations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such deviations are suitable for performing the disclosed methods.

[0056] As used herein, "nucleotide sequence", "DNA sequence" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. As used herein, the term "nucleic acid" is a single-stranded or double-stranded covalently linked nucleotide sequence, wherein the 3' and 5' ends on each nucleotide are connected by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized and manufactured in vitro, or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as DNA or RNA that has been methylated, or RNA that has been subjected to post-translational modifications, such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA) and peptide nucleic acids (PNA). The size of nucleic acids (also referred to herein as "polynucleotides") is usually expressed as the number of base pairs (bp) for double-stranded polynucleotides, or as the number of nucleotides (nt) in the case of single-stranded polynucleotides. One thousand bp or nt is equal to a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are usually referred to as "oligonucleotides" and may include primers for use in manipulating DNA, such as by polymerase chain reaction (PCR).

[0057] In the context of the present disclosure, the term "amino acid" is used in its broadest sense and is intended to include amino acids containing an amine (NH 2 ) and carboxyl (COOH) functional groups and a side chain (e.g., R group) specific to each amino acid. Amino acids generally refer to naturally occurring Lα-amino acids or residues. Commonly used single-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A = Ala; C = Cys; D = Asp; E = Glu; F = Phe; G = Gly; H = His; I = Ile; K = Lys; L = Leu; M = Met; N = Asn; P = Pro; Q =

[0058] Gln; R = Arg; S = Ser; T = Thr; V = Val; W = Trp; and Y = Tyr (Lehninger, AL, (1975) Biochemistry, 2nd edition, pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids that are not normally incorporated into proteins (such as norleucine), and chemically synthesized compounds with properties known in the art as characteristic of amino acids (such as β-amino acids). For example, analogs or mimetics of phenylalanine or proline that allow conformational restrictions of peptide compounds identical to natural Phe or Pro are included in the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5, p. 341, Academic Press, Inc., NY, 1983, which is incorporated herein by reference.

[0059] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, and to naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. Peptides may be prepared using recombinant techniques, such as by expressing recombinant or synthetic polynucleotides. Recombinantly produced peptides are typically substantially free of culture medium, e.g., culture medium accounts for less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

[0060] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. A protein may consist of a single polypeptide or may comprise a plurality of polypeptides assembled to form a multimer. A multimer may be a homo-oligomer or a hetero-oligomer. A protein may be a naturally occurring or wild-type protein or a modified or non-naturally occurring protein. A protein may differ from a wild-type protein, for example, by the addition, substitution or deletion of one or more amino acids.

[0061] The "variant" of protein covers peptides, oligopeptides, polypeptides, proteins and enzymes, which have amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type protein in question, and have biological and functional activities similar to the unmodified protein derived therefrom. As used herein, the term "amino acid identity" refers to the degree to which a sequence is identical on a comparison window on the basis of amino acids to amino acids. Therefore, "sequence identity percentage" is calculated by: comparing two sequences that are optimally aligned on a comparison window, determining the number of positions where identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce sequence identity percentage.

[0062] For all aspects and embodiments of the invention, a "variant" has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity with the amino acid sequence of the corresponding wild-type protein. The sequence identity can also be a fragment or portion of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a specific region, domain or subunit may share 80%, 90% or up to 99% sequence identity with the reference sequence.

[0063] The term "wild type" refers to a gene or gene product that is separated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the term "modified", "mutant" or "variant" refers to a gene or gene product that displays sequence modification (e.g., substitution, truncation or insertion), post-translational modification and / or functional properties (e.g., altered properties) compared to a wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties when compared to a wild-type gene or gene product. Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced by arginine (R) by replacing the codon (ATG) for methionine at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art. For example, non-natural amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system for expressing mutant monomers. Alternatively, it can be introduced by expressing mutant monomers in Escherichia coli (E. coli), and the mutant monomers are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-natural) analogs of those specific amino acids. If the mutant monomer is produced using partial peptide synthesis, it can also be produced by naked connection. Conservative substitution replaces amino acids with other amino acids with similar chemical structures, similar chemical properties or similar side chain volumes. The introduced amino acid can have polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge similar to the amino acid replaced by it. Alternatively, conservative substitution can introduce another aromatic or aliphatic amino acid to replace pre-existing aromatic or aliphatic amino acids. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 main amino acids as defined in Table 1 below. In the case where amino acids have similar polarity, the hydrophilicity scale of the amino acid side chain in Table 2 can also be referenced to determine this.

[0064] Table 1 - Chemical properties of amino acids

[0065]

[0066]

[0067] Table 2 - Hydrophilicity Scale

[0068]

[0069] Mutants or modified proteins, monomers or peptides can also be chemically modified in any manner and at any site. Preferably, the mutants or modified monomers or peptides are chemically modified by connecting the molecule to one or more cysteines (cysteine ​​connection), connecting the molecule to one or more lysines, connecting the molecule to one or more non-natural amino acids, enzymatically modifying the epitope, or modifying the end. Suitable methods for performing such modifications are well known in the art. The mutants of the modified proteins, monomers or peptides can be chemically modified by the connection of any molecule. For example, the mutants of the modified proteins, monomers or peptides can be chemically modified by the connection of a dye or a fluorophore.

[0070] Method of the present invention

[0071] The present invention provides a method for characterizing at least a portion of a telomere. A telomere is a region of a repetitive DNA sequence located at the end of a eukaryotic chromosome. Each chromosome has a telomere at each end. Telomeres protect the terminal region of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing the DNA repair system from mistaking the end of the DNA chain for a double-strand break. Chromosomes are structures formed by long DNA molecules containing all or part of the genetic material in eukaryotic cells. The segments of DNA known as subtelomeres usually separate telomeres from chromatin (i.e., genomic DNA) in chromosomes. The structure of a chromosome is typically telomere-subtelomere-chromatin-subtelomere-telomere.

[0072] The terms "part" and "portion" in at least a portion of a telomere are interchangeable. A portion may be any number of telomeres. At least a portion of a telomere is preferably at least about 5%, such as at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% of the telomere. The method is preferably used to characterize all of the telomere / the whole telomere.

[0073] The method is preferably used to characterize the entire telomere and the other part of the chromosome. The other part of the chromosome can be any other amount of the chromosome. The other part can be any one of the % of the rest of the chromosome discussed above about at least a portion of the telomere.

[0074] The method is preferably used to characterize (i) the entire telomere, (ii) the entire telomere and at least a portion of a subtelomere or the entire subtelomere, (iii) the entire telomere, the entire subtelomere and at least a portion of the chromatin or the entire chromatin, (iv) the entire telomere, the entire subtelomere, the entire chromatin and at least a portion of the opposite subtelomere or the entire opposite subtelomere, or (v) the entire telomere, the entire subtelomere, the entire chromatin, the entire opposite subtelomere and at least a portion of the opposite telomere or the entire opposite telomere. Chromatin is interchangeable with genomic DNA.

[0075] The method is preferably used to characterize (i), i.e. the entire telomere. The method is preferably used to characterize (ii). The method is preferably used to characterize the entire telomere and the entire subtelomere. The method is preferably used to characterize (iii). The method is preferably used to characterize the entire telomere, the entire subtelomere and the entire chromatin. The method is preferably used to characterize (iv). The method is preferably used to characterize the entire telomere, the entire subtelomere, the entire chromatin and the entire relative subtelomere. The method is preferably used to characterize (v). The method is preferably used to characterize the entire telomere, the entire subtelomere, the entire chromatin, the entire relative subtelomere and the entire relative telomere.

[0076] In any of (ii)-(v), the at least a portion of the subtelomere / chromatin / relative subtelomere / relative telomere may be any of the % discussed above with respect to at least a portion of the telomere.

[0077] Described method is preferably used to characterize whole chromosome (all of a chromosome / a whole chromosome).Method of the present invention preferably repeats at the other end of chromosome, and described method comprises two chains that characterize whole chromosome.Method of the present invention is preferably included in and carries out method of the present invention at the two ends of chromosome, and comprises two chains that characterize whole chromosome.Any method in method of the present invention can be used at each end of chromosome.The method used at each end can be identical or different.Described method is preferably identical.

[0078] The method preferably comprises characterizing in step (b) the following ligated non-protruding strands in the 5' to 3' direction from the end of the telomere: (i) a telomere; (ii) at least a portion of a subtelomere or the entire subtelomere; (iii) at least a portion of chromatin or the entire chromatin; (iv) at least a portion of an opposite subtelomere or the entire opposite subtelomere; or (v) at least a portion of an opposite telomere or the entire opposite telomere. Any of the embodiments discussed above for (i)-(v) are equally applicable to step (b) of the method.

[0079] At least a portion of a telomere, including any of (i)-(v) discussed above, can be of any length. For example, at least a portion of a telomere can be at least about 10, at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 nucleotides or nucleotide pairs in length. At least a portion of a telomere can be about 1000 or more nucleotides or nucleotide pairs in length, about 5000 or more nucleotides or nucleotide pairs in length, about 100000 or more nucleotides or nucleotide pairs in length, about 500,000 or more nucleotides or nucleotide pairs in length, about 1,000,000 or more nucleotides or nucleotide pairs in length, about 10,000,000 or more nucleotides or nucleotide pairs in length, about 100,000,000 or more nucleotides or nucleotide pairs in length, or about 200,000,000 or more nucleotides or nucleotide pairs in length, or the entire length of a chromosome.

[0080] The method is preferably used to characterize at least a portion of one or two telomeres on each chromosome in two or more different chromosomes. This allows to compare the characteristics of the telomeres on two or more different chromosomes. Two or more different chromosomes may come from identical cells, tissues, organisms or classification levels (such as genus or species), or may come from different cells, tissues or organisms or classification levels (such as genus or species). Cells, tissues, organisms or classification levels are usually eukaryotic. The method can be used to characterize at least a portion of one or two telomeres on each chromosome in two or more different chromosomes of any number, such as about 3 or more, about 4 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more or about 40 or more different chromosomes. The preferred quantity of different chromosomes includes but is not limited to 2, 4, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 22, 24, 26, 28, 30, 31, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80 and 82. In polyploid cells, tissues, organisms or classification levels, the quantity of different chromosomes may even be higher. The method is preferably used to characterize at least a portion of one or two telomeres on each chromosome in 46 different chromosomes. In these embodiments, the method comprises (a) ligating a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of a telomere, wherein the 3' end of the adapter specifically hybridizes to a first portion of the protruding strand and the 5' end of the adapter does not hybridize to an opposing portion of the protruding strand, and (b) using each telomere adapter to characterize the ligated non-protruding strand of at least a portion of each telomere in the 5' to 3' direction from the end of each telomere.

[0081] The method preferably comprises (a) sequencing at least a portion of the telomere, (b) measuring the length of at least a portion of the telomere, (c) performing telomere-to-telomere assembly on a chromosome or genome, (d) identifying telomere or chromosome fusions, (e) identifying one or more modifications in at least a portion of the telomere, (f) identifying at least a portion of the telomere as a variant, or (g) linking at least a portion of the telomere to a specific cell or tissue type. The method may comprise any number of (a)-(g) and combinations thereof.

[0082] The method preferably comprises (a). Methods for sequencing the ligated non-overhanging strand are discussed in more detail below.

[0083] The method preferably comprises (b).The lack of digestion in the method of the invention means that the length of at least a portion of a telomere or the entire telomere can be easily measured in one step.

[0084] The method preferably comprises (c). As described above, the method allows characterization of the entire chromosome from telomere to telomere. Also as discussed above, the method can be applied to two or more different chromosomes, which means that the entire genome can be characterized.

[0085] The method preferably comprises (d).The method may be used to identify telomere fusions by characterising at least a portion of a telomere.

[0086] The method preferably comprises (e). The one or more modifications preferably comprise one or more of the following: (i) methylation of one or more nucleotides; (ii) oxidation of one or more nucleotides; and (iii) damage to one or more nucleotides. The method may identify (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii). For example, at least a portion of a telomere may comprise one or more pyrimidine dimers. Such dimers are often associated with damage caused by ultraviolet light and are a major cause of cutaneous melanoma. Nanopore sequencing is capable of identifying methylated, oxidized and damaged nucleotides.

[0087] The method preferably comprises (f). The method may identify at least a portion of a telomere as a variant comprising one or more of: (i) a telomere deletion; (ii) a telomere addition; and / or (ii) a telomere substitution. The method may identify at least a portion of a telomere as a variant comprising (i), (ii), (iii), (i) and (ii), (ii) and (iii), or (i), (ii) and (iii). (i) may comprise the deletion of any number of nucleotides from the telomere. (i) may comprise a single deletion or multiple deletions. The deletion may be located in one or both strands. (ii) may comprise the addition of any number of nucleotides to the telomere. (ii) may comprise a single addition or multiple additions. The addition may be located in one or both strands. (iii) may comprise the substitution of any number of nucleotides within the telomere. The substitution may be located in one or both strands.

[0088] The method preferably comprises (g). If the method is performed in a specific cell, tissue, organism or classification level (such as genus or species), at least a portion of the telomere can be connected to the cell, tissue, organism or classification level (such as genus and species). As discussed above, the method of the present invention can be performed on different chromosomes from different cells, tissues or classification levels, and therefore at least a portion of each telomere can be connected to different cells, tissues or classification levels. The cell, tissue, organism or classification level is typically eukaryotic.

[0089] Telomere adapter

[0090] The method of the present invention comprises connecting a polynucleotide telomere adapter to the 5' end of the non-protruding strand at the end of the telomere in step (a). Methods for connecting polynucleotides are known in the art. The telomere adapter is formed by at least one polynucleotide, as discussed below. The polynucleotide telomere adapter is interchangeable with the telomere adapter.

[0091] Telomeres typically have 3' overhangs (see Figure 1 ). This means that the strand in the 5' to 3' direction usually protrudes at the end of the telomere. The strand in the 3' to 5' direction at the end of the telomere is usually non-protruding. "Non-protruding" and "not protruding" are interchangeable. The two strands at the end of the telomere are usually DNA.

[0092] Telomere adapters can be any type of polynucleotide. Polynucleotides (such as nucleic acids) are macromolecules comprising two or more nucleotides. Polynucleotides can be single-stranded or double-stranded. Double-stranded polynucleotides are made by hybridizing two single-stranded polynucleotides together. Polynucleotide telomere adapters can be single-stranded polynucleotides or double-stranded polynucleotides.

[0093] Polynucleotides can comprise any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. Nucleotides generally contain a nucleobase, a sugar and at least one phosphate group. The nucleobase and the sugar form a nucleoside. The nucleobase is generally heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).

[0094] The sugar is typically a pentose. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

[0095] Nucleotide is usually ribonucleotide or deoxyribonucleotide. Nucleotide usually contains monophosphate, diphosphate or triphosphate. Nucleotide can contain more than three phosphates, such as 4 or 5 phosphates. Phosphoric acid can be connected to the 5' or 3' side of nucleotide. Nucleotide includes but is not limited to adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate. Nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.

[0096] A nucleotide may be abasic (ie, lack a nucleobase). A nucleotide may also lack a nucleobase and a sugar (ie, be a C3 spacer).

[0097] The nucleotides in a polynucleotide can be linked to each other in any manner. The nucleotides are usually linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked by their nucleobases, as in pyrimidine dimers.

[0098] Polynucleotides can be nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotides can include an RNA chain hybridized with a DNA chain. Polynucleotides can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA) or other synthetic polymers with nucleotide side chains. The PNA backbone is composed of repeated N-(2-aminoethyl)-glycine units connected by peptide bonds. The GNA backbone is composed of repeated diol units connected by phosphodiester bonds. The TNA backbone is composed of repeated threose linked together by phosphodiester bonds. LNA is formed by ribonucleotides with additional bridges connecting 2' oxygen and 4' carbon in the ribose moiety as discussed above.

[0099] The polynucleotide is preferably DNA, RNA or a DNA or RNA hybrid, most preferably DNA. A DNA / RNA hybrid may comprise DNA and RNA on the same strand. Preferably, the DNA / RNA hybrid comprises a DNA strand hybridized to an RNA strand.

[0100] The backbone of a polynucleotide can be altered to reduce the likelihood of chain breaks. For example, DNA is known to be more stable than RNA under many conditions. The backbone of a polynucleotide chain can be modified to avoid damage caused by, for example, harsh chemicals such as free radicals. DNA or RNA containing non-natural or modified bases can be produced by amplifying natural DNA or RNA polynucleotides using appropriate polymerases in the presence of modified NTPs.

[0101] The telomere adapter can be of any length. For example, the telomere adapter can be at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, or at least about 50 nucleotides or nucleotide pairs in length.

[0102] Telomere adapters are typically single-stranded oligonucleotides. Oligonucleotides are short nucleotide polymers that typically have about 50 or fewer nucleotides, such as about 45 or fewer, about 42 or fewer, about 41 or fewer, about 40 or fewer, about 35 or fewer or about 30 or fewer nucleotides. The length of the telomere adapter is preferably about 15 to about 50 nucleotides, such as a length of about 20 to about 45 nucleotides. For example, the length of the oligonucleotide can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides or about 45 nucleotides. The length of the polynucleotide telomere adapter is most preferably about 30 nucleotides or about 41 nucleotides. The different regions of the telomeric adaptor are discussed in more detail below.

[0103] Telomere adapters are generally synthetic or semi-synthetic. For example, DNA or RNA can be pure synthesis, synthesized by conventional DNA synthesis methods such as phosphoramidite-based chemical methods. Synthetic polynucleotide subunits can be joined together by known means, such as connection or chemical bonds, to produce longer chains. Internal self-forming structures (e.g., hairpins, quadruplexes) can be designed into substrates, for example, by connecting appropriate sequences. Synthetic polynucleotides can be replicated and amplified for production by means known in the art (including PCR, incorporation into bacterial factories, etc.).

[0104] The polynucleotide telomeric adapter has a 5' to 3' directionality. The 3' end of the adapter specifically hybridizes with the first portion of the protruding strand. The 3' end can be any part / portion of the polynucleotide telomeric adapter, such as at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. The 3' end of the adapter can be any length, such as at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 10 nucleotides, or at least about 20 nucleotides in length.

[0105] The 3' end of the adapter is preferably about 18 or less nucleotides, such as about 16 or less, about 15 or less, about 14 or less, about 10 or less, about 8 or less, or about 7 or less nucleotides. The length of the 3' end is preferably about 4 to about 18 nucleotides, such as about 5 to about 16, about 6 to about 15, or about 7 to about 10 nucleotides in length. For example, the length of the 3' end can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 nucleotides. The length of the 3' end of the polynucleotide telomere adapter is most preferably about 7 nucleotides or about 18 nucleotides in length.

[0106] The 3' end of the telomere adaptor specifically hybridizes to the first portion of the protruding strand. The first portion of the protruding strand can be any of the lengths discussed above for the 3' end. The first portion of the protruding strand is preferably the same length as the 3' end of the telomere adaptor.

[0107] The 3' end "specifically hybridizes" to the first portion of the protruding strand when it hybridizes preferentially or with high affinity to the first portion of the protruding strand, but does not substantially hybridize, does not hybridize, or hybridizes with only low affinity to other polynucleotide sequences, particularly telomeres or other sequences in chromosomes. Conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, (1995)). Hybridization can be performed under low stringency conditions, for example, in the presence of a buffer solution of 30% to 35% formamide, 1M NaCl and 1% sodium dodecyl sulfate (SDS) at 37° C., followed by washing 20 times in 1× (0.1650M Na+) to 2× (0.33M Na+) standard sodium citrate (SSC) at 50° C. Hybridization can be performed under moderate stringency conditions, for example, in the presence of a buffer solution of 40% to 45% formamide, 1M NaCl and 1% SDS at 37° C., followed by washing in 0.5× (0.0825M Na+) to 1× (0.1650M Na+) SSC at 55° C. Hybridization can be performed under high stringency conditions, for example, in the presence of a buffer solution of 50% formamide, 1M NaCl, 1% SDS at 37° C., followed by washing in 0.1× (0.0165M Na+) SSC at 60° C. The 3' end "specifically hybridizes" if it hybridizes to the first portion of the protruding strand at a Tm that is at least 2°C higher than its melting temperature (Tm) for other polynucleotide sequences, such as at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, or at least 10°C. More preferably, the 3' end hybridizes to the first portion of the protruding strand at a Tm that is at least 2°C higher than its Tm for other polynucleotide sequences, such as at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 20°C, at least 30°C, or at least 40°C. Preferably, the 3' end hybridizes to the first portion of the protruding strand at a Tm that is at least 2°C, such as at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 20°C, at least 30°C, or at least 40°C higher than its Tm for a polynucleotide that differs from said first portion of the protruding strand by one or more nucleotides, such as by 1, 2, 3, 4, or 5 or more nucleotides. The 3' end typically hybridizes to the first portion of the protruding strand at a Tm of at least 90°C, such as at least 92°C or at least 95°C.Tm can be measured experimentally using known techniques, including use of DNA microarrays, or can be calculated using a publicly available Tm calculator, such as those available on the Internet.

[0108] The 3' end of the adapter typically comprises or consists of a sequence that is at least about 80% identical to the reverse complement of the sequence of the first portion of the protruding strand. The 3' end of the adapter preferably comprises or consists of a sequence that is at least about 85% identical or at least about 85.7% identical to the reverse complement of the sequence of the first portion of the protruding strand. The 3' end of the adapter more preferably comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 98% or at least about 99% identical to the reverse complement of the sequence of the first portion of the protruding strand. The 3' end of the adapter most preferably comprises or consists of a sequence that is the reverse complement of the sequence of the first portion of the protruding strand. Complementarity means that the 3' end of the adapter comprises or consists of a sequence that is 100% identical to the reverse complement of the sequence of the first portion of the protruding strand. Complementarity is typically determined using typical Watson-Crick base pairing. In a most preferred embodiment, the 3' end is single-stranded DNA and comprises or consists of a sequence that is the reverse complement of the sequence of the first portion of the protruding DNA strand located at the end of the telomere.

[0109] Human telomeres generally comprise multiple repetitions of the TTAGGG sequence. The 3' end of the telomere adapter preferably comprises at least one sequence that specifically hybridizes with one of the six possible sequences of the first part of the protruding chain or serves as its reverse complement. The six possible sequences in the 5' to 3' direction are generally TTAGGG, TAGGGT, AGGGTT, GGGTTA, GGTTAG and GTTAGG. The 3' end of the telomere adapter preferably comprises CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC. The 3' end of the telomere adapter preferably comprises two or more continuous repetitions of CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC. The 3' end of the telomere adapter may comprise any number of CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC, such as 3 or more, 4 or more or 5 or more continuous repetitions. The 3' end preferably comprises or consists of three consecutive repeats of CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC.

[0110] The method may include using more than one polynucleotide telomere adapter. Any number of telomere adapters may be used, such as about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, or about 10 or more. The method preferably includes contacting the telomere with a population of six telomere adapters before step (a), each of the six telomere adapters having a reverse complement specific hybridization with one of the six possible sequences of the first portion of the protruding strand or comprising its 3' end and a 5' end that is not hybridized with the relative portion of the protruding strand. The population includes sequences that specifically hybridize with all six possible sequences or serve as their reverse complement. The six possible sequences in the 5' to 3' direction are typically TTAGGG, TAGGGT, AGGGTT, GGGTTA, GGTTAG, and GTTAGG. The colonies of the six telomere adapters preferably each have a 3' end, and the end specifically hybridizes with one of the six possible sequences of the first part of the protruding chain (that is, one of CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC and CCTAAC) or as its reverse complement. The colony generally comprises an adapter, and the adapter comprises a sequence that specifically hybridizes with all six possible sequences or all six possible reverse complement sequences. The 3' end of each adapter can have two or more of one of its six possible reverse complement sequences (that is, CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC), such as 3 or more, 4 or more or 5 or more continuous repetitions. The 3' end of each adapter preferably has three continuous repetitions of CCCTAA, ACCCTA, AACCCT, TAACCC, CTAACC or CCTAAC. All adapters in the colony can have multiple continuous repetitions of one of its six possible reverse complement sequences and preferably have the same number of repetitions.

[0111] The population preferably comprises six telomeric adaptors, wherein the 3' end of one adaptor comprises or consists of CCCTAA, the 3' end of one adaptor comprises or consists of ACCCTA, the 3' end of one adaptor comprises or consists of AACCCT, the 3' end of one adaptor comprises or consists of TAACCC, the 3' end of one adaptor comprises or consists of CTAACC, and the 3' end of one adaptor comprises or consists of CCTAAC. The population preferably comprises six telomeric adaptors, wherein the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of CCCTAA, the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of ACCCTA, the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of AACCCT, the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of TAACCC, the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of CTAACC, and the 3' end of one adaptor comprises or consists of two or more (such as three) consecutive repeats of CCTAAC. The population preferably comprises six telomeric adaptors, wherein one adaptor comprises or consists of ACCCTAA at its 3' end, one adaptor comprises or consists of AACCCTA at its 3' end, one adaptor comprises or consists of TAACCCT at its 3' end, one adaptor comprises or consists of CTAACCC at its 3' end, one adaptor comprises or consists of CCTAACC at its 3' end, and one adaptor comprises or consists of CCCTAAC at its 3' end. All specific sequences are given in the 5' to 3' direction.

[0112] The 5' end of the polynucleotide telomeric adapter does / does not hybridize with the opposite portion of the protruding strand. This means that the 5' end of the adapter does / does not form a duplex with the opposite portion of the protruding strand and can be freely characterized or modified, as discussed in more detail below. The lack of hybridization can be measured as discussed above. The 5' end can be any portion of the polynucleotide telomeric adapter, such as at least about 50%, at least about 60%, at least 70%, at least about 80%, or at least about 85%. The 5' end of the adapter can be any length, such as at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or at least about 30 nucleotides in length.

[0113] The 5' end of the adapter is preferably 60 or less nucleotides, such as about 50 or less, about 40 or less, about 30 or less, or about 25 or less nucleotides. The length of the 5' end is preferably about 15 to about 35 nucleotides, such as about 18 to about 30 or about 20 to 25 nucleotides in length. For example, the length of the 5' end can be about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, or about 28 nucleotides. The length of the 5' end of the polynucleotide telomere adapter is most preferably about 23 nucleotides.

[0114] A skilled artisan is able to design a 5' end that is sufficiently mismatched with the opposing portion of the protruding strand so that it will not hybridize. The 5' end of the adaptor typically comprises or consists of a sequence that is less than about 20% identical to the reverse complement of the sequence of the opposing portion of the protruding strand. The 5' end of the adaptor preferably comprises or consists of a sequence that is less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% identical to the reverse complement of the sequence of the opposing portion of the protruding strand. The 5' end of the adaptor most preferably comprises or consists of a sequence that is 0% identical to (i.e., not complementary to) the reverse complement of the sequence of the opposing portion of the protruding strand. Complementarity, or lack thereof, is typically determined using typical Watson-Crick base pairing.

[0115] For human telomeres, the 5' end does not contain a repeat of CCCTAA. The preferred 5' end contains or consists of AGCAATACGTAACTGAACGAAGT (SEQ ID NO: 7). This is in the 5' to 3' direction. In embodiments using more than one polynucleotide telomere adapter, the adapters may have the same 5' end or different 5' ends. The nucleotides at the 5' end of the telomere adapter are preferably modified, for example, with a phosphate group.

[0116] The telomere adapter preferably comprises or consists of a sequence as shown in any one of SEQ ID NOs: 1-6. These sequences are shown in Tables 3 and Figure 1 middle.

[0117] The method preferably comprises, prior to step (a), contacting the telomeres with a population of six telomere adaptors, the population of six telomere adaptors comprising or consisting of the following sequences:

[0118] AGCAATACGTAACTGAACGAAGTACCCTAA(SEQ ID NO:1);

[0119] AGCAATACGTAACTGAACGAAGTAACCCTA(SEQ ID NO:2);

[0120] AGCAATACGTAACTGAACGAAGTTAACCCT(SEQ ID NO:3);

[0121] AGCAATACGTAACTGAACGAAGTCTAACCC(SEQ ID NO:4);

[0122] AGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 5); and

[0123] AGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 6).

[0124] These are shown in Table 3 and Table 3 of Example 1. Figure 1 middle.

[0125] The telomeric adaptor preferably comprises or consists of a sequence as shown in any one of SEQ ID NOs: 14-19. These sequences are shown below and in Table 14.

[0126] The method preferably comprises, prior to step (a), contacting the telomeres with a population of six telomere adaptors, the population of six telomere adaptors comprising or consisting of the following sequences:

[0127] AGCAATACGTAACTGAACGAAGTCCCTAACCCTAACCCTAA (SEQ ID NO: 14);

[0128] AGCAATACGTAACTGAACGAAGTTAACCCTAACCCTAACCC (SEQ ID NO: 15);

[0129] AGCAATACGTAACTGAACGAAGTCTAACCCTAACCCTAACC (SEQ ID NO: 16);

[0130] AGCAATACGTAACTGAACGAAGTCCTAACCCTAACCCTAAC (SEQ ID NO: 17);

[0131] AGCAATACGTAACTGAACGAAGTAACCCTAACCCTAACCCT (SEQ ID NO: 18); and

[0132] AGCAATACGTAACTGAACGAAGTACCCTAACCCTAACCCTA (SEQ ID NO: 19).

[0133] These are shown in Table 14.

[0134] Any of the telomere adaptors used in the present invention may comprise a click chemistry group for facilitating covalent attachment, as discussed below.

[0135] splint polynucleotide

[0136] Step (a) preferably further comprises hybridizing the splint polynucleotide to the 5' end of the telomere adaptor. This is possible because the 5' end of the telomere adaptor does not hybridize to the protruding strand at the end of the telomere. The splint polynucleotide can be any of the polynucleotides discussed above for the telomere adaptor. The splint polynucleotide can be any of the lengths discussed above for the 5' of the telomere adaptor. The length of the splint polynucleotide can be the same as the 5' end, or it can be a different length. The entire splint polynucleotide or a portion thereof can hybridize to the entire 5' end or a portion thereof.

[0137] The splint polynucleotide typically specifically hybridizes with the 5' end of the telomeric adapter. Specific hybridization is discussed above. The splint polynucleotide typically comprises or consists of a sequence that is at least 80% identical to the reverse complement of a portion of the sequence at the 5' end of the telomeric adapter. The splint polynucleotide preferably comprises or consists of a sequence that is at least about 85% identical, at least about 90%, at least about 95%, at least about 98% or at least about 99% identical to the reverse complement of a portion of the 5' end of the telomeric adapter. The splint polynucleotide preferably comprises or consists of a sequence that is complementary to the reverse complement of a portion of the 5' end of the telomeric adapter. Complementarity means that the splint polynucleotide comprises or consists of a sequence that is 100% identical to the reverse complement of a portion of the 5' end of the telomeric adapter. Complementarity is typically determined using typical Watson-Crick base pairing. The portion of the 5' end can be of any length, such as a length of at least about 5, at least about 10, at least about 15 or at least about 20 nucleotides.

[0138] The splint polynucleotide preferably comprises or consists of a sequence that specifically hybridizes to part or all of AGCAATACGTAACTGAACGAAGT (SEQ ID NO:7). A preferred splint polynucleotide comprises or consists of ACTTCGTTCAGTTACGTATTGCT (SEQ ID NO:8). This is in the 5' to 3' direction. SEQ ID NO:8 is the reverse complement of SEQ ID NO:7.

[0139] The splint polynucleotide is preferably compatible with the sequencing adapter. The splint polynucleotide is typically adaptable so that when hybridized to the 5' end of the telomeric adapter, it forms a 3' overhang. An example of this is shown in Figure 2The overhang can be any length, such as at least about one nucleotide, at least about two nucleotides, at least about three nucleotides, at least about four nucleotides, at least about five nucleotides, or at least about six nucleotides in length. The 3' overhang preferably specifically hybridizes with a portion of a sequencing adapter, such as an overhang on a sequencing adapter. Specific hybridization is discussed above. The 3' overhang is preferably complementary to a portion of a sequencing adapter, such as an overhang on a sequencing adapter. Suitable sequencing adapters are discussed in more detail below.

[0140] The splint polynucleotide preferably comprises or consists of ACTTCGTTCAGTTACGTATTGCTAGCAAT (SEQ ID NO: 9) or ACTTCGTTCAGTTACGTATTGCTA (SEQ ID NO: 10). These are shown in Table 5 and specifically hybridize to the 5' end having the sequence shown in SEQ ID NO: 7. The overhang on the sequencing adaptor preferably comprises ATTGCT.

[0141] The splint polynucleotide preferably comprises or consists of ACTTCGTTCGGTGACTTGAGGACAGCAAT (SEQ ID NO: 20). This is shown in Table 15. The overhang on the sequencing adaptor preferably comprises ATTGCT.

[0142] Sequencing adapters

[0143] Step (a) preferably further comprises connecting a sequencing adapter to the telomere adapter and, if present, to the splint polynucleotide, and step (b) preferably comprises characterizing the connected non-protruding strand in the 5' to 3' direction of the at least a portion of the telomere using the sequencing adapter. Step (a) preferably further comprises connecting a sequencing adapter to the telomere adapter, and step (b) preferably comprises characterizing the connected non-protruding strand in the 5' to 3' direction of the at least a portion of the telomere using the sequencing adapter. Step (a) preferably further comprises connecting a sequencing adapter to the telomere adapter and the splint polynucleotide, and step (b) preferably comprises characterizing the connected non-protruding strand in the 5' to 3' direction of the at least a portion of the telomere using the sequencing adapter. In all cases, the connection is preferably a covalent connection. The sequencing adapter may be connected or bonded to the telomere adapter and, if present, to the splint polynucleotide. The sequencing adapter is preferably attached to the telomeric adapter and, if present, the splint polynucleotide by ligation. The sequencing adapter may be attached to the telomeric adapter using click chemistry. Suitable click chemistry groups are discussed in more detail below.

[0144] More preferably, step (a) further comprises specifically hybridizing the 3' overhang formed by the splint polynucleotide hybridized to the telomeric adaptor to the overhang on the sequencing adaptor, and ligating (preferably covalently ligating) the sequencing adaptor to the telomeric adaptor and the splint polynucleotide. An example of this is shown in Figure 3 The sequencing adapter can be connected or ligated to the telomere adapter and the splint polynucleotide. The sequencing adapter is preferably attached to the telomere adapter and the splint polynucleotide by ligation.

[0145] Sequencing adapters typically comprise a polynucleotide chain that can be ligated to the end of a target polynucleotide. Target polynucleotides are typically intended for characterization according to the methods disclosed herein and include telomeric adapters, splint polynucleotides, and / or polynucleotide extensions.

[0146] Sequencing adapters can be added to both ends of the target polynucleotide. Alternatively, different adapters can be added to both ends of the target polynucleotide. An adapter can be added to only one end of the target polynucleotide. Methods for adding adapters to polynucleotides are known in the art. The adapter can be attached to the polynucleotide, for example, by ligation, by click chemistry, by labeling, by topoisomerization, or by any other suitable method.

[0147] The adapter may be synthetic or artificial. Typically, the adapter comprises a polymer as described herein. The adapter preferably comprises a polynucleotide. The adapter may comprise a single-stranded polynucleotide chain. The adapter may comprise a double-stranded polynucleotide. The sequencing adapter may comprise any of the polynucleotides discussed above for the telomere adapter, and include DNA, RNA, modified DNA (such as basic DNA), RNA, PNA, LNA, BNA and / or PEG. Typically, the adapter comprises a single-stranded and / or double-stranded DNA or RNA.

[0148] The sequencing adapter can be a Y adapter. The Y adapter is usually double-stranded and (a) comprises a region where the two chains are hybridized together at one end and (b) comprises a region where the two chains are not complementary at the other end. The non-complementary portion of the chain forms an overhang. The hybridization stem of the adapter is usually connected to the 5' end of the first chain of the double-stranded polynucleotide and the 3' end of the second chain of the double-stranded polynucleotide; or connected to the 3' end of the first chain of the double-stranded polynucleotide and the 5' end of the second chain of the double-stranded polynucleotide. The presence of the non-complementary region in the Y adapter gives the adapter a Y shape because the two chains are usually not hybridized to each other, unlike the double-stranded portion. The hybridization stem end of the Y adapter can also include a short overhang, allowing it to specifically hybridize and connect with a telomere adapter, a splint polynucleotide or a polynucleotide extension.

[0149] Some methods of the methods of the present invention use polynucleotide binding proteins to control the movement of telomere chains relative to nanopores. Polynucleotide binding proteins can bind to the overhangs of adapters such as Y adapters. Polynucleotide binding proteins can bind to double-stranded regions. Polynucleotide binding proteins can bind to single-stranded and / or double-stranded regions of adapters. A first polynucleotide binding protein can bind to a single-stranded region of such an adapter, and a second polynucleotide binding protein can bind to a double-stranded region of an adapter.

[0150] The sequencing adapter preferably comprises a membrane anchor or a pore anchor. The anchor may be linked to a polynucleotide that is complementary to and thus hybridizes to the overhang, which is bound to the polynucleotide binding protein.

[0151] One of the non-complementary strands of a sequencing adaptor, such as a Y adaptor, may comprise a leader sequence that is capable of threading into the nanopore when in contact with the nanopore.

[0152] The leader sequence typically comprises a polymer, such as a polynucleotide, for example, DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader sequence preferably comprises a single strand of DNA, such as a poly dT segment. The leader sequence can be any length, but is typically 10 to 150 nucleotides in length, such as 20 to 120, 30 to 100, 40 to 80 or 50 to 70 nucleotides in length.

[0153] The sequencing adapter can be a hairpin loop adapter. A hairpin loop adapter is an adapter comprising a single polynucleotide chain, wherein the ends of the polynucleotide chains can hybridize to each other or be hybridized to each other, and wherein the middle segment of the polynucleotide forms a loop. Suitable hairpin loop adapters can be designed using methods known in the art. Typically, the 3' end of the hairpin loop adapter is connected to the 5' end of the first chain of the double-stranded polynucleotide, and the 5' end of the hairpin loop adapter is connected to the 3' end of the second chain of the double-stranded polynucleotide; or the 5' end of the hairpin loop adapter is connected to the 3' end of the first chain of the double-stranded polynucleotide, and the 3' end of the hairpin loop adapter is connected to the 5' end of the second chain of the double-stranded polynucleotide. As explained in more detail below, the sequencing adapter can be connected to the target polynucleotide to characterize the target polynucleotide.

[0154] It will also be understood by those skilled in the art that when the adapter comprises a polynucleotide chain, the sequence of the adapter is generally not decisive and can be controlled or selected according to any polynucleotide to be characterized, such as polynucleotide binding proteins and other experimental conditions.Exemplary sequences are provided in the example only by way of illustration.For example, the adapter may include sequences such as SEQ ID NO:21-26 or 28-33 in WO2021 / 255476 (incorporated herein by reference in their entirety) or one or more sequences such as SEQ ID NO:21-26 or 28-33 in WO 2021 / 255476 (incorporated herein by reference in their entirety) have at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence similarity or identity of the polynucleotide sequence.The sequence of the adapter can generally be changed without negatively affecting the effectiveness of the method of the present invention.

[0155] The sequencing adapter may comprise a loading site for loading a polynucleotide binding protein. The loading site may be, for example, a single-stranded region that can be targeted by a polynucleotide binding protein. The loading site may be a region of the sequencing adapter to which an exogenous polynucleotide chain comprising a polynucleotide binding protein may bind to transfer the polynucleotide binding protein to a polynucleotide to be evaluated in the method of the present invention.

[0156] If present, the polynucleotide binding protein may be provided on a sequencing adaptor. WO 2015 / 110813 and WO 2020 / 234612 describe loading a polynucleotide binding protein onto a target polynucleotide such as an adaptor and are hereby incorporated by reference in their entirety.

[0157] Spacer

[0158] The polynucleotides used in the present invention, such as telomere adapters, sequencing adapters, splint polynucleotides or polynucleotide extensions, can include one or more spacers, such as about one to about 10 spacers, such as about 1 to about 5 spacers, such as about 1, 2, 3, 4 or 5 spacers. The spacer can include any suitable number of spacer units. The spacer generally provides an energy barrier that hinders the movement of the polynucleotide binding protein. For example, the spacer can hinder the movement of the polynucleotide binding protein by reducing the traction of the protein, such as using a baseless spacer. The spacer can physically prevent the movement of the protein, such as by introducing a bulky chemical group to physically hinder the movement of the polynucleotide binding protein.

[0159] One or more spacers are typically included in a polynucleotide or in a sequencing adapter to provide a unique signal when they pass through or across a nanopore. One or more spacers can be used to define or separate one or more regions of a polynucleotide; for example, to separate an adapter from a target polynucleotide.

[0160] Insulators can include linear molecules, such as polymers, such as polypeptides or polyethylene glycol (PEG). Generally, such insulators have structures different from the target polynucleotides. For example, if the target polynucleotide is DNA, insulators or each insulator generally do not include DNA. Specifically, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), insulators or each insulator preferably include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or synthetic polymers with nucleotide side chains. The spacer may comprise one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuridine, one or more trans-thymidine (trans-dT), one or more trans-dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more isodeoxycytidine (iso-dC), one or more isodeoxyguanosine (iso-dG), one or more C3 (OC3) bases. 3 H 6 OPO 3 ) groups, one or more photodegradable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 )OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, or one or more spacers 18 (iSp18) [(OCH 2 CH 2 ) 6 OPO 3 ] group; or one or more thiol groups. The spacer may contain any combination of these groups. Many of these groups may be selected from (Integrated DNA ) are commercially available. For example, C3, iSp9 and iSp18 spacers can be obtained from The spacer may contain any number of the above-mentioned groups as spacer units.

[0161] The spacer can include one or more chemical groups, such as one or more chemical side groups. One or more chemical groups can be connected to one or more nucleobases in the sequencing adapter. One or more chemical groups can be connected to the main chain of the sequencing adapter. Any number of suitable chemical groups can be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxin and / or anti-digoxin and dibenzylcyclooctyne groups.

[0162] Insulator can comprise one or more abasic nucleotides (that is, lacking the nucleotide of nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides.In abasic nucleotide, nucleobase can be replaced by-H(idSp) or-OH.By removing nucleobase from one or more adjacent nucleotides, abasic insulator can be inserted into target polynucleotide.For example, polynucleotide can be modified to include 3-methyladenine, 7-methylguanine, 1, N6-vinylidene adenine inosine or hypoxanthine, and nucleobase can be removed from these nucleotides using human alkyladenine DNA glycosidase (hAAG).Alternately, polynucleotide can be modified to include uracil, and nucleobase is removed with uracil-DNA glycosidase (UDG).One or more insulators preferably do not include any abasic nucleotides.

[0163] Suitable spacers can be designed or selected depending on the nature of the polynucleotide or sequencing adaptor, the polynucleotide binding protein, and the conditions under which the method is performed.

[0164] Label

[0165] The polynucleotides used in the present invention, such as telomere adapters, sequencing adapters, splint polynucleotides or polynucleotide extensions, can contain tags or tethers. For example, a polynucleotide can be bound to a tag on a nanopore, for example, via its adapter, and released at certain points during characterization of the polynucleotide by the nanopore, for example. Strong non-covalent binding (e.g., biotin / avidin) is still reversible and can be used in some embodiments of the methods described herein.

[0166] The nanopore tag and sequencing adapter pair can be configured such that the binding strength or affinity of a binding site on the polynucleotide (e.g., a binding site provided by an anchor or leader sequence of the adapter or by a capture sequence within the duplex stem of the adapter) to the tag on the nanopore is sufficient to maintain the connection between the nanopore and the polynucleotide until an applied force is placed thereon to release the bound polynucleotide from the nanopore.

[0167] The tag or tether is preferably uncharged. This ensures that the tag or tether is not pulled into the nanopore under the influence of a potential difference.

[0168] One or more molecules that attract or bind polynucleotides or adapters can be connected to a detector (e.g., a hole). Any molecule that hybridizes to an adapter and / or a target polynucleotide can be used. The molecule connected to the hole can be selected from a PNA tag, a PEG linker, a short oligonucleotide, a positively charged amino acid, and an aptamer. The holes that connect such molecules to them are known in the art. For example, the holes that connect short oligonucleotides to them are disclosed in Howarka et al. (2001) Nature Biotech. 19: 636-639 and WO 2010 / 086620, and the holes containing PEG connected to the lumen of the hole are disclosed in Howarka et al. (2000) Journal of the American Chemical Society (J.Am.Chem.Soc.) 122 (11): 2411-2416.

[0169] Short oligonucleotides attached to a detector (eg, a nanopore) comprising a sequence complementary to a sequence in a leader sequence or another single-stranded sequence in an adaptor can be used to enhance capture of target polynucleotides in the methods described herein.

[0170] The label or tether can include or can be an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA or morpholino). The length of the oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA or morpholino) can be about 10-30 nucleotides or about 10-20 nucleotides in length. The oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA or morpholino) used in the label or tether can have at least one end (e.g., 3' or 5' end) modified for conjugation with other modifications or solid substrate surfaces (including, for example, beads). The end modifier can add reactive functional groups that can be used for conjugation. The example of the functional group that can be added includes, but is not limited to, amino, carboxyl, thiol, maleimide, aminooxy and any combination thereof. The functional group can be combined with spacers (e.g., C3, C9, C12, spacers 9 and 18) of different lengths to increase the physical distance between the functional group and the end of the oligonucleotide sequence.

[0171] The tag or tether can include or be a morpholino oligonucleotide. The length of the morpholino oligonucleotide can be about 10-30 nucleotides or about 10-20 nucleotides in length. The morpholino oligonucleotide can be modified or unmodified. For example, the morpholino oligonucleotide can be modified on the 3' and / or 5' ends of the oligonucleotide. The examples of modifications on the 3' and / or 5' ends of the morpholino oligonucleotide include but are not limited to 3' affinity tags and functional groups for chemical connection (including, for example, 3'-biotin, 3'-primary amines, 3'-disulfide amides, 3'-pyridyl disulfide groups and any combination thereof); 5' end modifications (including, for example, 5'-primary amines and / or 5'-dabcyl), modifications for click chemistry (including, for example, 3'-azides, 3'-alkynes, 5'-azides, 5'-alkynes) and any combination thereof.

[0172] The tag or tether may further include a polymer joint, for example, to facilitate coupling to a detector, for example, a nanopore. Exemplary polymer joints include, but are not limited to, polyethylene glycol (PEG). The molecular weight of the polymer joint may be from about 500Da to about 10kDa (including end values), or from about 1kDa to about 5kDa (including end values). The polymer joint (e.g., PEG) may be functionalized with different functional groups, including, for example, but not limited to, maleimide, NHS ester, dibenzocyclooctyne (DBCO), azide, biotin, amine, alkyne, aldehyde, and any combination thereof. The tag or tether may further include a 1kDa PEG with a 5'-maleimide group and a 3'-DBCO group. The tag or tether may further include a 2kDa PEG with a 5'-maleimide group and a 3'-DBCO group. The tag or tether may further include a 3kDa PEG with a 5'-maleimide group and a 3'-DBCO group. The tag or tether may further comprise a 5 kDa PEG having a 5'-maleimide group and a 3'-DBCO group.

[0173] Other examples of tags or tethers include, but are not limited to, a His tag, biotin or streptavidin, an antibody that binds to the analyte, an aptamer that binds to the analyte, an analyte binding domain, such as a DNA binding domain (including, for example, a peptide zipper, such as a leucine zipper, a single-stranded DNA binding protein (SSB)), and any combination thereof.

[0174] Any method known in the art can be used to connect the label or tether to the outer surface of the nanopore, for example, on the cis side of the membrane. For example, one or more labels or tethers can be connected to the nanopore by one or more cysteines (cysteine ​​bonds), one or more primary amines (such as lysine), one or more non-natural amino acids, one or more histidines (His tags), one or more biotins or streptavidins, one or more antibody-based labels, one or more enzyme modifications of epitopes (including, for example, acetyltransferases), and any combination thereof. Suitable methods for carrying out such modifications are well known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), and Liu C.C. and Schultz PG, "Annuals of Biochemistry (Annu. Rev Biochem)", 2010, 79, 413-444. Figure 1 Any of the amino acids numbered 1-71 in .

[0175] Where one or more tags or tethers are attached to the nanopore via cysteine ​​bonds, one or more cysteines may be introduced into one or more monomers forming the nanopore by substitution. The nanopore may be chemically modified by attaching: (i) maleimides, including dibromomaleimides, such as: 4-phenylazomaleimide, 1.N-(2-hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1.3-maleimidopropionic acid, 1.1-4-aminophenyl-1H-pyrrole, 2,5, dione, 1.1-4-hydroxyphenyl-1H-pyrrole, 2,5, dione, N-ethylmaleimide, N-methoxy 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)maleimide, N-(2-aminoethyl)maleimide, 3-maleimido-PROXYL, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)maleimide )-maleimide, N-(2,4-dimethylphenyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-p-methylphenyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl 1-[2-oxo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, 1-benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimide, N-(4-nitrophenyl)maleimide;(ii) Iodoacetamides, such as 3-(2-iodoacetamide)-propyl, N-(cyclopropylmethyl)-2-iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2-trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4-(aminosulfonyl)phenyl)-2-iodoacetamide, N-(1,3-benzothiazol-2-yl)-2-iodoacetamide, N-(2,6-diethylphenyl)-2-iodoacetamide, (iii) bromoacetamides such as N-(4-(acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-cyanophenyl)acetamide, 2-bromo-N-(3-(trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)acetamide, 2-bromo-N-(3-(trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)acetamide, 2-bromo- )-3-methylbutanamide, N-benzyl-2-bromo-N-phenylpropionamide, N-(2-bromo-butyryl)-4-chloro-benzenesulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo-N-phenethyl-acetamide, 2-adamantan-1-yl-2-bromo-N-cyclohexyl-acetamide, 2-bromo-N-(2-methylphenyl)butanamide, monobromoacetanilide; (iv) disulfides, such as aldehyde thiol-2, aldehyde thiol-4, isopropyl disulfide , 1-(isobutyldisulfanyl)-2-methylpropane, dibenzyl disulfide, 4-aminophenyl disulfide, 3-(2-pyridyldithio)propionic acid, 3-(2-pyridyldithio)propionic acid hydrazide, 3-(2-pyridyldithio)propionic acid N-succinimidyl ester, am6amPDP1-βCD; and (v) thiols, such as: 4-phenylthiazole-2-thiol, Purpald, 5,6,7,8-tetrahydro-quinazoline-2-thiol. ;

[0176] The tag or tether can be connected to the nanopore directly or through one or more linkers. The hybrid linker described in WO 2010 / 086602 (which is incorporated herein by reference in its entirety) can be used to connect the tag or tether to the nanopore. Alternatively, a peptide linker can be used. The peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed so that it does not interfere with the function of the monomer and the hole. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16 serine and / or glycine amino acids. More preferred flexible linkers include (SG) 1 、(SG) 2 、(SG) 3 、(SG) 4 、(SG) 5 and (SG) 8, wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24 proline amino acids. More preferred rigid linkers include (P) 12 , where P is proline.

[0177] Suitable pore tags are also described in WO 2018 / 100370, which describes a non-hairpin method for characterizing double-stranded polynucleotides and is incorporated herein by reference in its entirety.

[0178] anchor

[0179] The polynucleotides used in the present invention, such as telomere adapters, sequencing adapters, splint polynucleotides or polynucleotide extensions, may comprise a membrane anchor. The anchor generally aids in characterizing the target polynucleotide according to the methods disclosed herein. For example, the membrane anchor may facilitate the positioning of the selected polynucleotide around the nanopore.

[0180] The anchor may be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. The hydrophobic anchor is preferably a lipid, a fatty acid, a sterol, a carbon nanotube, a polypeptide, a protein or an amino acid, such as cholesterol, palmitate or tocopherol. The anchor may comprise a thiol, biotin or a surfactant.

[0181] The anchor can be biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or fusion protein), Ni-NTA (for binding to polyhistidine or polyhistidine-tagged protein), or a peptide (such as an antigen).

[0182] The anchor preferably comprises a joint, or 2, 3, 4 or more joints. Preferred joints include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides and polypeptides. These joints can be linear, branched or cyclic. For example, the joint can be a cyclic polynucleotide. The adapter can hybridize with the complementary sequence on the cyclic polynucleotide joint. One or more anchors or one or more joints can contain components that can be cut or decomposed, such as restriction sites or photolabile groups. The joint can be functionalized with a maleimide group to connect to a cysteine ​​residue in a protein. Suitable joints are described in WO 2010 / 086602 (which is incorporated herein by reference in its entirety).

[0183] The anchor is preferably cholesterol or a fatty acyl chain. For example, any fatty acyl chain of 6 to 30 carbon atoms in length, such as hexadecanoic acid, may be used.

[0184] Examples of suitable anchors and methods of linking anchors to adaptors are disclosed in WO 2012 / 164270 and WO 2015 / 150786, which are incorporated herein by reference in their entireties.

[0185] The anchor may consist of or include the following: a hydrophobic modification to a polynucleotide or sequencing adapter. The hydrophobic modification may include a modified phosphate group contained in a polynucleotide or polynucleotide anchor. The hydrophobic modification may, for example, include a thiophosphate, such as a charge-neutralized alkyl thiophosphate (PPT) described in Jones et al., Journal of the American Chemical Society, 2021, 143, 22, 8305, the entire contents of which are hereby incorporated by reference. Suitable alkyl groups include, for example, C 1 -C 10 Alkyl such as C 2 -C 6 Alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, and hexyl. Incorporation of charge-neutralized alkyl phosphorothioates into polynucleotides allows the polynucleotides to be anchored to hydrophobic regions such as lipid bilayers.

[0186] Polynucleotide extension

[0187] In alternatives using splint polynucleotides, step (a) preferably further comprises connecting or covalently linking the polynucleotide extension to the telomere adapter. The polynucleotide extension is typically connected or covalently linked to the 5' end of the telomere adapter. The polynucleotide extension can be any polynucleotide in the polynucleotide discussed above about the telomere adapter. The polynucleotide extension can be any length, including any length in the length discussed above about the 5' of the telomere adapter. The polynucleotide extension is preferably covalently linked to the telomere adapter using click chemistry. Suitable click chemistry is known in the art and discussed herein.

[0188] Preferred polynucleotide extensions include GCTTGGGTGTTTAACC (SEQ ID NO: 11) or consist of it. This is used in the click adapters of Table 7. The polynucleotide extension may include or further include one or more, such as two or more, three or more or four or more universal nucleotides, such as Int 5-nitroindole (i5NiTInd). One or more universal nucleotides are typically located at the 3' end of the polynucleotide extension. One or more universal nucleotides can be used to connect or covalently link the polynucleotide extension to the telomere adapter.

[0189] The method of the present invention preferably comprises ligating a polynucleotide telomere adapter to the 5' end of the non-protruding strand located at the end of the telomere in step (a), wherein the 3' end of the adapter specifically hybridizes to the first portion of the protruding strand, and the 5' end of the adapter does not hybridize to the opposite portion of the protruding strand and wherein the telomere adapter comprises a polynucleotide extension located at the 5' end. Any of the embodiments discussed above with respect to telomere adapters and polynucleotide extensions also apply to such "extended" telomere adapter embodiments.

[0190] The extended telomere adapter preferably comprises or consists of a sequence shown in SEQ ID NO:11 covalently linked to any one of SEQ ID NO:1-6. The extended telomere adapter preferably comprises or consists of a sequence shown in SEQ ID NO:11 covalently linked to any one of SEQ ID NO:14-19. The two sequences are preferably covalently linked by one or more, such as two or more, three or more or four or more universal nucleotides, such as Int 5-nitroindole (i5NiTInd). The two sequences are more preferably covalently linked by 4 i5NiTInds. The extended telomere adapter preferably comprises or consists of a sequence shown in any one of SEQ ID NO:25-30. The extended telomere adapter preferably comprises a click chemistry group at the 5' end, such as DBCOTEG. Examples of these extended telomere adapters are shown in Table 7.

[0191] The method preferably comprises, prior to step (a), contacting the telomeres with a population of six extended telomere adaptors, the population of six extended telomere adaptors comprising or consisting of the following sequences:

[0192] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTACCCTAA (SEQ ID NO: 1);

[0193] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTAACCCTA (SEQ ID NO: 2);

[0194] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTTAACCCT (SEQ ID NO: 3);

[0195] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCTAACCC (SEQ ID NO: 4);

[0196] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 5); and

[0197] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 6).

[0198] The method preferably comprises, prior to step (a), contacting the telomeres with a population of six extended telomere adaptors, the population of six extended telomere adaptors comprising or consisting of the following sequences:

[0199] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTACCCTAA (SEQ ID NO: 25);

[0200] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTAACCCTA (SEQ ID NO: 26);

[0201] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTTAACCCT(SEQ ID NO:27);

[0202] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCTAACCC (SEQ ID NO: 28);

[0203] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 29); and

[0204] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 30), wherein N is Int 5-nitroindole (i5NiTInd).

[0205] The method preferably comprises, prior to step (a), contacting the telomeres with a population of six extended telomere adaptors, the population of six extended telomere adaptors comprising or consisting of the following sequences:

[0206] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCCTAACCCTAACCCTAA (SEQ ID NO: 14);

[0207] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTTAACCCTAACCCTAACCC (SEQ ID NO: 15);

[0208] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCTAACCCTAACCCTAACC (SEQ ID NO: 16);

[0209] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCTAACCTAACCCTAACCCTAAC (SEQ ID NO: 17);

[0210] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTAACCCTAACCCTAACCCT (SEQ ID NO: 18); and

[0211] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTACCCTAACCCTAACCCTA (SEQ ID NO: 19).

[0212] The two sequences in the extended adapter are preferably covalently linked by one or more, such as two or more, three or more or four or more universal nucleotides, such as Int 5-nitroindole (i5NiTInd). The two sequences in the extended adapter are more preferably covalently linked by 4 i5NiTInds. The extended telomere adapter preferably comprises a click chemistry group at its 5' end, such as DBCOTEG. Examples of these extended adapters are shown in Tables 7 and Figure 4 middle.

[0213] The polynucleotide extension preferably comprises a sequencing adapter, and step (b) preferably comprises characterizing the ligated non-protruding strand of the at least a portion of the telomere in the 5' to 3' direction from the end of the telomere using the sequencing adapter. Step (a) preferably further comprises connecting (such as covalently connecting) a sequencing adapter to the polynucleotide extension, and step (b) preferably comprises characterizing the ligated non-protruding strand of the at least a portion of the telomere in the 5' to 3' direction using the sequencing adapter. Step (a) more preferably further comprises specifically hybridizing the 5' protruding end on the sequencing adapter to the polynucleotide extension, and connecting (such as covalently connecting) the sequencing adapter to the polynucleotide extension. In any of these sequencing adapter embodiments, the polynucleotide extension can be connected or covalently connected to the telomere adapter, or the method can use an extended telomere adapter as defined above. The sequencing adapter can be connected or bonded to the polynucleotide extension. The sequencing adapter is preferably connected (attach) to the polynucleotide extension by ligation. The polynucleotide extension is preferably covalently linked to the sequencing adapter using click chemistry. The polynucleotide extension preferably comprises a click chemistry group at its 5' end, such as a DBCOTEG group, and is linked to the sequencing adapter using click chemistry. Other possible click chemistry groups are discussed in more detail below.

[0214] Biotin enrichment

[0215] The telomere adapter, including the extended telomere adapter, preferably comprises biotin. The biotin is preferably located at the 5' end of the telomere adapter or the extended telomere adapter. An example of this is shown in Figure 6 In such embodiments, step (a) preferably further comprises enriching the ligated non-protruding strand of the at least a portion of the telomere using the biotin. Suitable methods for biotin-based enrichment are known in the art. For example, the telomere adapter preferably comprises biotin, and the surface (such as a bead) comprises avidin and / or streptavidin, and the surface can be used to enrich the ligated non-protruding strand of at least a portion of the telomere.

[0216] Linker or spacer

[0217] Preferably there are one or more joints or spacers between the 3' end of the telomere adapter and the 5' end of the telomere adapter. The one or more joints or spacers are preferably flexible. The telomere adapter can include any number of one or more spacers, such as about one to about 10 spacers, such as about 1 to about 5 spacers, such as about 1, 2, 3, 4 or 5 spacers. The spacer can include any suitable number of spacer units. The spacer generally provides an energy barrier that hinders the movement of the polynucleotide binding protein. For example, the spacer can hinder the movement of the polynucleotide binding protein by reducing the traction of the protein, such as using a baseless spacer. The spacer can physically prevent the movement of the protein, such as by introducing a bulky chemical group to physically hinder the movement of the polynucleotide binding protein. Suitable spacers and their uses are discussed above with respect to sequencing adapters, and these spacers are also applicable to telomere adapters.

[0218] Bidirectional characterization

[0219] The method of the present invention preferably further comprises characterizing the unligated protruding strands of said at least a portion of said telomere in the 5' to 3' direction towards said end of said telomere. An example of this is shown in Figure 7 Any method can be used to characterize the unattached protruding strand, including any of the methods discussed below. The same method or different methods can be used to characterize the attached non-protruding strand and the unattached protruding strand. Preferably, the same method is used to characterize the attached non-protruding strand and the unattached protruding strand.

[0220] The method of the present invention preferably further comprises using a polymer-guided effector protein (such as a Cas9 protein) to generate a double-strand break at the opposite end of the at least a portion of the telomere relative to the telomere end and connecting a sequencing adapter to the opposite end, and using the sequencing adapter to characterize the unconnected protruding strand in the 5' to 3' direction toward the end of the telomere of at least a portion of the telomere. The sequencing adapter can be connected or bonded to the opposite end. The sequencing adapter can be any of the sequencing adapters discussed above. Connecting a Y sequencing adapter to a double-strand break is known in the art.

[0221] The present invention also provides a method for characterizing at least a portion of a telomere, the method comprising (a) connecting a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of the telomere, wherein the 3' end of the adapter is complementary to the first portion of the protruding strand and the 5' end of the adapter is not complementary to the opposite portion of the protruding strand, (b) using a polymer-guided effector protein (such as a Cas9 protein) to generate a double-strand break at the opposite end of at least a portion of the telomere relative to the telomere end, and connecting a sequencing adapter to the opposite end, and (c) using the telomere adapter to characterize the connected non-protruding strand of at least a portion of the telomere in the 5' to 3' direction from the end of the telomere, and using the sequencing adapter to characterize the unconnected protruding strand of at least a portion of the telomere in the 5' to 3' direction toward the end of the telomere.

[0222] Polymer-guided effector proteins

[0223] The polymer-guided effector protein can be any protein that binds to the opposite end of at least a portion of a telomere via a guide polymer. As a non-limiting example, the polymer-guided effector protein can be bound or linked to a guide oligonucleotide (such as an aptamer) or a guide polypeptide (such as an antibody) that binds to a portion of at least a portion of a telomere.

[0224] The polymer-guided effector protein is preferably a polynucleotide-guided effector protein. In this embodiment, the guide polymer is preferably a guide polynucleotide. The polynucleotide-guided effector protein can be any protein that binds or is connected to the guide polynucleotide, and the effector protein binds to the opposite end of at least a portion of the telomere, preferably to the target polynucleotide sequence at the opposite end of at least a portion of the telomere to which the guide polynucleotide binds. The polynucleotide-guided effector protein preferably comprises a target polynucleotide sequence recognition domain and at least one nuclease domain. The recognition domain binds to the guide polynucleotide (e.g., RNA) and the target polynucleotide (e.g., DNA). The polynucleotide-guided effector protein may include a nuclease domain that cuts one or both chains of a double-stranded polynucleotide, or may contain two nuclease domains, wherein the first nuclease domain is positioned to cut a chain of the target polynucleotide sequence, and the second nuclease domain is positioned to cut the complementary chain of the target polynucleotide sequence. The nuclease domain may be active or inactive. For example, one or both of the nuclease domains or the two nuclease domains may be inactivated by mutation.

[0225] The guide polynucleotide can be a guide RNA, a guide DNA, or a guide containing both DNA and RNA. The guide polynucleotide is preferably a guide RNA. Therefore, the effector protein guided by the polynucleotide is preferably an RNA-guided effector protein.

[0226] The RNA-guided effector protein can be any protein that binds to or is connected to the guide RNA. The RNA-guided effector protein generally binds to a region of the guide RNA that is not a region of the guide RNA that binds to the target polynucleotide sequence. For example, in the case where the guide RNA comprises crRNA and tracrRNA, the RNA-guided effector protein generally binds to tracrRNA, and the crRNA generally binds to the opposite end of at least a portion of the telomere, also referred to as the target polynucleotide sequence. The RNA-guided effector protein preferably also binds to the target polynucleotide sequence. The region of the guide RNA that binds to the target polynucleotide sequence may also bind to the RNA-guided effector protein. The RNA-guided effector protein generally binds to the double-stranded region of the target polynucleotide sequence. The region of the target polynucleotide sequence to which the RNA-guided effector protein binds is generally located near the sequence to which the guide RNA hybridizes. The guide RNA and the RNA-guided effector protein generally form a complex that then binds to the target polynucleotide sequence at a site determined by the sequence of the guide RNA.

[0227] RNA-guided effector proteins can bind to the upstream or downstream of the sequence bound by the guide RNA. For example, RNA-guided effector proteins can bind to the protospacer adjacent motif (PAM) in the DNA next to the sequence bound by the guide RNA. PAM is a short (less than 10, usually 2 to 6 base pairs) sequence, such as 5'-NGG-3' (where N is any base), 5'-NGA-3', 5'-YG-3' (where Y is pyrimidine), 5'TTN-3' or 5'-YTN-3'. Different RNA-guided effector proteins bind to different PAMs. RNA-guided effector proteins can bind to target polynucleotide sequences that do not contain PAMs, especially when the target is RNA or a DNA / RNA hybrid.

[0228] The RNA-guided effector protein is typically a nuclease, such as an RNA-guided endonuclease. The RNA-guided effector protein is typically a Cas protein. The RNA-guided effector protein may be Cas, Csn2, Cpf1, Csf1, Cmr5, Csm2, Csy1, Cse1, or C2c2. The Cas protein may be Cas3, Cas4, Cas8a, Cas8b, Cas8c, Cas9, Cas10, or Cas10d. Preferably, the Cas protein is Cas9. In the case where the target polynucleotide sequence comprises a double-stranded DNA region, Cas, Csn2, Cpf1, Csf1, Cmr5, Csm2, Csy1, or Cse1 is preferably used. In the case where the target polynucleotide sequence comprises a double-stranded RNA region, C2c2 is preferably used.

[0229] DNA-guided effector proteins, such as proteins from the RecA family, can be used for target DNA. Examples of proteins from the RecA family that can be used are RecA, RadA, and Rad51. The nuclease activity of RNA-guided endonucleases can be invalidated. One or more of the catalytic nuclease sites of the RNA-guided endonucleases can be inactivated. For example, in the case where the RNA-guided endonucleases comprise two catalytic nuclease sites, one or both of the catalytic sites can be inactivated. Typically, one of the catalytic sites will cut a strand of the polynucleotide that it specifically binds, and the other catalytic site will cut the relative strand of the polynucleotide. Therefore, RNA-guided endonucleases can cut two strands, one strand, or no strands of the double-stranded region of the target polynucleotide. RNA-guided endonucleases preferably cut two strands of at least a portion of a telomere.

[0230] The polynucleotide-guided effector protein is preferably Cas9. Cas9 has a bi-lobed multi-domain protein structure, which includes target recognition and nuclease lobes. The recognition lobe binds to the guide RNA and DNA. The nuclease lobe contains HNH and RuvC nuclease domains, which are positioned to cut the complementary and non-complementary strands of the target DNA. The structure of Cas 9 is described in detail in the following: Nishimasu, H. et al., (2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156, 935-949. The relevant PDB reference for Cas9 is 5F9R (Crystal Structure of Catalytically Active Streptococcus pyogenes CRISPR-Cas9, Complexed with Single Guide RNA and Double-stranded DNA for Target DNA Cutting).

[0231] The Cas9 may be an 'enhanced specificity' Cas9 that exhibits reduced off-target binding compared to wild-type Cas9. An example of such an 'enhanced specificity' Cas9 is Streptococcus pyogenes Cas9D10A / H840A / K848A / K1003A / R1060A. ONLP12296 is the amino acid sequence of Streptococcus pyogenes Cas9D10A / H840A / K848A / K1003A / R1060A with a C-terminal double-stranded tag with a TEV-cleavable linker.

[0232] The catalytic site of the RNA-guided endonuclease can be inactivated by mutation. The mutation can be a substitution, insertion or deletion mutation. For example, one or more, such as 2, 3, 4, 5 or 6 amino acids can be substituted or inserted into the catalytic site, or missing from the catalytic site. The mutation is preferably a substitution insertion, and if there is a single amino acid at the catalytic site, it is more preferably a substitution. The technician will be able to easily identify the catalytic site of the RNA-guided endonuclease and the mutation that inactivates it. For example, in the case where the RNA-guided endonuclease is Cas9, a catalytic site can be inactivated by a mutation at D10, and another can be inactivated by a mutation at H640.

[0233] An inactive ('dead') polynucleotide-guided effector protein that does not cleave the target polynucleotide sequence and therefore shows no directional bias. An active ('live') polynucleotide-guided effector protein that cleaves the target polynucleotide sequence may still only bind to one of the two ends of the cleavage site and therefore may show some directional bias.

[0234] The polymer-directed effector protein may be specifically modified for use in the methods of the invention. The protein may comprise an anchor capable of coupling to a membrane, such as cholesterol. The polymer-directed effector protein preferably has a binding moiety capable of coupling to a surface to which it is attached. The surface is preferably the surface of a bead.

[0235] Wizard polymer

[0236] The guide polymer is capable of binding to the opposite end of at least a portion of the telomere and mediating the binding of the effector protein guided by the polymer to the opposite end of at least a portion of the telomere. The guide polymer may have any structure that enables it to bind to the opposite end of at least a portion of the telomere, also referred to as the target polynucleotide sequence. It may be any polymer in the polymers discussed above for the telomere adapter. The guide polymer is preferably an oligonucleotide, a polynucleotide, a polypeptide, a protein, an oligosaccharide or a polysaccharide. The guide polypeptide or protein is preferably a zinc finger binding protein, a transcription activator-like effector (TALE), a transcription factor, a restriction enzyme, a DNA binding protein or enzyme, an antibody or an antibody fragment. Suitable antibody fragments are known in the art and include, but are not limited to, Fab, Fab', (Fab')2, FV, scFv, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, double scFv, mini antibodies, Fab2 and Fab3. The guide oligonucleotide is preferably an aptamer.

[0237] The guide polymer can be bound to the polymer-guided effector protein. In this context, the guide polymer can be bound to the polymer-guided effector protein, can be bound by the polymer-guided effector protein, or both. The guide polymer can be linked to the polymer-guided effector protein. Suitable methods for linking the guide polymer to the polymer-guided effector protein are known in the art, such as using streptavidin / biotin. The polymer-guided effector is preferably covalently linked to the guide polymer. Covalent linkage, such as using click chemistry, is discussed in WO 2018 / 060740 (which is incorporated herein by reference in its entirety).

[0238] The guide polymer is preferably a guide polynucleotide. In this case, the polymer-guided effector protein is preferably a polynucleotide-guided effector protein. The guide polynucleotide preferably comprises a sequence capable of binding to the opposite end of at least a portion of the telomere or specifically hybridizing with the target polynucleotide sequence. The guide polynucleotide preferably comprises a nucleotide sequence that binds to a sequence at the opposite end of at least a portion of the telomere and a nucleotide sequence that binds to the polynucleotide-guided effector protein. The guide polynucleotide preferably comprises a nucleotide sequence that specifically hybridizes to a sequence at the opposite end of at least a portion of the telomere and a nucleotide sequence that binds to the polynucleotide-guided effector protein. The guide polynucleotide may have any structure that enables it to specifically bind / hybridize to the opposite end of at least a portion of the telomere and bind to the polynucleotide-guided effector protein.

[0239] The guide polynucleotide typically specifically hybridizes to a sequence of about 20 nucleotides in the target polynucleotide sequence. The sequence to which the guide polynucleotide binds can be about 10 to about 40, such as about 15 to about 30, preferably about 18 to about 25, such as about 19, 20, 21, 22, 23 or 24 nucleotides. The guide polynucleotide is typically complementary to a strand at the opposite end of at least a portion of a telomere. The guide polynucleotide preferably comprises a nucleotide sequence of about 10 to about 40, such as about 15 to about 30, preferably about 18 to about 25, such as about 19, 20, 21, 22, 23 or 24 nucleotides, which is complementary to the sequence of the target polynucleotide sequence or a sequence therein. The degree of complementarity is preferably exact.

[0240] The guide polynucleotide is preferably a guide RNA. The guide RNA can be complementary to a region 5' relative to the PAM in the target polynucleotide sequence. This is preferred where the target polynucleotide comprises DNA, especially where the RNA effector protein is Cas9 or Cpf1. The guide RNA can be complementary to a region flanked by guanines in the target polynucleotide sequence. This is preferred where the target polynucleotide comprises RNA, especially where the RNA effector protein is C2c2.

[0241] The guide RNA may have any structure that enables it to bind to a target polynucleotide sequence and an RNA-guided effector protein. The guide RNA may include crRNA and tracrRNA that bind to a sequence in the target polynucleotide sequence. TracrRNA is typically bound to an RNA-guided effector protein. The typical structure of a guide RNA is known in the art. For example, crRNA is typically single-stranded RNA, and tracrRNA typically has a double-stranded region in which one chain is connected to the 3' end of the crRNA, and a portion of a hairpin loop is formed at the 3' end of the chain that is not connected to the crRNA. The crRNA and tracrRNA can be transcribed in vitro as a single sgRNA. The guide RNA is preferably an sgRNA.

[0242] The guide RNA may contain other components, such as additional RNA bases or DNA bases or other nucleobases. The RNA and DNA bases in the guide RNA may be natural bases or modified bases. Guide DNA may be used instead of guide RNA, and DNA-guided effector proteins may be used instead of RNA-guided effector proteins. In the case where the target polynucleotide is RNA, guide DNA and DNA-guided effector proteins may be preferably used.

[0243] Characterization

[0244] Step (b) comprises characterizing the ligated non-protruding strand of at least a portion of the telomere in the 5' to 3' direction from the end of the telomere using the telomere adapter. The method preferably also comprises characterizing the unligated protruding strand of at least a portion of the telomere in the 5' to 3' direction toward the end of the telomere using a sequencing adapter. Any characterization method may be used. The method preferably uses next generation sequencing (NGS).

[0245] The connected non-protruding chains and / or the unconnected protruding chains are preferably moved relative to a detector such as a nanopore. The detector may be selected from the following: (i) a zero-mode waveguide; (ii) a field effect transistor, optionally a nanowire field effect transistor; (iii) an AFM tip; (iv) a nanotube, optionally a carbon nanotube; and (v) a nanopore. Preferably, the detector is a nanopore.

[0246] The ligated non-protruding strands and / or unligated protruding strands can be characterized in the methods of the present invention in any suitable manner. The ligated non-protruding strands and / or unligated protruding strands are preferably characterized by detecting ionic current or optical signals as they move relative to the nanopore. This is described in more detail herein. The methods are applicable to these and other methods of characterizing polynucleotides.

[0247] In another non-limiting example, the connected non-protruding strand and / or the unconnected protruding strand are characterized by detecting a polynucleotide processing reaction, such as a byproduct of a sequencing reaction while synthesis. The method may therefore involve detecting the product of sequentially adding (poly) nucleotides to a nucleic acid chain by an enzyme, such as a polymerase. The product may be a change in one or more properties of an enzyme, such as a change in the conformation of the enzyme. Such methods may therefore include subjecting an enzyme such as a polymerase or a reverse transcriptase to a polynucleotide under the following conditions: in response to a template chain nucleic acid base encountered in sequence and / or a natural or analog base specified by the incorporation template (i.e., an incorporation event), so that the template-dependent incorporation of a nucleotide base into a growing oligonucleotide chain causes a conformational change in the enzyme, detects a conformational change in the enzyme in response to such an incorporation event, and thereby detects the sequence of the template chain. In such methods, the polynucleotide chain may be moved according to the method of the present invention. Such methods may involve using methods known to those skilled in the art, such as the method described in US2017 / 0044605, to detect and / or measure incorporation events.

[0248] In another embodiment, the byproducts can be labeled so that when nucleotides are added to the synthetic nucleic acid strand complementary to the template strand, the phosphate-labeled substance is released, and, for example, the phosphate-labeled substance is detected using a detector as described herein. The polynucleotides characterized in this way can be moved according to the methods herein. Suitable markers can be optical markers detected using nanopores or zero-mode waveguides or by Raman spectroscopy or other detectors. Suitable markers can be non-optical markers detected using nanopores or other detectors.

[0249] In another approach, the nucleoside phosphates (nucleotides) are not labeled and the natural byproduct species are detected after the addition of nucleotides to a synthetic nucleic acid strand complementary to the template strand. A suitable detector may be an ion-sensitive field effect transistor or other detector.

[0250] These and other detection methods are suitable for use in the methods described herein.A detector can be used to make any suitable measurements as the polynucleotide moves relative to the detector.

[0251] Nanopore characterization

[0252] Preferably the nanopore is used to characterize the ligated non-protruding strands and / or the unligated protruding strands.

[0253] The method preferably comprises (i) contacting the ligated non-protruding strand and / or the unligated protruding strand with a nanopore such that the ligated non-protruding strand and / or the unligated protruding strand moves relative to the nanopore, and (ii) performing one or more measurements as the ligated non-protruding strand and / or the unligated protruding strand moves relative to the nanopore, wherein the measurement results indicate one or more properties of the ligated non-protruding strand and / or the unligated protruding strand, and thereby characterize the ligated non-protruding strand and / or the unligated protruding strand. The one or more properties are preferably selected from (i) the length of the ligated non-protruding strand and / or the unligated protruding strand, (ii) the identity of the ligated non-protruding strand and / or the unligated protruding strand, (iii) the sequence of the ligated non-protruding strand and / or the unligated protruding strand, (iv) the secondary structure of the ligated non-protruding strand and / or the unligated protruding strand, and (v) whether the ligated non-protruding strand and / or the unligated protruding strand is modified. The ligated non-protruding strands and / or the unligated protruding strands may be modified with one or more proteins or with one or more markers, tags or spacers by methylation, by oxidation, by damage. One or more properties of the ligated non-protruding strands and / or the unligated protruding strands are preferably measured by electrical and / or optical measurements. The electrical measurements are preferably current measurements, impedance measurements, tunneling measurements or field effect transistor (FET) measurements.

[0254] The method more preferably comprises (i) contacting the ligated non-protruding strands and / or the unligated protruding strands with a nanopore such that the ligated non-protruding strands and / or the unligated protruding strands move through the nanopore, and (ii) measuring a current moving through the nanopore as the ligated non-protruding strands and / or the unligated protruding strands move through the nanopore, wherein the current indicates one or more properties of the ligated non-protruding strands and / or the unligated protruding strands, and thereby characterizes the ligated non-protruding strands and / or the unligated protruding strands. The one or more properties may be any of those described above.

[0255] Preferably a polynucleotide binding protein is used to control the movement of the ligated non-protruding strands and / or unligated protruding strands relative to or through the nanopore. The use of such proteins in nanopore sequencing is known. Examples of suitable proteins are discussed in more detail below.

[0256] The present invention also provides a method for characterizing at least a portion of a telomere, the method comprising (a) ligating a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of the telomere, wherein the 3' end specifically hybridizes with a first portion of the protruding strand and the 5' end of the adapter does not hybridize with the opposite portion of the protruding strand, (b) using a polymer-guided effector protein (such as a Cas9 protein) to generate a double-stranded break at the opposite end of the at least a portion of the telomere relative to the telomere end, and ligating a sequencing adapter to the opposite end, (c) contacting the telomere adapter with a nanopore, such that The connected non-protruding strand moves relative to the nanopore, and one or more measurements are taken when the connected non-protruding strand moves relative to the nanopore, wherein the measurement results indicate one or more characteristics of the connected non-protruding strand, and thereby characterize the connected non-protruding strand, and (d) a sequencing adapter is contacted with the nanopore so that the unconnected protruding strand moves relative to the nanopore, and one or more measurements are taken when the unconnected protruding strand moves relative to the nanopore, wherein the measurement results indicate one or more characteristics of the unconnected protruding strand, and thereby characterize the unconnected protruding strand. The connected non-protruding strand and / or the unconnected protruding strand preferably move through the nanopore. The method preferably comprises measuring the current moving through the nanopore, wherein the current indicates one or more characteristics of the connected non-protruding strand and the unconnected protruding strand. Preferably, a polynucleotide binding protein is used to control the movement of the connected non-protruding strand and the unconnected protruding strand relative to the nanopore / through the nanopore. The telomere adapter and the sequencing adapter can be any of those adapters discussed above.

[0257] Any suitable nanopore can be used. The nanopore is preferably a transmembrane pore. A transmembrane pore is a structure that passes through the membrane to some extent. It allows hydrated ions driven by an applied potential to flow across or within the membrane. The transmembrane pore usually passes through the entire membrane so that hydrated ions can flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not necessarily pass through the membrane. It may be closed at one end. For example, the pore can be a hole, gap, channel, groove or slit in the membrane, and the hydrated ions can flow along the membrane or into the membrane.

[0258] The nanopore generally has a first opening and a second opening. The first opening is generally a cis opening, and the second opening is generally a trans opening. However, the first opening may be a trans opening, and the second opening may be a cis opening. Any polynucleotide binding protein used in the method of the present invention is generally provided at the first opening of the nanopore and thus controls the movement of the target polynucleotide in the direction from the second opening of the nanopore toward the first opening of the nanopore.

[0259] Any transmembrane pore may be used in the methods of the invention. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami pores are disclosed in WO2013 / 083983.

[0260] Nanopore is preferably a transmembrane protein hole. Transmembrane protein hole is a polypeptide or polypeptide set that allows hydrated ions (such as polynucleotides) to flow from one side of the membrane to the other side of the membrane. In the method of the present invention, transmembrane protein hole can form a hole that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side. Transmembrane protein hole preferably allows polynucleotides to flow from one side of a membrane (such as a triblock copolymer membrane) to the other side. Transmembrane protein hole allows polynucleotides to move through the hole.

[0261] The nanopore may be a transmembrane protein pore, which is a monomer or an oligomer. The pore is preferably composed of several repeated subunits, such as at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 or at least about 16 subunits. The pore is preferably a hexamer, heptamer, octamer or non-polymeric pore. The pore may be a homo-oligomer or a hetero-oligomer.

[0262] A transmembrane protein pore may comprise a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β-barrel or channel or a transmembrane α-helical bundle or channel.

[0263] Typically, the barrel or channel of a transmembrane protein pore comprises amino acids that promote interaction with an analyte, such as a target polynucleotide (as described herein). These amino acids are preferably located near the contraction of the barrel or channel. Transmembrane protein pores typically comprise one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically promote interaction between a hole and a nucleotide, polynucleotide or nucleic acid.

[0264] The nanopore may be a transmembrane protein pore derived from a β-barrel pore or an α-helical bundle pore. The β-barrel pore comprises a barrel or channel formed by a β-strand. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin and leukocidin, and outer membrane proteins / porins of bacteria such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC or MspD, CsgG, outer membrane porins F (OmpF), outer membrane porins G (OmpG), outer membrane phospholipase A and Neisseria autotransporter (NalP) and other pores such as lysin. The α-helical bundle pore comprises a barrel or channel formed by an α-helix. Suitable α-helical bundle pores include, but are not limited to, inner membrane proteins and α outer membrane proteins such as WZA and ClyA toxins.

[0265] The nanopore may be a transmembrane pore derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, and the hemolytic protein fragaceatoxin C (FraC).

[0266] The nanopore may be a transmembrane protein pore derived from CsgG, for example derived from CsgG from E. coli strain K-12 substrain MC4100. Such pores are oligomeric and typically comprise 7, 8, 9 or 10 monomers derived from CsgG. The pore may be a homo-oligomeric pore derived from CsgG comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from CsgG comprising at least one monomer that is different from the other monomers. Examples of suitable pores derived from CsgG are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318 and WO 2019 / 002893 (all incorporated herein by reference in their entirety).

[0267] The nanopore may be a transmembrane pore derived from lysenin. Examples of suitable pores derived from lysenin are disclosed in WO 2013 / 153359, which is incorporated herein by reference in its entirety.

[0268] The nanopore may be a transmembrane pore derived from or based on α-hemolysin (α-HL). The wild-type α-hemolysin pore is formed by 7 identical monomers or subunits (i.e., it is a heptamer). The α-hemolysin pore may be α-hemolysin-NN or a variant thereof. The variant preferably comprises N residues at positions E111 and K147.

[0269] The nanopore may be a transmembrane protein pore derived from Msp (eg, derived from MspA). Examples of suitable pores derived from MspA are disclosed in WO 2012 / 107778, which is incorporated herein by reference in its entirety.

[0270] The nanopore may be a transmembrane pore derived from or based on ClyA.

[0271] membrane

[0272] The detector or nanopore is typically present in a membrane.Any suitable membrane may be used.

[0273] The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids, which have both hydrophilic and lipophilic properties. Amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers can have unique properties that polymers formed by individual subunits do not have. Block copolymers can be engineered so that one of the monomer subunits is hydrophobic (i.e., lipophilic) in an aqueous medium, while the other subunits are hydrophilic. In this case, block copolymers can have amphiphilic properties and can form a structure that simulates a biofilm. The block copolymers may be diblock (which consists of two monomer subunits), but may also be built up from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers may be triblock, tetrablock or pentablock copolymers. The membrane may be a triblock copolymer membrane.

[0274] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed so that lipids form monolayers. These lipids are generally found in extreme microorganisms, thermophilic microorganisms, halophilic microorganisms, and acidophilic microorganisms that survive in harsh biological environments. Its stability is believed to be derived from the fusogenic properties of the final double layer. It is simple to construct block copolymer materials that simulate these biological entities by producing triblock polymers with the general motif hydrophilicity-hydrophobicity-hydrophilicity. This material can form a monomeric membrane that behaves similarly to a lipid bilayer and covers a series of stage performances from vesicles to lamellar membranes. The membranes formed by these triblock copolymers maintain several advantages over biological lipid membranes. Because the triblock copolymers are synthesized, the accurate construction can be carefully controlled to provide the correct chain length and properties required to form a membrane and interact with holes and other proteins.

[0275] Block copolymers can also be constructed from subunits that are not classified as lipid submaterials, for example, hydrophobic polymers can be prepared from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsections of the block copolymers can also have low protein binding properties, which allows the production of membranes that are highly resistant when exposed to raw biological samples. This head group unit can also be derived from non-classical lipid head groups.

[0276] Compared to biological lipid membranes, triblock copolymer membranes also have increased mechanical and environmental stability, such as much higher operating temperature or pH ranges.The synthetic nature of block copolymers provides a platform for tailoring polymer-based membranes for a wide range of applications.

[0277] The membrane may be one of the membranes disclosed in International Application No. WO2014 / 064443 or WO2014 / 064444 (both International Applications are incorporated herein by reference in their entirety).

[0278] The amphiphilic molecule can be chemically modified or functionalized to facilitate coupling to the polynucleotide. The amphiphilic layer can be a single layer or a double layer. The amphiphilic layer is generally planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported.

[0279] Amphiphilic membranes are usually naturally mobile, essentially at a rate of about 10 -8 cm2 -1 The lipid diffusion rate is 2.0, which acts as a two-dimensional liquid. This means that the pore and the coupled polynucleotide can move normally within the amphiphilic membrane.

[0280] The membrane can be a lipid bilayer. The lipid bilayer is a model of a cell membrane and is used as an excellent platform for a range of experimental studies. For example, a lipid bilayer can be used for in vitro studies of membrane proteins by single channel recording. Alternatively, a lipid bilayer can be used as a biosensor for detecting the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734 and WO 2006 / 100484 (the documents are incorporated herein by reference in their entirety).

[0281] Methods for forming lipid bilayers are known in the art.Lipid bilayers are generally formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69: 3561-3566).

[0282] The lipid bilayer can be formed as described in WO 2009 / 077734 (which is incorporated herein by reference in its entirety). In this method, the lipid bilayer is formed by dry lipids. The lipid bilayer can be formed across an opening, as described in WO 2009 / 077734.

[0283] The film may include a solid layer. The solid layer may be formed of both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials (such as Si 3 N 4 、A1 2 O 3 and SiO), organic and inorganic polymers (such as polyamides), plastics (such as ) or elastomers (such as two-component addition-cured silicone rubber) and glass. The solid layer can be formed of graphene. Suitable graphene layers are disclosed in WO 2009 / 035647 (which is incorporated herein by reference in its entirety). If the membrane comprises a solid layer, the pores are generally present in the amphiphilic membrane or layer, which is contained within the solid layer, such as holes, holes, gaps, channels, grooves or gaps within the solid layer. A skilled person can prepare a suitable solid / amphiphilic hybrid system. Suitable systems are disclosed in WO2009 / 020682 and WO 2012 / 005857 (which are incorporated herein by reference in their entirety). Any of the amphiphilic membranes or layers discussed above can be used.

[0284] The methods disclosed herein are generally performed using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell into which a pore is inserted. The methods are generally performed using an artificial amphiphilic layer (e.g., an artificial triblock copolymer layer). The layer may contain other transmembrane and / or intramembrane proteins and other molecules in addition to the pore. Suitable equipment and conditions are discussed below. The methods of the invention are generally performed in vitro.

[0285] Polynucleotide binding protein

[0286] As will be appreciated by those skilled in the art, any suitable polynucleotide binding protein may be used in the methods and products of the invention.The polynucleotide binding protein may be any protein capable of binding to a polynucleotide and controlling its movement relative to a detector such as a nanopore.

[0287] In more detail, polynucleotide binding proteins such as helicases can generally operate in at least two active modes (when provided with all the necessary components for facilitating movement, such as ATP and Mg). 2+ The invention relates to controlling the movement of DNA in two modes: an active mode (when the necessary components for promoting movement are not provided; or when the polynucleotide binding protein is modified to prevent the active mode).

[0288] When provided with all the necessary components for facilitating movement, the polynucleotide binding protein can move along a polynucleotide such as DNA in a 5'-3' direction or a 3'-5' direction. Many polynucleotide binding proteins process polynucleotides such as DNA in a 5'-3' direction. Polynucleotide binding proteins that control the movement of polynucleotides in this manner are generally suitable for use in the methods of the present invention.

[0289] In some embodiments, the polynucleotide binding protein is used to control the movement of the polynucleotide relative to the nanopore, and the polynucleotide is used to control the movement of the polynucleotide relative to the nanopore. However, when the polynucleotide binding protein is not provided with the necessary components for promoting movement, or is modified to prevent it from actively controlling the movement of the polynucleotide relative to the nanopore, it can still passively control the movement of the polynucleotide relative to the nanopore. For example, the polynucleotide binding protein can be combined with the polynucleotide and act as a brake that slows down the movement of the polynucleotide when the polynucleotide is pulled into the hole by the field applied (for example, by the first force in the method of the present invention). Under the "non-active" mode, it is usually insignificant whether DNA is 3' or 5' is captured downwards (that is, in the 5'-3' direction or in the 3'-5' direction, moving through the nanopore), because the applied force provides the power for moving the polynucleotide through the nanopore. However, in such embodiments, the polynucleotide binding protein can still control the movement of the polynucleotide relative to the nanopore, for example, by acting as a brake. When in the non-active mode, the mobile control of the polynucleotide by the polynucleotide binding protein can be described in a variety of ways (including ratchet, sliding and braking). Generally, the method of the present invention does not include the use of the polynucleotide binding protein operated in the passive mode. However, when a polynucleotide binding protein is used, it may be a polynucleotide binding protein operating in a passive mode.

[0290] Some methods of the invention may comprise using a polynucleotide binding protein as a pause moiety to hinder the movement of a polynucleotide chain through a nanopore. A polynucleotide binding protein may be a protein that binds to a polynucleotide but does not have polynucleotide processing capabilities, i.e., it is not a polynucleotide binding protein.

[0291] Polynucleotide handling enzymes are polypeptides that can interact with polynucleotides. Enzymes can modify polynucleotides by cutting polynucleotides to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The enzyme can modify polynucleotides by moving the polynucleotides toward or to a specific position. As used herein, polynucleotide binding proteins can be or can be derived from polynucleotide handling enzymes. Polynucleotide binding proteins can be or can be derived from polynucleotide handling enzymes.

[0292] The polynucleotide binding protein may be derived from a member of any of the Enzyme Classification (EC) Groups: 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3.1.31.

[0293] Typically, the polynucleotide binding protein is a helicase, a polymerase, an exonuclease, a topoisomerase, or a variant thereof.

[0294] The polynucleotide binding protein may be modified to prevent the polynucleotide binding protein from detaching from the polynucleotide. Therefore, the target polynucleotide is preferably not detached from the polynucleotide binding protein.

[0295] As used herein, the term "disengagement" refers to the dissociation of a polynucleotide binding protein and a target polynucleotide. Therefore, a polynucleotide binding protein can be modified to prevent it from dissociating from a target polynucleotide, such as dissociating into a reaction medium. It is important to distinguish the potential "disengagement" of a polynucleotide binding protein from the "disintegration" of a polynucleotide binding protein and a target polynucleotide. As used herein, "disintegration" refers to the instantaneous release of a target polynucleotide into the active site (described in more detail herein) of a polynucleotide binding protein, but does not mean to disengage. Therefore, for example, a polynucleotide binding protein can be modified to prevent a polynucleotide binding protein from disengaging from a polynucleotide, but does not prevent a polynucleotide binding protein from disintegrating from a polynucleotide. When disintegrating, a polynucleotide binding protein still engages with a target polynucleotide. For example, a polynucleotide binding protein can still engage with a target polynucleotide (that is, it can be prevented from disengaging from a target polynucleotide) because it is topologically closed around the target polynucleotide. The polynucleotide binding site can still be free to bind or disintegrate a target polynucleotide, so that a polynucleotide binding protein can bind or disintegrate a target polynucleotide, and a polynucleotide binding protein still engages with a target polynucleotide simultaneously. When the polynucleotide binding protein is unbound from the target polynucleotide, it may be able to move on (e.g., along) the target polynucleotide under the applied force and may be able to re-bind to the target polynucleotide. When engaged on the target polynucleotide but unbound from the target polynucleotide, the polynucleotide binding protein cannot dissociate from the target polynucleotide.

[0296] Polynucleotide binding protein can be adjusted in any suitable manner to prevent from disengaging.For example, polynucleotide binding protein can be loaded on polynucleotide, and then modified to prevent it from disengaging from polynucleotide.Alternatively, polynucleotide binding protein can be modified to prevent it from disengaging from polynucleotide before being loaded on polynucleotide.Methods known in the art can be used, such as the method discussed in WO 2014 / 013260 (hereby incorporated by reference in its entirety), and with particular reference to describing modified polynucleotide binding protein such as helicase to prevent it from disengaging from polynucleotide chain to realize modification of polynucleotide binding protein and / or polynucleotide binding protein to prevent it from disengaging from polynucleotide.For example, polynucleotide binding protein can be modified by processing with tetramethyl azodicarbonamide (TMAD).Various other closed parts are described in WO 2021 / 255476 (which is incorporated herein by reference in its entirety).

[0297] The polynucleotide decomposition opening of given polynucleotide binding protein can be determined by reference to its structure, for example, with reference to its X-ray crystal structure. The X-ray crystal structure can be obtained in the presence and / or absence of polynucleotide substrates. Standard packages known in the art can be used to infer or confirm the position of the polynucleotide decomposition opening in a given polynucleotide binding protein by molecular modeling. The polynucleotide decomposition opening can be produced instantaneously by the movement of one or more parts of polynucleotide binding protein, for example, one or more domains.

[0298] The polynucleotide binding protein can be modified by closing the polynucleotide decombination opening. The polynucleotide decombination opening can be closed with a closing portion. Therefore, closing the polynucleotide decombination opening can prevent the polynucleotide binding protein from disengaging from the polynucleotide. For example, the polynucleotide binding protein can be modified by covalently closing the polynucleotide decombination opening. However, as explained above, closing the polynucleotide decombination opening does not necessarily prevent the target polynucleotide from decombining with the polynucleotide binding site of the polynucleotide binding protein. The preferred protein for addressing in this way is a helicase.

[0299] The polynucleotide binding protein can be modified with a closing moiety for (i) topologically closing the polynucleotide binding site of the polynucleotide binding protein around the target polynucleotide and (ii) promoting the disassociation of the target polynucleotide from the polynucleotide binding site of the polynucleotide binding protein and / or delaying the reassociation of the target polynucleotide from the polynucleotide binding site of the polynucleotide binding protein. The polynucleotide binding protein can be modified in any suitable manner to facilitate the attachment of such a closing moiety.

[0300] The closing portion may comprise a bifunctional cross-linking portion. The closing portion may comprise a bifunctional cross-linking agent. The bifunctional cross-linking agent may connect at two points on the polynucleotide binding protein and close the polynucleotide debinding opening of the polynucleotide binding protein, thereby preventing the polynucleotide from dissociating from the polynucleotide binding protein while allowing the polynucleotide to debind from the polynucleotide binding site of the polynucleotide binding protein.

[0301] The closed part can be connected to any suitable position on the polynucleotide binding protein. For example, the closed part can cross-link two amino acid residues of the polynucleotide binding protein. Generally, at least one amino acid cross-linked by the closed part is cysteine ​​or a non-natural amino acid. Cysteine ​​or a non-natural amino acid can be introduced into the polynucleotide binding protein by replacing or modifying the naturally occurring amino acid residues of the polynucleotide binding protein. The method for introducing non-natural amino acids is well known in the art and includes, for example, natural chemical connection with a synthetic polypeptide chain containing such non-natural amino acids. The method for introducing cysteine ​​into the polynucleotide binding protein is also within the capabilities of those skilled in the art, such as using the technology disclosed in the following references: Sambrook et al., "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., "Current Protocols in Molecular Biology" (Supplement 114), John Wiley & Sons, New York (2016).

[0302] The length of the closed portion may be approximately to about The length of the closed portion can be calculated based on static bond lengths or, more preferably, using molecular dynamics simulations. The length can be, for example, about to about As promised to about For example, about 8 to about As about 10 to about or about For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or

[0303] Polynucleotide binding proteins suitable for closure using a closure moiety as described above are discussed in more detail herein. The polynucleotide binding protein is preferably a helicase, such as the Dda helicase described herein.

[0304] The polynucleotide binding protein may be or may be derived from an exonuclease. Suitable enzymes include, but are not limited to, exonuclease I from E. coli, exonuclease III from E. coli, RecJ from T. thermophilus and bacteriophage lambda exonuclease, TatD exonuclease and variants thereof.

[0305] The polynucleotide binding protein may be a polymerase. The polymerase may be 3173 DNA polymerase (which can be obtained from Company), SD polymerase (available from In one embodiment, the enzyme is Phi29 DNA polymerase or a variant thereof. Modified forms of Phi29 polymerase that can be used in the present invention are disclosed in U.S. Pat. No. 5,576,204.

[0306] The polynucleotide binding protein can be a topoisomerase. In one embodiment, the topoisomerase is a member of any of the partial classification (EC) groups 5.99.1.2 and 5.99.1.3. The topoisomerase can be a reverse transcriptase, which is an enzyme capable of catalyzing the formation of cDNA from an RNA template. They can be obtained, for example, from the New England and Commercially available.

[0307] The polynucleotide binding protein is preferably a helicase. Any suitable helicase can be used according to the method of the present invention. For example, the enzyme used according to the present disclosure or each enzyme can be independently selected from Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase and Dda helicase or its variant. Monomeric helicase can include several domains connected together. For example, TraI helicase and TraI subgroup helicase can contain two RecD helicase domains, a release enzyme domain and a C-terminal domain. These domains usually form a monomeric helicase that can work and can not form an oligomer. The specific example of suitable helicase includes Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1 and TraI. These helicases usually act on single-stranded DNA. The example of the helicase that can move along the two chains of double-stranded DNA includes FtfK and a six-polymerase complex, or a multi-subunit complex, such as RecBCD. The polynucleotide binding protein is preferably a Dda (DNA-dependent ATPase) helicase.

[0308] Hel308 helicase is described in publications such as WO 2013 / 057495, the entire contents of which are incorporated by reference. RecD helicase is described in publications such as WO 2013 / 098562, the entire contents of which are incorporated by reference. XPD helicase is described in publications such as WO 2013 / 098561, the entire contents of which are incorporated by reference. Dda helicase is described in publications such as WO 2015 / 055981 and WO 2016 / 055777, the entire contents of which are incorporated by reference.

[0309] The helicase can be Trwc Cba or a variant thereof, Hel308 Mbu or a variant thereof, or Dda or a variant thereof. The variant can differ from the native sequence in any of the ways discussed herein. An exemplary variant of Dda comprises E94C / A360C. Another exemplary variant of Dda comprises E94C / A360C, and then (ΔM1)G1G2 (i.e., a deletion of M1, and then an addition of G1 and G2).

[0310] Disclaimer

[0311] The 3' end of the telomere adapter preferably does not contain one or more universal nucleotides. Universal nucleotides are nucleotides that hybridize or bind to all nucleotides in the template polynucleotide to some extent. Universal nucleotides are preferably nucleotides that hybridize or bind to nucleotides containing nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C) to some extent. Compared with the hybridization or binding strength with other nucleotides, universal nucleotides can hybridize or bind to some nucleotides with greater intensity. For example, universal nucleotides (I) containing nucleosides, 2'-deoxyinosine will show a preferential pairing order of IC>IA>IG approximately=IT. If universal nucleotides replace nucleotide species in a population, polymerase will replace the nucleotide species with universal nucleotides. For example, if a polymerase contacts a population of free dAMP, dTMP, dCMP and universal nucleotides, it will replace dGMP with universal nucleotides.

[0312] The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formyl indole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). The universal nucleotide more preferably comprises one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O'-methylinosine, 4-nitroindole 2'-deoxynucleoside, 4-nitroindole nucleoside, 5-nitroindole 2'-deoxynucleoside, 5-nitroindole nucleoside, 6-nitroindole 2'-deoxynucleoside, 6-nitroindole nucleoside, 3-nitropyrrole 2'-deoxynucleoside, 3-nitropyrrole nucleoside, a non-cyclic sugar analog of hypoxanthine, nitroindole nucleoside, The universal nucleotides are preferably 2'-deoxyinosine. The universal nucleotides are preferably 2'-deoxyinosine. The universal nucleotides are preferably IMP or dIMP. The universal nucleotide is most preferably dPMP (2'-deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2,6-diaminopurine monophosphate).

[0313] The method of the present invention preferably does not comprise restriction digestion. The method of the present invention preferably does not comprise at least a portion of amplified telomeres or polymerase chain reaction (PCR). The method of the present invention preferably does not comprise (i) restriction digestion and / or (ii) at least a portion of amplified telomeres or polymerase chain reaction (PCR). The method of the present invention preferably does not comprise (i) restriction digestion, (ii) at least a portion of amplified telomeres, and (iii) or polymerase chain reaction (PCR). These embodiments provide the advantages discussed above.

[0314] Telomere Source

[0315] The method of the invention may be performed on any suitable sample. The sample is typically a sample known to contain or suspected to contain at least a portion of a telomere.

[0316] The sample may be a biological sample. The present invention may be performed in vitro on a sample obtained or extracted from any organism or microorganism. Telomeres are generally present only in eukaryotic organisms. At least a portion of a telomere may be from any eukaryotic organism, including any of the eukaryotic organisms listed below.

[0317] The sample is preferably a fluid sample. The sample generally comprises a body fluid. The body fluid may be obtained from a human or an animal. The human or animal may suffer from, be suspected of suffering from, or be at risk of suffering from a disease. The sample may be urine, lymph, saliva, mucus, semen, or amniotic fluid, but preferably whole blood, plasma, or serum. Typically, the sample is of human origin, but alternatively, it may be from another mammal, such as from commercially raised animals, such as horses, cattle, sheep, or pigs, or alternatively may be a pet, such as a cat or dog.

[0318] Alternatively, samples of plant origin are typically obtained from commercial crops such as cereals, legumes, fruits or vegetables, for example wheat, barley, oats, rapeseed, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton, tea or coffee.

[0319] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water, such as drinking water, sea water or river water, and reagents for laboratory tests.

[0320] The sample may be processed prior to analysis, for example by centrifugation or passing through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately after it is taken. The sample may also be stored, preferably at -70°C, prior to analysis.

[0321] The sample typically comprises genomic DNA. The sample may comprise T cell DNA.

[0322] At least a portion of the telomere can be from a common organism, such as a plant or an animal. At least a portion of the telomere is typically obtained from a human or an animal, such as urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. At least a portion of the telomere can be obtained from a plant, such as a cereal, a legume, a fruit, or a vegetable.

[0323] General approach

[0324] As stated above, the methods of the invention may be performed using any suitable detector, and therefore any suitable apparatus for detecting polynucleotides may be used.

[0325] Any device suitable for nanopore sensing can be used to carry out the method of the present invention. For example, the device can include a chamber containing an aqueous solution and a barrier that divides the chamber into two sections. The barrier generally has an opening in which a membrane containing a transmembrane pore is formed. Transmembrane pores are described herein.

[0326] The method can be carried out using the apparatus described in WO 2008 / 102120, WO 2010 / 122293 or WO 00 / 28312 (the documents are incorporated herein by reference in their entirety). In short, the binding of molecules (e.g., target polynucleotides) in the channel of the hole will have an impact on the open channel ion flow through the hole, which is the essence of the "molecular sensing" of the hole channel. The change of the open channel ion flow can be measured by the change of the current using a suitable measurement technique. The degree of reduction of the ion flow measured by the reduction of the current is related to the size of the obstacle in or near the hole. Therefore, the binding of the molecules of interest (e.g., target polynucleotides) in or near the hole provides a detectable and measurable event, thereby forming the basis of a "biosensor". Detecting the presence of biomolecules can be applied to personalized drug development, medicine, diagnosis, life science research, environmental monitoring, and security and / or defense industry.

[0327] When used to characterize a polynucleotide, the presence, absence, or one or more characteristics of a target polynucleotide are determined. The method can be used to determine the presence, absence, or one or more characteristics of at least one target polynucleotide. The method can involve determining the presence, absence, or one or more characteristics of two or more target polynucleotides. The method can include determining the presence, absence, or one or more characteristics of any number of target polynucleotides (such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more target polynucleotides). Any number of characteristics of one or more target polynucleotides can be determined, such as 1, 2, 3, 4, 5, 10 or more characteristics. Characteristics suitable for detection in the methods provided herein include the identity or sequence of the polynucleotides, the length of the polynucleotides, whether the polynucleotides are modified, etc. In some embodiments, the method of the present invention is a method for sequencing at least a portion of a telomere. In some embodiments, the sequence of at least a portion of a telomere can be determined in real time by comparing the real-time signal or base call with a known reference. Exemplary methods for determining polynucleotide sequences are described in WO 2016 / 059427 (which is incorporated herein by reference in its entirety).

[0328] When used to characterize a polynucleotide, the method may involve measuring the ionic current flowing through the hole, usually by measuring the current. Alternatively, the ionic current flowing through the hole may be measured optically, as disclosed in Heron et al.: Journal of the American Chemical Society, Vol. 9, 131, No. 5, 2009. Therefore, the device may also include a circuit capable of applying an electric potential and measuring the electrical signal across the membrane and the hole. A patch clamp or a voltage clamp may be used to perform the characterization method. The characterization method preferably involves the use of a voltage clamp.

[0329] The method may involve measuring an optical signal as described in Chen et al., Nature Communications (2018) 9:1733, the entire contents of which are hereby incorporated by reference. For example, single-molecule surface enhanced Raman spectroscopy (SERS) may be locally enabled using nanopores such as optically engineered nanopore structures (e.g., plasmonic nanoslits) to allow characterization of polynucleotides by direct Raman spectroscopy detection.

[0330] The method can be performed on silicon-based arrays of wells, wherein each array comprises 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0331] The method can be related to measuring the electric current flowing through the hole. The method is usually carried out when the voltage is applied across the membrane and the hole. The voltage used is usually +2V to -2V, usually -400mV to +400mV. The voltage used is preferably in the range with a lower limit and an upper limit, and the lower limit is selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV, and the upper limit is independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more preferably in the range of 100mV to 240mV, and most preferably in the range of 120mV to 220mV. By using the applied potential increased, the discrimination between different nucleotides can be increased by a hole.

[0332] The method is generally carried out in the presence of any charge carrier, such as a metal salt, for example an alkali metal salt; a halogen salt, for example a chloride salt such as an alkali metal chloride salt. The charge carrier may include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) is generally used. KCl is preferred. The salt may be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration may be in a saturated state. The salt concentration may be 3M or less, and is typically 0.1M to 2.5M, 0.3M to 1.9M, 0.5M to 1.8M, 0.7M to 1.7M, 0.9M to 1.6M, or 1M to 1.4M. The salt concentration is preferably 150mM to 1M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow identification of currents indicative of binding / no binding against the background of normal current fluctuations.

[0333] The method is typically performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in an aqueous solution in the chamber. Any suitable buffer may be used. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The method is typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0334] The method can be carried out at a temperature of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is typically carried out at room temperature. The method is optionally carried out at a temperature that supports enzyme function, such as at about 37°C.

[0335] Any protein described herein, such as a protein pore, can be prepared synthetically or by recombinant means. For example, the pore can be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the pore can be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When the protein is produced by synthetic means, such amino acids can be introduced during production. The pore can also be altered after synthesis or recombinant production.

[0336] Any protein described herein, such as a protein pore, can be produced using standard methods known in the art. The polynucleotide sequence encoding the pore or construct can be derived and replicated using standard methods in the art. The polynucleotide sequence encoding the pore or construct can be expressed in a bacterial host cell using standard techniques in the art. The pore can be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0337] The pores can be produced on a large scale from protein producing organisms after purification by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system and Gilson HPLC system.

[0338] Polynucleotide telomeric adaptor

[0339] The present invention also provides a polynucleotide telomere adapter, wherein the 3' end of the adapter specifically hybridizes with the first portion of the protruding strand located at the end of the telomere, and the 5' end of the adapter is not complementary to the opposite portion of the protruding strand located at the end of the telomere. The telomere adapter can be any of the telomere adapters defined above with respect to the method of the present invention, including extended telomere adapters. The telomere adapter can further comprise a splint polynucleotide and / or a sequencing adapter.

[0340] The present invention also provides a population of six telomere adapters, each of which has a 3' end that specifically hybridizes with one of the six possible sequences of the first portion of the protruding strand located at the end of the telomere and a 5' end that is not complementary to the opposite portion of the protruding strand located at the end of the telomere. The population can be any of the populations defined above with respect to the method of the present invention, including extended telomere adapters.

[0341] The population preferably comprises six telomere adaptors comprising or consisting of the following sequences:

[0342] AGCAATACGTAACTGAACGAAGTACCCTAA(SEQ ID NO:1);

[0343] AGCAATACGTAACTGAACGAAGTAACCCTA(SEQ ID NO:2);

[0344] AGCAATACGTAACTGAACGAAGTTAACCCT(SEQ ID NO:3);

[0345] AGCAATACGTAACTGAACGAAGTCTAACCC(SEQ ID NO:4);

[0346] AGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 5); and

[0347] AGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 6).

[0348] These are shown in Table 3 and Table 3 of Example 1. Figure 1 middle.

[0349] The population preferably comprises six telomere adaptors comprising or consisting of the following sequences:

[0350] AGCAATACGTAACTGAACGAAGTCCCTAACCCTAACCCTAA (SEQ ID NO: 14); AGCAATACGTAACTGAACGAAGTTAACCCTAACCCTAACCC (SEQ ID NO: 15); AGCAATACGTAACTGAACGAAGTCTAACCCTAACCCTAACC (SEQ ID NO: 16);

[0351] AGCAATACGTAACTGAACGAAGTAACCCTAACCCTAACCCT (SEQ ID NO: 18); and

[0352] AGCAATACGTAACTGAACGAAGTACCCTAACCCTAACCCTA (SEQ ID NO: 19).

[0353] These are shown in Table 14.

[0354] The population preferably comprises six extended telomere adaptors comprising or consisting of the following sequences:

[0355] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTACCCTAA (SEQ ID NO: 1);

[0356] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTAACCCTA (SEQ ID NO: 2);

[0357] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTTAACCCT (SEQ ID NO: 3);

[0358] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCTAACCC (SEQ ID NO: 4);

[0359] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 5); and

[0360] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 6).

[0361] The population preferably comprises six extended telomere adaptors comprising or consisting of the following sequences:

[0362] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTACCCTAA (SEQ ID NO: 25);

[0363] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTAACCCTA (SEQ ID NO: 26);

[0364] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTTAACCCT(SEQ ID NO:27);

[0365] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCTAACCC (SEQ ID NO: 28);

[0366] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCCTAACC (SEQ ID NO: 29); and

[0367] GCTTGGGTGTTTAACCNNNNAGCAATACGTAACTGAACGAAGTCCCTAAC (SEQ ID NO: 30), wherein N is Int 5-nitroindole (i5NiTInd).

[0368] The population preferably comprises six extended telomere adaptors comprising or consisting of the following sequences:

[0369] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCCTAACCCTAACCCTAA (SEQ ID NO: 14);

[0370] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTTAACCCTAACCCTAACCC (SEQ ID NO: 15);

[0371] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCTAACCCTAACCCTAACC (SEQ ID NO: 16);

[0372] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTCCTAACCTAACCCTAACCCTAAC (SEQ ID NO: 17);

[0373] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTAACCCTAACCCTAACCCT (SEQ ID NO: 18); and

[0374] SEQ ID NO: 11 covalently linked to AGCAATACGTAACTGAACGAAGTACCCTAACCCTAACCCTA (SEQ ID NO: 19).

[0375] The two sequences in the extended adapter are preferably covalently linked by one or more, such as two or more, three or more or four or more universal nucleotides, such as Int 5-nitroindole (i5NiTInd). The two sequences in the extended adapter are more preferably covalently linked by 4 i5NiTInds. The extended telomere adapter preferably comprises a click chemistry group at its 5' end, such as DBCOTEG. Examples of these extended adapters are shown in Tables 7 and Figure 4 middle.

[0376] The telomere adaptors in the population may each further comprise a splint polynucleotide and / or a sequencing adaptor.

[0377] Kits and Systems

[0378] The present invention also provides a kit for characterizing at least a portion of a telomere, the kit comprising (a) one or more polynucleotide telomere adapters of the present invention or a group of six telomere adapters of the present invention, and (b) one or more splint polynucleotides or one or more polynucleotide extensions. The one or more telomere adapters or the group of six telomere adapters can be any of the telomere adapters or groups defined above for the method of the present invention. The one or more splint polynucleotides or the one or more polynucleotide extensions can be any of the splint polynucleotides or polynucleotide extensions defined above for the method of the present invention. The one or more telomere adapters and the one or more polynucleotide extensions preferably comprise a click chemistry group.

[0379] The kit of the present invention preferably further comprises one or more sequencing adapters. The one or more sequencing adapters may be any of the sequencing adapters discussed above with respect to the method of the present invention.

[0380] The kit preferably further comprises a polymer-guided effector protein and one or more guide polymers. The polymer-guided effector protein and the one or more guide polymers can be any of the polymer-guided effector proteins and guide polymers discussed above with respect to the methods of the present invention. The polymer-guided effector protein is preferably a Cas9 protein. The one or more guide polymers are preferably one or more guide RNAs.

[0381] The present invention also provides a system for performing the method of the present invention. The system is used to characterize at least a portion of a telomere. The system comprises (a) one or more polynucleotide telomere adapters of the present invention or a population of six telomere adapters of the present invention, and (b) a nanopore. Any of the embodiments discussed above with respect to the method of the present invention is also applicable to the system of the present invention.

[0382] Nanopores are preferably present in the membrane. Suitable membranes are discussed above. The system may include any of the membranes disclosed above, such as an amphiphilic layer, a triblock copolymer membrane, or a solid-state membrane. The membrane is typically part of an array of membranes, each of which preferably includes nanopores. The array may be any of the arrays described in WO 2018 / 060740 (which is incorporated herein by reference in its entirety).

[0383] The system is preferably adjusted to apply a voltage across the membrane and perform one or more electrical measurements. Suitable adjustments are discussed in WO 2018 / 060740 (which is incorporated herein by reference in its entirety).

[0384] The system may further comprise a polynucleotide binding protein. The kit may further comprise microparticles. Any of the embodiments discussed above with respect to the method of the invention are also applicable to the system of the invention.

[0385] The system may further comprise one or more splint polynucleotides, one or more polynucleotide extensions and / or one or more sequencing adaptors.Any of the embodiments discussed above with respect to the methods of the invention are equally applicable to the systems of the invention.

[0386] System or test kit can additionally comprise one or more other reagents or instruments that enable any embodiment in the above-mentioned embodiment to be implemented.Such reagent or instrument include one or more of the following: suitable buffer (aqueous solution), a device for obtaining a sample from a subject (such as a container or instrument comprising a needle), a device for amplification and / or expression of polynucleotides, a film as defined above or voltage or patch clamp device. Reagent can be present in a system or test kit in a dry state so that a fluid sample is used for resuspending reagent. System or test kit can also optionally comprise instructions that enable the system or test kit to be used in the method described herein or details about which organism the method can be used for. System or test kit can include magnet or electromagnet. System or test kit can optionally include nucleotides.

[0387] The following examples illustrate the present invention. It should be understood that although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for methods according to the present invention, various changes or modifications may be made in form and detail without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments and should not be construed as limiting the present application. The present application is limited only by the claims.

[0388] Example 1

[0389] This example demonstrates the assembly of telomeric adapters as described herein, and their use in combination with sequencing adapters for nanopore sequencing. Sequencing adapters are attached via a ligation reaction.

[0390] Telomeric adaptors assembly and ligation to chromosome ends

[0391] Assemble telomeric adapters using the sequences in Table 3 below (eg Figure 1 shown).

[0392] Table 3 - T1 to T6 telomere adapters used in Example 1:

[0393]

[0394] Underlined bases hybridize to the G-rich strand.

[0395] 1) 1 μM T1 to T6 were mixed in equal volumes to prepare "Telo-Mix".

[0396] 2) The ligation reaction solution is assembled as shown in Table 4 below:

[0397]

[0398] 3) Incubation was performed at 35°C for 16 hours followed by heat inactivation of T4 at 65°C for 10 minutes.

[0399] This protocol includes an optional step of digesting the adaptor-ligated DNA:

[0400] 1) Add 4 μL EcoRI to 200 μL ligation reaction.

[0401] 2) Incubate at 37°C for 15 minutes.

[0402] Bonding of "splint" oligonucleotides

[0403] The splint oligonucleotides were ligated as follows ( Figure 2 shown).

[0404] 1) Purify the above assembly reaction solution using 0.4x SPRI beads.

[0405] 2) Elute the DNA in 190 μL DI water (37°C, 15 min, with occasional mixing).

[0406] 3) Add 10 μL of NaCl (1 M) solution to make the final NaCl concentration 50 mM.

[0407] 4) Add 2 μL of 10 μM "splint" oligonucleotide to make the final "splint" concentration 100 nM.

[0408] Table 5:

[0409]

[0410] Underlined bases can hybridize to nanopore sequencing adaptors.

[0411] 5) Incubate at 50°C for 1 hour.

[0412] 6) Purify the reaction solution using 0.4x SPRI beads.

[0413] 7) Elute in 30 μL of water (37°C, 15 minutes, with occasional mixing).

[0414] Ligation of Nanopore Sequencing Adapters

[0415] 1) Assemble the sequencing adapter ligation reaction using the Adapter Mix II Expansion kit from Oxford Nanopore Technologies as follows ( Figure 3 ):

[0416] Table 6:

[0417]

[0418]

[0419] 2) Incubate at room temperature for 20 minutes.

[0420] 3) The reaction solution was cleaned up with 20 μL of AXP beads and LFB.

[0421] 4) Elution in 15 μL of elution buffer (37° C., 15 minutes).

[0422] Nanopore sequencing was performed using a GridION device from Oxford Nanopore Technologies Ltd equipped with a FLO-MIN106 flow cell.

[0423] Example 2

[0424] This example demonstrates the assembly of telomeric adapters as described herein, and their use in combination with sequencing adapters for nanopore sequencing. Sequencing adapters are ligated via "click" chemistry.

[0425] The telomeric adapter was assembled using the following sequence ( Figure 4 )

[0426] 1) Click on all telomeric adaptors with 5' DBCOTEG (Table 7):

[0427]

[0428] Underlined bases hybridize to the G-rich strand.

[0429] 2) 1 μM T1 to T6 were mixed in equal volumes to prepare "Telo Mix".

[0430] 3) The ligation reaction solution was assembled as follows (Table 8):

[0431]

[0432] 4) Incubate at 35°C for 16 hours, followed by heat inactivation of T4 at 65°C for 10 minutes.

[0433] 5) Add 1 μL of NEB exonuclease I to the reaction solution.

[0434] 6) Incubate at 37°C for 15 minutes, then incubate at 80°C for 15 minutes.

[0435] This protocol may include an optional step to digest the "Telo Mix" ligated DNA:

[0436] 1) Add 4 μL EcoRI to 200 μL ligation reaction.

[0437] 2) Incubate at 37°C for 15 minutes.

[0438] Addition of "click" nanopore sequencing adapters

[0439] 1) Purify the above reaction solution using 0.4x SPRI beads.

[0440] 2) Elute in 12 μL elution buffer (37°C, 15 minutes)

[0441] 3) Add 1 μL of sequencing adapter RAP-T from the kit SQK-PCS111 from Oxford Nanopore Technologies.

[0442] 4) Incubate at room temperature for 10 minutes.

[0443] Nanopore sequencing was performed using a GridION device from Oxford Nanopore Technologies Ltd equipped with a FLO-MIN106 flow cell.

[0444] Example 3

[0445] This example demonstrates the assembly of biotinylated telomeric adapters as described herein, and their use in combination with sequencing adapters for nanopore sequencing. Sequencing adapters are attached via a ligation reaction.

[0446] Biotinylated telomeric adaptors can be ligated to chromosome ends, allowing sequencing of both strands.

[0447] Assemble the biotinylated telomeric adaptor using the following sequence ( Figure 6 shown).

[0448] 1) Biotinylated telomeric adaptors (Table 9):

[0449]

[0450] Underlined bases hybridize to the G-rich strand.

[0451] 1) 1 μM T1 and T2 were mixed in equal volumes to prepare "Telo-Mix".

[0452] 2) The bonding reaction solution is assembled as follows (Table 10):

[0453]

[0454] 3) Incubate at 35°C for 16 hours.

[0455] 4) Follow Streptavidin Biotinylated pull-down protocol. Telomeric strands were eluted into 48 μL of water by heating the beads at 50 °C.

[0456] At this point, the telomeric reads of interest are enriched in the supernatant, and the telomeric adaptors have been ligated to the beads, separated from the free-floating strands with telomeric overhangs. The protocol now proceeds with processing the supernatant according to the ONT "Genomic DNA Ligation (SQK-LSK110)" protocol.

[0457] 5) Use FFPE DNA Repair Mix and Ultra TM II end repair / dA-tailing module fills in telomere overhangs (Table 11):

[0458]

[0459] Using a thermal cycler, the reaction solution was incubated at 20°C for 5 minutes and at 65°C for 5 minutes.

[0460] 6) The reaction solution was purified using 1x SPRI beads and 70% ethanol.

[0461] 7) Elute the DNA in 61 μL DI water (room temperature, 10 minutes with occasional mixing).

[0462] 8) Assemble the sequencing adapter ligation reaction solution (Table 12) using the sequencing adapters from the LSK109-112 kit from Oxford Nanopore Technologies Ltd. as follows:

[0463]

[0464]

[0465] 9) Incubate the reaction solution at room temperature for 10 minutes.

[0466] 10) Clean up the reaction solution with 40 μL AXP beads and LFB.

[0467] 11) Elution in 15 μL of elution buffer (37° C., 15 minutes).

[0468] The ends are attached by ligation with dA tails and ONT sequencing adaptors. Sequencing was performed using a GridION device from Oxford Nanopore Technologies equipped with a FLO-MIN106 flow cell.

[0469] Example 4

[0470] This example demonstrates the analysis of data obtained from the experiments described in Example 1.

[0471] Base calls were performed on data output in Fast5 file format using Bonito basecaller* (Oxford Nanopore Technologies Ltd.) with a telomere-trained model.

[0472] *https: / / github.com / nanoporetech / bonito.

[0473] Potential telomeric reads were identified in base space using a noise-removing repeat finder with a minimum motif repeat length of 500 bp. Reads were sorted using a custom script, with the order of the reads predetermined by the nature of the chemistry.

[0474] Reads were mapped to CHM13 v1.1, which added an alternative subtelomeric assembly with a mini-mapping aligner, allowing reads from subtelomeric regions to be anchored into the P and Q arms.

[0475] Mapped reads must have telomeric and subtelomeric alignments. At least 20% of the reads align to a reference to be considered in future steps.

[0476] When a read had multiple mapped alignments, a single alignment was chosen by selecting the longest read coverage and identity.

[0477] Methylation was interpreted using the Remora tool (Oxford Nanopore Technologies Ltd.); https: / / github.com / nanoporetech / remora.

[0478] The results are shown in Figure 8-13 middle.

[0479] Example 5

[0480] Table 13 below shows the total number of telomere reads, and the length of subtelomeres that are important for uniquely mapped reads, obtained by a variety of different methods, including restriction digestion methods and the "Teloseq" method described herein. MobI+AluI has the shortest subtelomere length, which makes uniquely mapped reads challenging, and this becomes apparent by the percentage mapped.

[0481] Table 13:

[0482]

[0483]

[0484] Example 6

[0485] This example demonstrates the use of a custom base caller to identify and map telomeric sequences enriched using the methods of the invention.

[0486] The Bonito base calling model was trained to optimize specifically for telomeric repeat sequences and reads were filtered using a noise-removing repeat finder ( Fig.14 a). Isolated telomeric reads were aligned to the CHM13v2.0 reference and alternative subtelomeric assemblies using minimap2. Alignment to subtelomeric regions was used to anchor reads to specific chromosome arms. Various telomere enrichment methods were compared using the human cell line HG002 ( Fig.14 b) The "Teloseq" method of the present invention was compared with other methods, including restriction digestion using AluI and MboI, which do not cut telomeres, Cas9-based sequencing using guide RNAs targeting subtelomeric regions, and whole genome sequencing. Fig.14 b shows that the "Teloseq" method of the present invention enables the identification of the largest number of telomeric sequences.

[0487] Example 7

[0488] Telomere sequencing was performed using the techniques described above, with sequencing data obtained using an R10 flow cell from Oxford Nanopore Technologies Ltd.

[0489] The Bonito base calling model was trained to optimize specifically for telomeric repeat sequences, using a stringent filtering phase to isolate telomeric motifs. Isolated telomeric reads were aligned to custom references using minimap2, including: CHM13 v2.0, HG002, and an alternative subtelomeric assembly that is nearly digested and retains the P and Q arms ( Fig.15 a).

[0490] Using the human cell line HG002, we captured 23,236 reads of telomeric motifs containing at least 50 bp of continuous repeats, of which 18,519 telomeric reads were uniquely mapped to chromosome arms ( Fig.15 b). Mapped telomere reads showed a median telomere length of 3,826 bp ( Fig.15 c).

[0491] Example 8

[0492] This example demonstrates phasing of telomeric reads, which can provide valuable telomeric variant information.

[0493] HG002 telomeric reads were uniquely mapped to the phased HG002 assembly of 88 autosomal haploidized chromosome arms, successfully aligning to 86 arms. In chromosome 18.q, telomeric SNP variants were observed only in the maternal copy, highlighted in dark grey ( Fig.16 a). When a mapping quality threshold of 30 and above is applied, the coverage of each haploidized chromosome arm is approximately 200-fold ( Fig.16 b). It was observed that the mapping quality of some chromosomes was low, such as that of 1.p and 21.p. The light-colored boxes represent paternal haploidized chromosomes, where no uniquely mapped telomere alignment to the reference was identified (chr13, chr22).

[0494] Example 9

[0495] This example demonstrates the mapping of haploidized telomeric reads to provide high-resolution telomere length measurements.

[0496] HG002 telomeric reads were uniquely mapped to the phased HG002 assembly to assess telomere length ( Fig.17). Without phasing, the average P arm telomere length was 3,768bp, while the average Q arm telomere length was 4,062bp. On average, the Δ between maternal and paternal telomere lengths was 91. However, when the haplotype was split by chromosome arms, there was a significant difference between the paternal haploid arms (Δ523). The paternal P arm average was 3,690bp, while the paternal Q arm average was 4,213bp. There was no significant difference between the maternal haploid arm telomere lengths (Δ73). The maternal P arm average was 3,850bp, while the maternal Q arm average was 3,923bp.

[0497] Example 10

[0498] Example 1 was repeated using the telomere adaptors shown in Table 14 and the splint polynucleotides in Table 15.

[0499] Table 14 - Telomere adapters used in Example 10:

[0500]

[0501] Table 15 - Splint polynucleotides used in Example 10:

[0502]

Claims

1. A method for characterizing at least a portion of a telomere, the method comprising: (a) ligating a polynucleotide telomere adaptor to the 5' end of a non-overhanging strand located at the end of the telomere, wherein the 3' end of the adaptor specifically hybridizes to a first portion of the overhanging strand and the 5' end of the adaptor does not hybridize to an opposing portion of the overhanging strand; and (b) characterizing the ligated non-protruding strand of the at least a portion of the telomere in the 5' to 3' direction from the end of the telomere using the telomere adaptor.

2. The method of claim 1, wherein characterizing at least a portion of the telomere comprises (a) sequencing at least a portion of the telomere, (b) measuring the length of at least a portion of the telomere, (c) performing telomere-to-telomere assembly of a chromosome or genome, (d) identifying telomere or chromosome fusions, (e) identifying one or more modifications in at least a portion of the telomere, (f) identifying at least a portion of the telomere as a variant, or (g) linking at least a portion of the telomere to a specific cell or tissue type.

3. A method according to claim 1 or 2, wherein the method is used to characterize (i) the entire telomere, (ii) the entire telomere and at least a portion of a subtelomere or the entire subtelomere, (iii) the entire telomere, the entire subtelomere and at least a portion of the chromatin or the entire chromatin, (iv) the entire telomere, the entire subtelomere, the entire chromatin and at least a portion of the relative subtelomere or the entire relative subtelomere, or (v) the entire telomere, the entire subtelomere, the entire chromatin, the entire relative subtelomere and at least a portion of the relative telomere or the entire relative telomere.

4. The method according to any one of the preceding claims, wherein the method is used to characterize at least a portion of one or two telomeres on each of two or more different chromosomes.

5. The method according to any one of the preceding claims, wherein step (a) further comprises hybridizing a splint polynucleotide to the 5' end of the telomeric adaptor.

6. The method of claim 5, wherein the splint polynucleotide is compatible with a sequencing adaptor.

7. A method according to any of the preceding claims, wherein step (a) further comprises ligating a sequencing adapter to the telomere adapter and, if present, to the splint polynucleotide, and step (b) comprises characterizing the ligated non-protruding strand in the 5' to 3' direction of at least a portion of the telomere using the sequencing adapter.

8. The method according to any one of claims 1 to 4, wherein step (a) further comprises ligating or covalently attaching the polynucleotide extension to the telomere adaptor, or step (a) uses a telomere adaptor, and the telomere adaptor further comprises a polynucleotide extension at its 5' end.

9. The method of claim 8, wherein the polynucleotide extension comprises a sequencing adapter, or step (a) further comprises covalently linking a sequencing adapter to the polynucleotide extension, and step (b) comprises using the sequencing adapter to characterize the ligated non-protruding strand of at least a portion of the telomere in the 5' to 3' direction from the end of the telomere.

10. The method of claim 8 or 9, wherein the polynucleotide extension is covalently linked to the telomere adaptor or covalently linked to the sequencing adaptor using click chemistry.

11. The method of any of the preceding claims, wherein the telomeric adaptor comprises biotin.

12. The method of claim 11, wherein step (a) further comprises enriching the ligated non-protruding strand of the at least a portion of the telomere using the biotin.

13. The method of any of the preceding claims, wherein the 3' end of the telomeric adaptor is at least 5 or at least about 7 nucleotides in length.

14. The method according to any one of the preceding claims, wherein there are one or more linkers or spacers between the 3' end of the telomere adaptor and the 5' end of the telomere adaptor.

15. The method of claim 14, wherein the linker is a flexible linker or a spacer.

16. A method according to any of the preceding claims, wherein the method comprises contacting the telomere with a population of six telomere adaptors prior to step (a), each of the six telomere adaptors having a 3' end that specifically hybridizes to one of six possible sequences of the first portion of the protruding chain and a 5' end that does not hybridize to the relative portion of the protruding chain.

17. The method of any one of the preceding claims, wherein the ligated non-protruding strands are characterized using a nanopore.

18. The method according to any one of the preceding claims, wherein the method does not comprise (i) restriction digestion and / or (ii) amplification of the at least a portion of the telomere or polymerase chain reaction (PCR).

19. The method according to any one of the preceding claims, wherein the method further comprises characterizing the unligated protruding strands of the at least a portion of the telomere in the 5' to 3' direction towards the end of the telomere.

20. The method of claim 19, wherein the method comprises using a polymer-guided effector protein to generate a double-stranded break at an opposite end of the at least a portion of the telomere relative to the telomere end and ligating a sequencing adapter to the opposite end, and using the sequencing adapter to characterize the unligated protruding strand of the at least a portion of the telomere in the 5' to 3' direction toward the end of the telomere.

21. The method according to any one of claims 1 to 18, wherein the method is repeated at the other end of the chromosome and the method comprises characterizing both strands of the entire chromosome.

22. A method for characterizing at least a portion of a telomere, the method comprising (a) ligating a polynucleotide telomere adapter to the 5' end of a non-protruding strand located at the end of the telomere, wherein the 3' end specifically hybridizes to a first portion of the protruding strand and the 5' end of the adapter does not hybridize to an opposite portion of the protruding strand, (b) using a polymer-guided effector protein to generate a double-stranded break at an opposite end of at least a portion of the telomere relative to the telomere end and ligating a sequencing adapter to the opposite end, and (c) using the telomere adapter to characterize the ligated non-protruding strand of at least a portion of the telomere in a 5' to 3' direction from the end of the telomere, and using the sequencing adapter to characterize the unligated protruding strand of at least a portion of the telomere in a 5' to 3' direction toward the end of the telomere.

23. A polynucleotide telomere adaptor, wherein the 3' end of the adaptor specifically hybridizes to a first portion of a protruding strand at the end of a telomere, and the 5' end of the adaptor does not hybridize to an opposing portion of the protruding strand at the end of the telomere.

24. The polynucleotide telomere adaptor of claim 23, wherein the adaptor is as defined in claim 11, 13 or 14.

25. A population of six telomere adaptors, each of the six telomere adaptors having a 3' end that specifically hybridizes to one of six possible sequences of a first portion of a protruding strand located at the end of a telomere and a 5' end that does not hybridize to a relative portion of the protruding strand located at the end of the telomere.

26. A population according to claim 25, wherein each linker is defined as in claim 11, 13 or 14.

27. A kit for characterizing at least a portion of a telomere, the kit comprising (a) one or more polynucleotide telomere adapters according to claim 23 or 24 or a group of six telomere adapters according to claim 25 or 26, and (b) one or more splint polynucleotides or one or more polynucleotide extensions.

28. The kit of claim 27, wherein the telomere adaptor and the one or more polynucleotide extensions comprise click chemistry groups.

29. The kit of claim 27 or 28, wherein the kit further comprises one or more sequencing adapters.

30. The kit according to any one of claims 27 to 29, wherein the kit further comprises a polymer-guided effector protein and one or more guide polymers.

31. A system comprising (a) one or more polynucleotide telomere adapters according to claim 23 or 24 or a population of six telomere adapters according to claim 25 or 26, and (b) a nanopore.

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