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WO2024249061A1 - Détection d'un nucléotide méthylé à l'aide d'un extincteur - Google Patents

Détection d'un nucléotide méthylé à l'aide d'un extincteur Download PDF

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WO2024249061A1
WO2024249061A1 PCT/US2024/029126 US2024029126W WO2024249061A1 WO 2024249061 A1 WO2024249061 A1 WO 2024249061A1 US 2024029126 W US2024029126 W US 2024029126W WO 2024249061 A1 WO2024249061 A1 WO 2024249061A1
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Prior art keywords
quencher
polynucleotide
methylated
coupled
nucleotide
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Rebekah KARADEEMA
Gaetano SPECIALE
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Illumina Inc
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Illumina Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the enzyme 5-MTase may add a methyl group to the 5-position of cytosine to form 5-methylcytosine (5-mC) in a manner such as described in Deen et al., “Methyltransferase-directed labeling of biomolecules and its applications,” Angewandte Chemie International Edition 56: 5182-5200 (2017), the entire contents of which are incorporated by reference herein.
  • Other enzyme(s) may oxidize the cytosine’s methyl group to form the 5-mC derivative 5-hydroxymethylcytosine (5-hmC), and may oxidize the 5-hmC further to form the 5-mC derivatives 5-formylcytosine (5-fC) and 5- carboxylcytosine (5-caC).
  • 5-mC and 5-hmC may be referred to as epigenetic markers, and it can be desirable to detect them in a genomic sequence.
  • 5-mC is proposed to have diverse roles in 1 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO regulation of gene expression, parental imprinting, and molecular etiology of human diseases including cancer.
  • cfDNA circulating cell-free DNA
  • Two broad categories of approaches have been developed to measure DNA methylation.
  • Enrichment strategies select methylated DNA fragments using a 5-mC-specific antibody, methylation-sensitive restriction enzymes, or methylation-induced changes in DNA duplex stability.
  • the methylated DNA fragments then can be measured in relation to a non- enriched sample by qPCR or other standard nucleic acid quantitation strategies.
  • Methylation assays based on chemical transformation begin by treating the sample with a chemical or enzymatic reagent that creates a difference in base pairing between methylated and non- methylated cytosine residues.
  • the current golden standard method for detecting 5-mC and 5- hmC is bisulfite sequencing, which converts any unmethylated C in the sequence to uracil (U), but does not convert 5-mC or 5-hmC to the corresponding uracil derivatives.
  • U uracil
  • 5-hmC uracil
  • PCR polymerase chain reaction
  • the uracil is amplified as thymidine (T)
  • T thymidine
  • the unmethylated C is sequenced as T.
  • the 5-mC and 5-hmC are amplified as C, and as such are sequenced as C.
  • any Cs in the sequence may be identified as corresponding to 5-mC or 5-hmC because they had not been converted to U.
  • Such a scheme may be referred to as a “three-base” sequencing scheme because any unmethylated C is converted to T.
  • this type of scheme reduces sequence complexity and may lead to reduced sequencing quality, lower mapping rates, and relatively uneven coverage of the sequence.
  • [0007] Despite the importance of DNA methylation in the etiology of many human diseases, and the identification of hundreds of methylation biomarkers for cancer and other disorders, only a small number of methylation-based diagnostic assays have been adopted for use in the clinic. A major reason for this discrepancy is the relative difficulty of measuring cytosine methylation as compared to SNPs and other DNA sequence changes.
  • Cytosine methylation is a relatively minor chemical change in the structure of the nucleobase, and on its own does not change the pattern of hydrogen bond donors and acceptors that govern specific base pairing.
  • Examples provided herein are related to detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide. Compositions and methods for performing such detection are disclosed. [0009] Some examples herein provide a method for detecting a first methylated nucleotide in a polynucleotide.
  • the method may include coupling a first quencher to the first methylated nucleotide.
  • the method may include adding fluorescently labeled nucleotides and a polymerase to a primer hybridized to the polynucleotide.
  • the method may include using the first quencher to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides.
  • the method may include using the reduced fluorescence caused by the first quencher to detect the first methylated nucleotide.
  • the first methylated nucleotide is selected from the group consisting of 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxylcytosine (5-caC) and 6-methyladenine (6-mA).
  • coupling the first quencher to the first methylated nucleotide includes: oxidizing the 5-mC, 5-hmC, or 5-fC to 5-carboxylcytosine (5-caC); reacting the 5- carboxyl group with a first molecule to form a first product.
  • the first product includes the first quencher.
  • the method further includes coupling the first quencher to the first product.
  • a ten-eleven translocation (TET) dioxygenase is used to oxidize the 5-mC, 5-hmC, or 5-fC to 5-caC.
  • a chemical reagent is used to oxidize the 5-mC, 5-hmC, or 5-fC.
  • the method further includes activating the 5-carboxyl group of the 5-caC before reacting the 5-carboxyl group with the first molecule.
  • the 5- carboxyl group of the 5-caC is activated using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- morpholinium chloride (DMTMM) or 1-ethyl-3-(3'- (dimethylamino)propyl)carbodiimide (EDC).
  • the first molecule includes a nucleophile.
  • the first molecule includes an azirine.
  • the first methylated nucleotide is 6-mA. 3 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO [0015]
  • the first methylated nucleotide is 5-mC.
  • coupling the first quencher to the first methylated nucleotide includes: reacting the 5-methyl group of the 5-mC with a first molecule to form a first product; and reacting the first product with a second molecule to couple the first quencher to the first product.
  • reacting the methyl group of the 5-mC with the first molecule includes using CMD1 to couple the first molecule to the 5-methyl group.
  • the first product includes a diol.
  • the second molecule includes a boronate.
  • coupling the first quencher to the first methylated nucleotide includes: oxidizing the 5-mC to 5-hmC; and reacting the 5-hydroxymethyl group of the 5- hmC with a first molecule to form a first product; and reacting the first product with a second molecule to couple the first quencher to the first product.
  • a ten-eleven translocation (TET) dioxygenase is used to oxidize the 5-mC to the 5-hmC.
  • the TET dioxygenase includes ccTET.
  • reacting the hydroxymethyl group of the 5-hmC with the first molecule includes using Mha.I to couple the first molecule to the hydroxymethyl group.
  • the first molecule includes an aminothiol.
  • the second molecule includes an N-hydroxysuccinimide (NHS) ester, an isocyanate, or an isothiocyanate.
  • the first methylated nucleotide is 5-hmC.
  • coupling the quencher to the first methylated nucleotide includes: reacting the 5- hydroxymethyl group of the 5-hmC with a first molecule to form a first product; and reacting the first product with a second molecule to couple the first quencher to the first product.
  • reacting the hydroxymethyl group of the 5-hmC with the first molecule includes using Mha.I to couple the first molecule to the hydroxymethyl group.
  • the first molecule includes an aminothiol.
  • the second molecule includes an N-hydroxysuccinimide (NHS) ester, an isocyanate, or an isothiocyanate.
  • the polynucleotide includes a second methylated nucleotide
  • the method further may include: coupling a second quencher to the second methylated nucleotide; using the second quencher to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides; and using the reduced fluorescence caused by the second quencher to detect the second methylated nucleotide.
  • the polynucleotide is coupled to a substrate.
  • the polynucleotide is within a cluster of polynucleotide amplicons coupled to the substrate.
  • the polynucleotide amplicons of the cluster also respectively include first methylated nucleotides
  • the method further may include coupling the first quencher to the first methylated nucleotides of the respective amplicons; adding fluorescently labeled nucleotides to primers respectively hybridized to the amplicons; using the first quencher to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides; and using the reduced fluorescence caused by the first quencher to detect the first methylated nucleotides in the respective amplicons.
  • the polynucleotide may include a methylated nucleotide coupled to a quencher.
  • the methylated nucleotide is directly coupled to the quencher.
  • the methylated nucleotide is indirectly coupled to the quencher.
  • the methylated nucleotide is selected from the group consisting of: 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5- carboxylcytosine (5-caC), and 6-methyladenine (6-mA).
  • the methylated nucleotide is 5-caC. In some examples, the 5-caC is coupled to the quencher via a carboxyl group of the 5-caC. In some examples, the 5-caC is coupled to the quencher via an azirine. [0024] In some examples, the methylated nucleotide is 6-mA. [0025] In some examples, the methylated nucleotide is 5-mC. In some examples, the 5-mC is coupled to the quencher via a diol. In some examples, the 5-mC is coupled to the quencher via a boronate. [0026] In some examples, the methylated nucleotide is 5-hmC.
  • the 5- hmC is coupled to the quencher via an aminothiol. In some examples, the 5-hmC is coupled to the quencher via an N-hydroxysuccinimide (NHS) ester, an isocyanate, or an isothiocyanate.
  • NHS N-hydroxysuccinimide
  • the polynucleotide is coupled to a substrate. In some examples, the polynucleotide is within a cluster of polynucleotide amplicons coupled to the substrate.
  • the polynucleotide amplicons of the cluster also include first methylated nucleotides and quenchers respectively coupled thereto.
  • FIGS.1A-1H schematically illustrate example compositions and operations in a process flow for detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide.
  • FIGS.2A-2C illustrate plots of example fluorescence intensity as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including a methylated nucleotide having a quencher coupled thereto, for different types of systems.
  • FIGS.3A-3E schematically illustrate example compositions and operations in a process flow for detecting different methylated nucleotides using different quenchers respectively coupled to the different methylated nucleotides.
  • FIG.4 illustrates a plot of example fluorescence intensities as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including different methylated nucleotides having different quenchers respectively coupled thereto.
  • FIG.5 schematically illustrates example compositions and operations in a process flow for detecting methylated nucleotides within a cluster of amplicons.
  • FIG.6 illustrates a flow of operations in an example method for detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide.
  • FIGS.7A-7F schematically illustrate example compositions and operations in a process flow for producing clonal clusters that preserve the CpG methylation state of a target polynucleotide. 6 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO [0036]
  • FIG.8 shows an example sequence of the human template-dependent DNA (cytosine- 5)-methyltransferase 1 (DNMT1, SEQ ID NO:1).
  • Examples provided herein are related to detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide. Compositions and methods for performing such detection are disclosed. [0038] Provided herein is detection of nucleotide methylation in which a quencher coupled to a methylated nucleotide generates a signal indicative of the methylated nucleotide. In a manner such as described in greater detail below, the quencher may be coupled to any of a variety of methylated nucleotides that may occur within a polynucleotide.
  • Such methylated nucleotide may be naturally occurring within the polynucleotide, or may be the product of a reaction with a nucleotide within the polynucleotide.
  • a primer may be hybridized to the polynucleotide, and the primer may be extended using a polymerase to add fluorescently labeled nucleotides.
  • the intensity of the fluorescent signal from the nucleotides being added may have relatively high level at locations that are sufficiently spatially separated from the quencher, may decrease to a minimum at a location adjacent to the quencher.
  • the fluorescent signals from the various nucleotides being added may be used to determine the sequence of the polynucleotide, e.g., using sequencing-by-synthesis as is known in the art.
  • the location of the quencher (that is, where the signal may be most reduced) may be correlated to the location and identity of the methylated nucleotide at which the signal is most reduced.
  • the present methylation detection may be performed directly on the sequencer without the need for user-based library preparation, which has traditionally used chemistry to convert either mC or unmethylated C to T, which otherwise may result in difficulties in read mapping such as explained above.
  • the present methylation detection is compatible, among other things, with PCR-free library preparations.
  • the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.”
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
  • the terms “substantially,” “approximately,” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they may refer to less than or equal to ⁇ 10%, such as less than or equal to ⁇ 5%, such as less than or equal to ⁇ 2%, such as less than or equal to ⁇ 1%, such as less than or equal to ⁇ 0.5%, such as less than or equal to ⁇ 0.2%, such as less than or equal to ⁇ 0.1%, such as less than or equal to ⁇ 0.05%.
  • hybridize is intended to mean noncovalently associating a first polynucleotide to a second polynucleotide along the lengths of those polymers to form a double-stranded “duplex.” For instance, two DNA polynucleotide strands may associate through complementary base pairing. The strength of the association between the first and second polynucleotides increases with the complementarity between the sequences of nucleotides within those polynucleotides. The strength of hybridization between polynucleotides may be characterized by a temperature of melting (Tm) at which 50% of the duplexes disassociate from one another.
  • Tm temperature of melting
  • pairs of bases may be “opposite” to each other, and the bases of that pair may be said to “associate” with each other.
  • bases of a given pair are complementary to each other, those bases also may be said to “hybridize” to one another.
  • the bases may be said to “disassociate” from each other.
  • nucleotide is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase.
  • a nucleotide that lacks a nucleobase may be referred to as “abasic.”
  • Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof.
  • nucleotides examples include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxy
  • nucleotide also is intended to encompass any nucleotide analogue (also referred to as a modified base) which is a type of nucleotide that includes a modified nucleobase, sugar and/or phosphate moiety compared to naturally occurring nucleotides.
  • Example modified nucleobases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2- aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2- thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8- halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl 9 SMRH:4879-1594-7704.2 IP-2313
  • modified bases may include targets and/or fluorophores in a manner such as described elsewhere herein.
  • certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5'-phosphosulfate.
  • Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.
  • polynucleotide refers to a molecule that includes a sequence of nucleotides that are bonded to one another.
  • a polynucleotide is one nonlimiting example of a polymer.
  • polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof.
  • a polynucleotide may be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or may include a mixture of a single stranded and double stranded sequences of nucleotides.
  • Double stranded DNA includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa.
  • Polynucleotides may include non-naturally occurring DNA, such as enantiomeric DNA.
  • the precise sequence of nucleotides in a polynucleotide may be known or unknown.
  • a gene or gene fragment for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag
  • genomic DNA genomic
  • a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides.
  • a polymerase can bind a primed single stranded target polynucleotide, and can sequentially add nucleotides to the growing primer to form a “complementary copy” polynucleotide having a sequence that is complementary to that of the target polynucleotide.
  • Another polymerase, or the same polymerase then can form a copy of the target nucleotide by forming a complementary copy of that complementary copy polynucleotide.
  • DNA polymerases may bind to the target polynucleotide and then move down the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing polynucleotide strand (growing amplicon).
  • DNA polymerases may synthesize complementary DNA molecules from DNA templates and RNA polymerases may synthesize RNA molecules from DNA templates (transcription). Polymerases may use a short RNA or DNA strand (primer), to begin strand growth.
  • Some polymerases may displace the strand upstream of the site where they are adding bases to a chain. Such polymerases may be said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase.
  • Example polymerases having strand displacing activity include, without limitation, the large fragment of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase or sequencing grade T7 exo-polymerase. Some polymerases degrade the strand in front of them, effectively replacing it with the growing chain behind (5' exonuclease activity).
  • primer refers to a polynucleotide to which nucleotides may be added via a free 3' OH group.
  • the primer length may be any suitable number of bases long and may include any suitable combination of natural and non-natural nucleotides.
  • a target polynucleotide may include an “adapter” that hybridizes to (has a sequence that is complementary to) a primer, and may be amplified so as to generate a complementary copy polynucleotide by adding nucleotides to the free 3' OH group of the primer.
  • a primer may be coupled to a substrate.
  • a “capture primer” refers to a primer that may be used to seed and/or to amplify a polynucleotide that includes an adapter which is substantially complementary to the capture primer.
  • substrate refers to a material used as a support for compositions described herein.
  • Example substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organo-silicate (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof.
  • POSS polyhedral organic silsesquioxanes
  • CMOS complementary metal oxide semiconductor
  • An example of POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp.776-778, which is incorporated by reference in its entirety.
  • substrates used in the present application include silica-based 11 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO substrates, such as glass, fused silica, or other silica-containing material.
  • substrates may include silicon, silicon nitride, or silicone hydride.
  • substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, and poly(methyl methacrylate).
  • Example plastics materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates.
  • the substrate is or includes a silica-based material or plastic material or a combination thereof.
  • the substrate has at least one surface comprising glass or a silicon-based polymer.
  • the substrates may include a metal. In some such examples, the metal is gold.
  • the substrate has at least one surface comprising a metal oxide.
  • the surface comprises a tantalum oxide or tin oxide.
  • Acrylamides, enones, or acrylates may also be utilized as a substrate material or component.
  • Other substrate materials may include, but are not limited to gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers and copolymers.
  • the substrate and/or the substrate surface may be, or include, quartz.
  • the substrate and/or the substrate surface may be, or include, semiconductor, such as GaAs or ITO.
  • Substrates may comprise a single material or a plurality of different materials. Substrates may be composites or laminates. In some examples, the substrate comprises an organo-silicate material. Substrates may be flat, round, spherical, rod-shaped, or any other suitable shape. Substrates may be rigid or flexible. In some examples, a substrate is a bead or a flow cell. [0050] In some examples, a substrate includes a patterned surface. A “patterned surface” refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions may be features where one or more capture primers are present. The features can be separated by interstitial regions where capture primers are not present.
  • the pattern may be an x-y format of features that are in rows and columns. In some examples, the pattern may be a repeating arrangement of features and/or interstitial regions. In some examples, the pattern may be a random arrangement of features and/or interstitial regions.
  • substrate includes an array of wells (depressions) in a surface. The wells may be provided by substantially vertical sidewalls. Wells may be fabricated as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and 12 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO microetching techniques.
  • the features in a patterned surface of a substrate may include wells in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic or other suitable material(s) with patterned, covalently-linked gel such as poly(N-(5-azidoacetamidylpentyl) acrylamide- co-acrylamide) (PAZAM).
  • PAZAM poly(N-(5-azidoacetamidylpentyl) acrylamide- co-acrylamide)
  • the process creates gel pads used for sequencing that may be stable over sequencing runs with a large number of cycles.
  • the covalent linking of the polymer to the wells may be helpful for maintaining the gel in the structured features throughout the lifetime of the structured substrate during a variety of uses.
  • the gel need not be covalently linked to the wells.
  • silane free acrylamide SFA which is not covalently attached to any part of the structured substrate, may be used as the gel material.
  • a structured substrate may be made by patterning a suitable material with wells (e.g.
  • a gel material e.g., PAZAM, SFA or chemically modified variants thereof, such as the azidolyzed version of SFA (azido-SFA)
  • polishing the surface of the gel coated material for example via chemical or mechanical polishing, thereby retaining gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions on the surface of the structured substrate between the wells.
  • Primers may be attached to gel material.
  • a solution including a plurality of target polynucleotides may then be contacted with the polished substrate such that individual target polynucleotides will seed individual wells via interactions with primers attached to the gel material; however, the target polynucleotides will not occupy the interstitial regions due to absence or inactivity of the gel material.
  • Amplification of the target polynucleotides may be confined to the wells because absence or inactivity of gel in the interstitial regions may inhibit outward migration of the growing cluster.
  • the process is conveniently manufacturable, being scalable and utilizing conventional micro- or nano-fabrication methods.
  • a patterned substrate may include, for example, wells etched into a slide or chip.
  • the pattern of the etchings and geometry of the wells may take on a variety of different shapes and sizes, and such features may be physically or functionally separable from each other.
  • Particularly useful substrates having such structural features include patterned substrates that 13 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO may select the size of solid particles such as microspheres.
  • An example patterned substrate having these characteristics is the etched substrate used in connection with BEAD ARRAY technology (Illumina, Inc., San Diego, CA).
  • a substrate described herein forms at least part of a flow cell or is located in or coupled to a flow cell.
  • Flow cells may include a flow chamber that is divided into a plurality of lanes or a plurality of sectors.
  • Example flow cells and substrates for manufacture of flow cells that may be used in methods and compositions set forth herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).
  • the term “plurality” is intended to mean a population of two or more different members. Pluralities may range in size from small, medium, large, to very large. The size of small plurality may range, for example, from a few members to tens of members.
  • Medium sized pluralities may range, for example, from tens of members to about 100 members or hundreds of members.
  • Large pluralities may range, for example, from about hundreds of members to about 1000 members, to thousands of members and up to tens of thousands of members.
  • Very large pluralities may range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions and up to or greater than hundreds of millions of members. Therefore, a plurality may range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges.
  • Example polynucleotide pluralities include, for example, populations of about 1 ⁇ 10 5 or more, 5 ⁇ 10 5 or more, or 1 ⁇ 10 6 or more different polynucleotides. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality may be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.
  • target polynucleotide is intended to mean a polynucleotide that is the object of an analysis or action. The analysis or action includes subjecting the polynucleotide to amplification, sequencing and/or other procedure.
  • a target polynucleotide may include nucleotide sequences additional to a target sequence to be analyzed.
  • a target polynucleotide may include one or more adapters, including an adapter that functions as a primer binding site, that flank(s) a target polynucleotide sequence that is to be analyzed. 14 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO [0057]
  • the terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise.
  • methylated nucleotide refers to a nucleotide that includes a methyl group (-CH 3 or -Me) or a derivatized methyl group.
  • methylcytosine or “mC” refers to cytosine in DNA (namely, 2'-deoxycytosine) that includes a methyl group (-CH3 or -Me), or a derivative of methylcytosine.
  • methyladenine or “mA” refers to adenine that includes a methyl group, or is a derivative of methyladenine.
  • a “derivative” of a methylated nucleotide refers to a nucleotide having a methyl group or a derivatized methyl group.
  • a nonlimiting example of a derivatized methyl group is an oxidized methyl group.
  • a nonlimiting example of an oxidized methyl group is hydroxymethyl (-CH2OH).
  • An mC derivative having a hydroxymethyl group may be referred to as hydroxymethylcytosine or hmC.
  • an oxidized methyl group is formyl group (-CHO).
  • An mC derivative having a formyl group may be referred to as formylcytosine or fC.
  • Another nonlimiting example of an oxidized methyl group is carboxyl (-COOH).
  • An mC derivative having a carboxyl group may be referred to as carboxylcytosine or caC.
  • the methyl group may be located at the 5 position of the cytosine, in which case the mC may be referred to as 5-mC.
  • the oxidized methyl group may be located at the 5 position of the cytosine, in which case the hmC may be referred to as 5-hmC, the fC may be referred to as 5-fC, or the caC may be referred to as 5-caC.
  • the methyl group of methyladenine may be located at the 6 position of the adenine, in which case the mA may be referred to as 6mA.
  • fluorophore is intended to mean an element that emits light at a first wavelength (“emission,” or “fluorescence”) responsive to excitation with light at a second wavelength (“optical excitation,” or “excitation light”) that is different from the first wavelength.
  • the light emitted by a fluorophore may be referred to as “fluorescence” and may be detected by suitable optical circuitry.
  • the light emitted by a fluorophore may have an “emission lifetime” that characterizes the intensity as a function of time with which the fluorophore fluoresces after optical excitation.
  • a fluorophore may be or 15 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO include a molecule such as an organic dye, a fluorescent protein, or a particle such as a quantum dot.
  • Example organic dyes include xanthene derivatives (such as fluorescein and rhodamine and their derivatives), cyanine and its derivatives, squaraine derivatives and ring- substituted squaraines, squaraine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, tetrapyrrole derivatives, and dipyrromethene derivatives.
  • Example fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP).
  • the fluorophore may include a quantum dot.
  • quantum dot it is meant a particle including about 100 to about 100,000 atoms and a diameter of about 2 to about 10 nm, and that emits light in response to excitation light or energy transfer.
  • Quantum dots may include, or may consist essentially of, inorganic atoms.
  • Quantum dots may include atoms from groups II-IV, groups III-V, or groups IV-VI of the period table, and may include a core having a first composition that is covered by a shell having a second, different composition.
  • Cadmium (Cd) may be included in the core and/or in the shell.
  • a quantum dot includes a CdSe core covered by a ZnS shell, and may be referred to as a CdSe/ZnS core/shell quantum dot.
  • a quantum dot includes a CdSe core covered by a CdS shell, and may be referred to as a CdSe/ZnS core/shell quantum dot.
  • Quantum dots may have relatively narrow emission peaks, and may have a brighter emission and a higher signal to noise ratio as 16 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO compared to organic dyes (e.g., may be about 10-20 times brighter than organic dyes).
  • Quantum dots also may be relatively stable because their inorganic composition may inhibit the effect of photobleaching. Quantum dots also may have a significantly longer fluorescence time (e.g., about 10-40 ns) as compared to that of inorganic dyes (e.g., a few nanoseconds).
  • the term “quencher” is intended to mean an element that, when in sufficient proximity to a fluorophore, reduces or inhibits fluorescence from that fluorophore.
  • a quencher may be or include a molecule.
  • Example commercially available quenchers include DABCYL (dimethylaminoazobenzenesulfonic acid, Jena Bioscience GMBH), Black Hole Quencher dyes (BHQ, LGC Biosearch Technologies), IOWA BLACK® FQ (Integrated DNA Technologies), IOWA BLACK® RQ (Integrated DNA Technologies), and IRDYE® QC-1 (LI-COR Biosciences).
  • DABCYL dimethylaminoazobenzenesulfonic acid
  • JHQ Black Hole Quencher dyes
  • IOWA BLACK® FQ Integrated DNA Technologies
  • IOWA BLACK® RQ Integrated DNA Technologies
  • IRDYE® QC-1 LI-COR Biosciences
  • Fluorescence may be detected using any suitable optical detection circuitry, which may include an optical detector to generate an electrical signal based on the light received from the fluorophore, and electronic circuitry to determine, using the electrical signal, that light was received from the fluorophore.
  • the optical detector may include an active-pixel sensor (APS) including an array of amplified photodetectors configured to generate an electrical signal based on light received by the photodetectors.
  • APSs may be based on complementary metal oxide semiconductor (CMOS) technology known in the art.
  • CMOS-based detectors may include field effect transistors (FETs), e.g., metal oxide semiconductor field effect transistors (MOSFETs).
  • CMOS-SPAD single-photon avalanche diode
  • FLIM fluorescence lifetime imaging
  • the optical detector may include a photodiode, such as an avalanche photodiode, charge-coupled device (CCD), cryogenic photon detector, reverse- biased light emitting diode (LED), photoresistor, phototransistor, photovoltaic cell, photomultiplier tube (PMT), quantum dot photoconductor or photodiode, or the like.
  • the optical detection circuitry further may include any suitable combination of hardware and software in operable communication with the optical detector so as to receive the electrical signal therefrom, and configured to detect the fluorescence based on such signal, e.g., based 17 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO on the optical detector detecting light from the fluorophore.
  • the electronic circuitry may include a memory and a processor coupled to the memory. The memory may store instructions for causing the processor to receive the signal from the optical detector and to detect the fluorophore using such signal.
  • the instructions can cause the processor to determine, using the signal from the optical detector, that fluorescence is emitted within the field of view of the optical detector and to determine, using such determination, that a fluorophore is present.
  • To “measure” fluorescence is intended to mean to determine a relative or absolute amount of the fluorescence that is detected.
  • the amount of fluorescence may be measured relative to a baseline amount of fluorescence, or as an absolute amount of fluorescence.
  • the amount of fluorescence from one or more fluorophores may be correlated to the amount of a modified base, in a polynucleotide, that is hybridized to the methylated nucleotide.
  • the memory of the electronic circuitry described above may store instructions causing the processor to monitor the level of the electrical signal at one or more times, and to correlate such level(s) to an amount of the methylated nucleotide.
  • Compositions and methods for detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide [0064] Provided herein are example assays for detecting methylated nucleotides that may be used for targeted, highly multiplexed, quantitative measurement of methylated nucleotides at any respective location(s) within a target polynucleotide.
  • quenchers respectively may be coupled to suitable methylated nucleotides within the target polynucleotide, and the presence of the methylated nucleotides may be detected via the quenchers’ reducing fluorescence intensity from fluorescently labeled nucleotides that are added to a primer hybridized to the target polynucleotide.
  • quenchers respectively may be coupled to different types of methylated nucleotides, thus providing for a wide variety of epigenetic assays to be performed concurrently with one another using the same or similar workflow as may be used to sequence the target polynucleotide itself.
  • FIGS.1A-1H schematically illustrate example compositions and operations in a process flow for detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide.
  • the composition illustrated in FIG.1A includes target polynucleotide 18 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO 110, e.g., a fragment of single-stranded DNA or RNA.
  • Polynucleotide 110 may include sugar-phosphate backbone 111 and bases 112.
  • polynucleotide 110 may be significantly longer than is suggested in FIG.1A, and that the polynucleotide may be in any suitable fluid or may be coupled to a substrate. It may be desired to assay whether any locations within polynucleotide 110 may include one or more methylated nucleotides.
  • polynucleotide 110 includes a methylated nucleotide 113, and a plurality of cytosines 114, in addition to other bases the types of which are not specifically illustrated.
  • polynucleotide 110 is illustrated as including a single methylated nucleotide 113.
  • polynucleotide 110 may include a plurality of methylated nucleotides of the same type as one another or of different types.
  • methylated nucleotide 113 may be selected from the group consisting of mC, hmC, fC, caC, and 6mA.
  • a quencher may be coupled to a methylated nucleotide 113 and used in such a manner that the methylated nucleotide 113 may be distinguished from other nucleotides.
  • a quencher may be coupled to methylcytosine 113 and used in such a manner that the methylcytosine 113 may be distinguished from cytosines 114.
  • a quencher may be coupled to 6mA and used in such a manner that the 6mA may be distinguished from adenines in the polynucleotide.
  • FIG.1B illustrates polynucleotide 110’ which includes a methylated nucleotide 113 (e.g., 5-mC, 5- hmC, 5-fC, 5-caC, or 6-mA) coupled to quencher 115.
  • quenchers are not coupled to cytosines 114 (or other unmethylated nucleotides).
  • methylated nucleotide 113 may be directly coupled to the quencher 115, while in other examples, the methylated nucleotide is indirectly coupled to the quencher. In either circumstance, quencher 115 selectively may be coupled to the methylated nucleotide 113 as compared to the unmethylated nucleotides 114.
  • methylated nucleotides and nonlimiting manners of selectively coupling quenchers to such nucleotides, are described in greater detail below.
  • any suitable number of different quenchers selectively may be coupled to any suitable number of different methylated nucleotides and used to detect such nucleotides in a manner such as described in greater detail below.
  • a primer 116 may be hybridized to polynucleotide 110’.
  • the primer may be extended using a plurality of fluorescently labeled nucleotides, and the 19 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO sequence of nucleotides added to the primer is complementary to the sequence of nucleotides in polynucleotide 110’.
  • the respective colors of fluorescence from the labels of such nucleotides may be used to determine the sequence of polynucleotide 110’, e.g., using a one- channel, two-channel, or four-channel process similar to that known in the art.
  • the present processes also may be used to detect the presence of methylated nucleotides within the target polynucleotide.
  • a polymerase (not specifically illustrated) has added a first fluorescently labeled nucleotide 117 to the 3 ⁇ end of primer 116 that is complementary to nucleotide 118 within polynucleotide 110’.
  • the fluorophore of nucleotide 117 is excited, and responsive to such excitation emits fluorescence at a particular wavelength and intensity, as intended to be suggested by the plot illustrated in FIG.1D.
  • Suitable optical detection circuitry (not specifically illustrated) detects the fluorescence from the fluorophore of nucleotide 117.
  • nucleotide 117 may be determined from the wavelength of the fluorescence in a manner such as known in the art. Additionally, as provided herein, information about the distance of nucleotide 117 from quencher 115, and thus information about the distance of nucleotide 118 from quencher 115, may be determined from the intensity of the fluorescence. In the nonlimiting example illustrated in FIG.1D, nucleotide 117 is sufficiently far from quencher 115 that the intensity of fluorescence from the label of such nucleotide is substantially not affected by the presence of quencher 115.
  • the intensity of fluorescence from labeled nucleotide 117 may be substantially the same as it would be if polynucleotide 110’ was not coupled to any quenchers.
  • primer 116 is further extended using a polymerase to add additional fluorescently labeled nucleotides to the 3 ⁇ -end of the primer, some of the nucleotides may be added to a location that is sufficiently close to quencher 115 that quencher may reduce fluorescence from such nucleotides. Additionally, the amount by which quencher 115 reduces the fluorescence from such nucleotides may be used to detect methylated nucleotide 113.
  • fluorescence from labeled nucleotide 119 may be reduced compared to the fluorescence from labeled nucleotide 117 because nucleotide 119 is closer to quencher 115 than is nucleotide 117.
  • Such reduction in fluorescence is intended to 20 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO be suggested by the darkened shading of nucleotide 119 and the reduced intensity of fluorescence illustrated in the plot of FIG.1E.
  • nucleotide 119 and thus the identity of complementary nucleotide 120, may be determined from the wavelength of the fluorescence in a manner such as known in the art.
  • fluorescence from labeled nucleotide 121 which is complementary to and added opposite to nucleotide 122, may be reduced compared to the fluorescence from labeled nucleotide 119 because nucleotide 121 is even closer to quencher 115 than is nucleotide 119.
  • Such reduction in fluorescence is intended to be suggested by the further darkened shading of nucleotide 121 and the further reduced intensity of fluorescence illustrated in the plot of FIG.1F.
  • nucleotide 121 and thus the identity of complementary nucleotide 122, may be determined from the wavelength of the fluorescence in a manner such as known in the art. Additionally, as illustrated in FIG.1G, fluorescence from labeled nucleotide 123, which is complementary to and added opposite to nucleotide 113, may be reduced compared to the fluorescence from labeled nucleotide 121 because nucleotide 123 is even closer to quencher 115 than is nucleotide 121. Such reduction in fluorescence is intended to be suggested by the further darkened shading of nucleotide 123 and the further reduced intensity of fluorescence illustrated in the plot of FIG.1G.
  • labeled nucleotide 123 is directly opposite the methylated nucleotide 113 to which quencher 115 is coupled, fluorescence from labeled nucleotide 123 may be expected to be reduced more strongly than the fluorescence from labeled nucleotides that are added further away from the quencher.
  • the identity of nucleotide 123, and thus the identity of complementary nucleotide 113 (that is, methylated nucleotide 113), may be determined from the wavelength of the fluorescence in a manner such as known in the art.
  • the fluorescence from such nucleotides may increase with their distance from methylated nucleotide 113, and thus from quencher 115.
  • fluorescence from labeled nucleotide 124 which is complementary to and added opposite to nucleotide 125, may be increased compared to the fluorescence from labeled nucleotide 123 because nucleotide 124 is farther from quencher 115 than is nucleotide 123.
  • Such reduction in fluorescence is intended to be suggested by the lightened shading of nucleotide 124 and the increased intensity fluorescence illustrated in the plot of FIG.1H.
  • nucleotide 124 may be determined from the wavelength 21 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO of the fluorescence in a manner such as known in the art.
  • labeled nucleotide 124 may be spaced by a similar distance from quencher 115 as is nucleotide 121 (e.g., both nucleotide 121 and nucleotide 124 may be located opposite the nucleotides which are directly adjacent to methylated nucleotide 113).
  • fluorescence from labeled nucleotides may further increase as a function of distance from quencher 115.
  • Methylated nucleotide 113 may be detected using the reduced fluorescence caused by the first quencher.
  • FIG.2A illustrates a plot of example fluorescence intensity 200 as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including a methylated nucleotide having a quencher coupled thereto.
  • Each “cycle” referred to in FIG.2A corresponds to addition of a single nucleotide to primer 116.
  • FIG.2A illustrates one example relationship between the intensity of fluorescence from different fluorescently labeled nucleotides (e.g., nucleotides 117, 119, 121, 123, 124 respectively described with reference to FIGS.1D-1H), and the respective distances of such nucleotides from quencher 114.
  • nucleotides 117, 119, 121, 123, 124 respectively described with reference to FIGS.1D-1H
  • nucleotides that are sufficiently far from quencher 115 may fluoresce with an intensity that is substantially unaffected by the presence of quencher, e.g., may have a fluorescence intensity at approximately baseline 210 illustrated in FIG.2A.
  • nucleotides that are sufficiently close to quencher may fluoresce with intensities that generally decrease as a function of distance from the quencher (that is, nucleotides added in cycles that are sufficiently close to the cycle in which labeled nucleotide 123 is added opposite methylated nucleotide 113).
  • the fluorescence from labeled nucleotide 123, added opposite methylated nucleotide 113 may be expected to be the lowest of all the labeled nucleotides because it is the closest to quencher 115.
  • Minimum 202 in FIG.2A is intended to correspond to the fluorescence intensity from labeled nucleotide 123, and it may be seen that the fluorescence intensity 200 increases on either side of such minimum.
  • FIG.2A may suggest that the fluorescence intensity 200 varies smoothly as a function of cycle number, in an actual implementation the fluorescence intensity may vary in a more complicated manner. For example, cycle numbers have integer values, and thus the fluorescence intensity may vary in a stepwise manner from one cycle to another rather than as a continuum as is suggested in FIG.2A.
  • the fluorescence intensity may vary as 22 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO a function of the particular fluorophore(s) that respectively are coupled to different nucleotides, because different fluorophores may interact differently than one another with a given quencher. Additionally, some nucleotides may never be coupled to a fluorophore, and instead are detected through the absence of fluorescence; illustratively, in some commercial instruments G is “dark” because it is not coupled to a fluorophore, and is detected through an absence of fluorescence.
  • fluorophore in a one-channel system such as known in the art, only a single type of fluorophore may be used, and such fluorophore may be coupled to different types of nucleotides at different times than one another using chemistry.
  • the fluorophore may be coupled to A and T at a first time during which detection circuitry obtains a first image, then the fluorophore may be chemically cleaved from A and chemically coupled to C and the detection circuitry may obtain a second image at a second time.
  • the nucleotide is identified as A; if the second image and not the first image include fluorescence from the label, then the nucleotide is identified as C; if both the first image and second image include fluorescence from the label, then the nucleotide is identified as T; and if neither the first image nor the second image includes fluorescence from the label, then the nucleotide is identified as G.
  • the fluorophores attached to the various nucleotides may physically and chemically interact in a similar manner with quencher 115.
  • the fluorophores may be of different types than one another (e.g., in the case of a two-channel or four-channel system), but may interact similarly as one another with the quencher 115. In either circumstance, it may be expected that fluorescence from each fluorescent label may similarly decrease as a function of proximity to quencher 115.
  • FIG.2B illustrates a plot of example fluorescence intensity 213 as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including a methylated nucleotide having a quencher coupled thereto, for an example one-channel system in which A, T, and C are coupled to the same type of fluorophore as one another at different times than one another, while G is not coupled to any fluorophores.
  • the fluorescence intensities may generally follow the same overall trend 210 as a function of cycle number as does intensity 200 23 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO described with reference to FIG.2A, and the intensities may be approximately at baseline 211 when the fluorophores are sufficiently far from quencher 115.
  • the fluorescence intensity 213 measured during a given cycle may be discontinuous with fluorescence intensities measured during the previous and subsequent cycles.
  • the detection circuitry may not detect any fluorescence – including at minimum 212 of trend 210, where G is added opposite to the methylated nucleotide in a manner such as described with reference to FIG.1G.
  • a similar trend 210 may be observed in two-channel or four-channel system, in which there are different types of fluorophores which all interact with the quencher similarly as one another.
  • different nucleotides may be coupled to different fluorophores than one another, and such fluorophores may emit fluorescence at different wavelengths from which the identities of the nucleotides respectively may be determined.
  • the present quencher 115 is coupled to methylated nucleotides in a two-channel system or four-channel system
  • different types of fluorophores respectively attached to the various nucleotides may physically and chemically interact in different manners with quencher 115. Accordingly, while it may be expected that fluorescence from a given type of label may similarly decrease as a function of proximity to quencher 115 in a manner such as described with reference to FIG.2B, fluorescence from other types of labels may decrease as a different function of proximity to quencher 115.
  • FIG.2C illustrates plot of example fluorescence intensity 223 from a first type of fluorophore, and fluorescence intensity 223’ from a second type of fluorophore, as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including a methylated nucleotide having a quencher coupled thereto, for an example two-channel or four-channel system in which A is coupled to the first type of fluorophore and G is coupled to the second time of fluorophore.
  • the fluorescence intensities from the first and second fluorophores respectively may generally follow different overall trends 220, 220’ as a function of cycle number, similarly as does intensity 200 described with reference to FIG.2A.
  • the trend 220 may be a different function of cycle number than is trend 220’, because the first fluorophore may interact differently with quencher 115 than does the second fluorophore.
  • the fluorescence intensity 223 or 223’ measured during a given cycle may be discontinuous with fluorescence intensities measured during the previous and subsequent cycles, and may have 24 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO different magnitudes because the fluorophores have different intrinsic fluorescent intensities than one another, as reflected by the different baselines 221, 221’ for the two fluorophores, and/or may have different responses to proximity to the quencher than one another.
  • the detection circuitry may detect a level of fluorescence at minimum 222’ of trend 220.
  • the minimum 222 of trend 220 may have a different value than minimum 222’, and as such one or more of the fluorescence intensities 223 may in some circumstances be approximately the same as intensity 223’ at minimum 222’.
  • appropriate processing circuitry may be used to determine the location of the minimum and thus to determine the location of the methylated nucleotide to which the quencher is coupled.
  • a base calling algorithm may be generated by using the present operations to sequence known DNA sequences including methylated nucleotides respectively coupled to quenchers (e.g., within any suitable number of one or more CpG sites, where “CpG” refers to a region of a polynucleotide in which C is followed by G in the 5 ⁇ to 3 ⁇ direction).
  • the measured reductions in fluorescence caused by the quenchers may be recorded, and bioinformatically used to create a base calling algorithm that may be used to detect similar reductions in fluorescence and correlate such reductions to the respective presence of methylated nucleotides coupled to quenchers.
  • a base calling algorithm that may be used to detect similar reductions in fluorescence and correlate such reductions to the respective presence of methylated nucleotides coupled to quenchers.
  • FIGS.3A- 3E schematically illustrate example compositions and operations in a process flow for detecting different methylated nucleotides using different quenchers respectively coupled to the different methylated nucleotides.
  • a first type of methylated nucleotide 313, e.g., methylcytosine (mC) is coupled to a first type of quencher (Q 1 ) 315
  • a second type of methylated nucleotide 314, e.g., 25 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO hydroxymethylcytosine (hmC) is coupled to a second type of quencher (Q2) 317 that is different from the first type of quencher.
  • the first and second methylated nucleotides 313, 314 may be selected from the group consisting of mC, hmC, fC, caC, and 6mA.
  • Nonlimiting examples of operations and compositions for selectively coupling different types of methylated nucleotides to different types of quenchers are provided elsewhere herein.
  • a polymerase (not specifically illustrated) may be used to add fluorescently labeled nucleotides to primer 316 in a manner similar to that described with reference to FIGS.1B- 1H.
  • Different types of nucleotides may be coupled to different types of fluorophores that are selected to respectively interact with the different types of quenchers.
  • a first type of nucleotide may be coupled to a first type of fluorophore F1 that interacts with first quencher Q 1 in a manner that decreases its fluorescence intensity as a function of proximity to Q1, in a manner similar to that described with reference to FIGS.1A- 1H and 2A-2C.
  • a second type of nucleotide (illustratively, A) may be coupled to a second type of fluorophore F2 that interacts with second quencher Q2 in a manner that decreases its fluorescence intensity as a function of proximity to Q 2 , also in a manner similar to that described with reference to FIGS.1A-1H and 2A-2C.
  • first fluorophore F 1 and second quencher Q 2 may be significantly weaker than the interaction between first fluorophore F1 and first quencher Q1.
  • interaction between second fluorophore F 2 and first quencher Q 1 may be significantly weaker than the interaction between second fluorophore F2 and second quencher Q2. Accordingly, in some examples, any reduction in fluorescence of first fluorophore F 1 substantially may be attributed to its proximity to first quencher Q1, and any reduction in fluorescence of second fluorophore F 2 substantially may be attributed to its proximity to second quencher Q 2 .
  • a polymerase (not specifically illustrated) may have added a T, labeled with first fluorophore F 1 , to primer 316. At this time, first fluorophore F1 may be sufficiently far from first quencher Q1 as substantially not to experience a decrease in fluorescence.
  • the polymerase (or another polymerase) may have added an A, labeled with second fluorophore F 2 , to primer 316. At this time, second fluorophore F 2 may be sufficiently far from second quencher Q2 as substantially not to experience a decrease in fluorescence.
  • the fluorophores F 1 , F 2 of some of the T and A 26 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO nucleotides may come into sufficient proximity to respective quenchers Q1, Q2 to experience a reduction in fluorescence.
  • a polymerase (not specifically illustrated) may have added another T, the first fluorophore F1 of which may be sufficiently close to first quencher Q 1 as to experience a decrease in fluorescence, as intended to be suggested by the darkened shading.
  • the polymerase (or another polymerase) may have added an A, the second fluorophore F2 of which may be sufficiently close to second quencher Q2 as to experience a decrease in fluorescence, as intended to be suggested by the darkened shading.
  • the first quencher Q1 may reduce fluorescence from first fluorophore F1 more than it reduces fluorescence from second fluorophore F 2 , such that the reduction in fluorescence of F1 is primarily a function of distance from Q1 and is significantly less of a function of distance from Q 2 .
  • the second quencher Q 2 may reduce fluorescence from second fluorophore F2 more than it reduces fluorescence from first fluorophore F2, such that the reduction in fluorescence of F 2 is primarily a function of distance from Q 2 and is significantly less of a function of distance from Q1.
  • first quencher Q1 may be selected so as to reduce at least twice as much fluorescence from first fluorophore F 1 than it does from second fluorophore F2, or at least three times as much fluorescence from first fluorophore F1 than it does from second fluorophore F 2 , or at least four times as much fluorescence from first fluorophore F1 than it does from second fluorophore F2, or at least five times as much fluorescence from first fluorophore F 1 than it does from second fluorophore F 2 , or at least ten times as much fluorescence from first fluorophore F1 than it does from second fluorophore F 2 .
  • second quencher Q 2 may be selected so as to reduce at least twice as much fluorescence from second fluorophore F2 than it does from first fluorophore F 1 , or at least three times as much fluorescence from second fluorophore F 2 than it does from first fluorophore F1, or at least four times as much fluorescence from second fluorophore F2 than it does from first fluorophore F1, or at least five times as much fluorescence from second fluorophore F 2 than it does from first fluorophore F 1 , or at least ten times as much fluorescence from second fluorophore F2 than it does from first fluorophore F1.
  • FIG.4 illustrates a plot of example fluorescence intensities as a function of the number of fluorescent nucleotides added to a primer hybridized to a polynucleotide including different methylated nucleotides having different quenchers respectively coupled thereto.
  • Trace 410 illustrated in FIG.4 corresponds to the overall trend of fluorescence 27 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO intensity from first fluorophore F1
  • trace 420 corresponds to the overall trend of fluorescence intensity from second fluorophore F 2 .
  • first fluorophores that are sufficiently far from first quencher Q1315 may fluoresce with an intensity that is substantially unaffected by the presence of both of quenchers Q1 and Q2, e.g., may have a fluorescence intensity at approximately a baseline (not specifically labeled) similar to that described with reference to FIGS.2A-2C.
  • first fluorophores F1 that are sufficiently close to first quencher Q1 may fluoresce with intensities that generally decrease as a function of distance from the quencher (that is, fluorophores added in cycles that are sufficiently close to the cycle in which the nucleotide is added opposite first methylated nucleotide 313).
  • the fluorescence from any first fluorophore F1 that is added opposite methylated nucleotide 313 may be expected to be the lowest of all the nucleotides coupled to the first fluorophore, at minimum 402, because it is the closest to quencher 315.
  • second fluorophores that are sufficiently far from second quencher Q2317 may fluoresce with an intensity that is substantially unaffected by the presence of both of quenchers Q 1 and Q 2 , e.g., may have a fluorescence intensity at approximately a baseline (not specifically labeled) similar to that described with reference to FIGS.2A-2C.
  • second fluorophores F 2 that are sufficiently close to second quencher Q2 may fluoresce with intensities that generally decrease as a function of distance from the quencher (that is, fluorophores added in cycles that are sufficiently close to the cycle in which the nucleotide is added opposite second methylated nucleotide 314).
  • the fluorescence from any second fluorophore that is added opposite methylated nucleotide 314 may be expected to be the lowest of all the nucleotides coupled to the first fluorophore, at minimum 403, because it is the closest to quencher 317.
  • trace 410 28 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO substantially does not include any features corresponding to a reduction in fluorescence of first fluorophore F 1 by second quencher Q 2 317
  • trace 420 substantially does not include any features corresponding to a reduction in fluorescence of second fluorophore F2 by first quencher Q 1 315.
  • trace 410 may include a first minimum 402 corresponding to interaction between the first fluorophore and the first quencher, and a second minimum (not specifically illustrated, but located at the same cycle number as minimum 403) corresponding to interaction between the first fluorophore and the second quencher. Because the interaction between the first fluorophore and the first quencher is stronger than the interaction between the first fluorophore and the second quencher, the first minimum of trace 410 may be deeper than the second minimum of trace 410.
  • the first minimum 402 of trace 410 may be expected to be at least twice as deep as the second minimum of trace 410.
  • trace 420 may include a first minimum (not specifically illustrated, but located at the same cycle number as minimum 402) corresponding to interaction between the second fluorophore and the first quencher, and a second minimum 403 corresponding interaction between the second fluorophore and the second quencher.
  • the second minimum 403 of trace 420 may be deeper than the first minimum of trace 420.
  • the second quencher reduces fluorescence of the second fluorophore at least twice as much as the first quencher reduces fluorescence of the second fluorophore
  • the second minimum 403 of trace 420 may be expected to be at least twice as deep as the first minimum of trace 420.
  • the respective locations of the first and second methylated nucleotides 313, 314 within the polynucleotide being analyzed may be determined in a manner similar to that described with reference to FIGS.2B-2C.
  • the fluorescence intensity measured during a given cycle may be discontinuous with fluorescence intensities measured during the previous and subsequent cycles.
  • the 29 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO fluorescence intensities measured during different cycles may have different magnitudes because the fluorophores have different intrinsic fluorescent intensities than one another.
  • appropriate processing circuitry may be used to determine the respective locations of minima within the fluorescence intensities from respective fluorophores F1, F2, and thus to determine the respective locations of the methylated nucleotides 313, 314 to which the first and second quenchers 315, 317 respectively are coupled.
  • a base calling algorithm may be generated by using the present operations to sequence known DNA sequences including methylated nucleotides respectively coupled to different quenchers (e.g., within any suitable number of one or more CpG sites).
  • the measured reductions in fluorescence caused by the different quenchers may be recorded, and bioinformatically used to create a base calling algorithm that may be used to detect similar reductions in fluorescence and correlate such reductions to the respective presence of methylated nucleotides coupled to different quenchers.
  • different types of methylated nucleotides may be coupled to a respective, different type of quencher.
  • the identity of a methylated nucleotide may be inferred from the reduction in fluorescence caused by the presence of the particular type of quencher coupled to that methylated nucleotide.
  • minimum 402 in trace 410 may be used to determine that methylcytosine was present at the location corresponding to minimum 402.
  • second quencher Q2 because fluorescence from the second fluorophore F2 selectively is inhibited by second quencher Q2, and second quencher Q2 selectively is coupled to hydroxymethylcytosine, minimum 403 in trace 420 may be used to determine that hydroxymethylcytosine was present at the location corresponding to minimum 403.
  • Quenchers for use in selectively reducing fluorescence from respective fluorophores may be selected using the teachings provided herein. For example, a wide variety of fluorophores are used in commercially available sequencing by synthesis equipment. Quenchers may be selected based on spectral overlap between the fluorophores and the quenchers, and desire behavior.
  • some quenchers may have a relatively narrow absorption spectrum and may be used to quench only a certain type or certain types of 30 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO fluorophores that are used for sequencing by synthesis, while other quenchers may have a relatively broad absorption spectrum and may be used to quench some or all types of fluorophores that are used for sequencing by synthesis.
  • any suitable number of different types of methylated nucleotides may be coupled to respective types of quenchers, and the respective reductions in fluorescence caused by such quenchers may be used to determine the presence and identify of the nucleotide.
  • the methylated nucleotide(s) may include any suitable combination of one or more of 5-methylcytosine (5-mC), 5- hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxylcytosine (5-caC), or 6- methyladenine (6-mA).
  • only one type of methylated nucleotide is coupled to a quencher.
  • two different types of methylated nucleotides are coupled to respective, different quenchers.
  • three different types of methylated nucleotides are coupled to respective, different quenchers.
  • four different types of methylated nucleotides are coupled to respective, different quenchers.
  • the polynucleotide being sequenced, and within which methylated nucleotides are identified, may be sequenced in any suitable manner.
  • the polynucleotide being sequenced is coupled to a substrate.
  • the polynucleotide may be located within a cluster of polynucleotide amplicons coupled to the substrate.
  • the polynucleotide amplicons of the cluster also respectively include first methylated nucleotides.
  • the quencher may be coupled to the methylated nucleotides of the respective amplicons in a manner such as provided herein, e.g., with reference to FIG.1B for a single quencher, or with reference to FIG.3A for multiple types of quenchers.
  • Fluorescently labeled nucleotides may be added to primers respectively hybridized to the amplicons, e.g., in a manner such as described with reference to FIGS.1D-1H or 3B-3E.
  • the quencher may be used to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides, e.g., in a manner such as described with reference to FIGS.1E-1H or 3D-3E.
  • FIG.5 schematically illustrates example compositions and operations in a process flow for detecting methylated nucleotides within a cluster of amplicons.
  • operation 1 illustrated in FIG.5 a cluster of amplicons of a polynucleotide is generated, wherein at least some of the amplicons include the methylated nucleotide.
  • substantially all of the amplicons include the methylated nucleotide.
  • DNMT1 enzyme may be used to maintain symmetric methylation on the flow cell during clustering, that is, may be used to methylate cytosine at locations that correspond to methylcytosine in the original polynucleotide.
  • the amplicons are illustrated as including a single methylated nucleotide, e.g., a single methylcytosine. However, it will be appreciated that the amplicons may include a plurality of methylated nucleotides of the same type as one another or of different types.
  • FIG.8 shows an example sequence of the human template-dependent DNA (cytosine-5)-methyltransferase 1 (DNMT1, SEQ ID NO:1).
  • DNMT1 preferentially identifies hemi-methylated CpG dinucleotide sites.
  • a hemi-methylated CpG dinucleotide also referred to as a hemi-methylated site, describes a situation where a cytosine of a CpG dinucleotide is methylated on one strand but the cytosine of the complementary CpG dinucleotide on the other strand is not methylated.
  • DNMT1 methylates the cytosine of the complementary CpG dinucleotide, converting the hemi-methylated site to CpG dinucleotides on both strands.
  • wild-type DNMT1 is unable to survive the high temperatures encountered during PCR cycling.
  • DNMT1-based methyl-CpG amplification can be used either with an engineered thermostable DNMT1 or the addition of fresh DNMT1 following each PCR cycle.
  • DNA methyltransferase enzymes are commercially available, for example from Sigma AldrichTM (catalog no. SRP0126) and from Active MotifTM (catalog no.31404).
  • a standard sequencing-by-synthesis read 1 procedure is performed to determine the sequence of the amplicons.
  • primers may be hybridized to the amplicons, and a polymerase used to add fluorescently labeled nucleotides (FFNs) to the primer.
  • FPNs fluorescently labeled nucleotides
  • the sequence of the polynucleotide is complementary to the sequence of fluorescently labeled nucleotides that are added.
  • the extended primer is removed using denaturing.
  • a quencher then may be coupled to the methylated nucleotides of the amplicons, e.g., using any suitable combination of operations provided herein.
  • any 5-mC in the amplicons is oxidized to 5-caC.
  • Such oxidation optionally may be performed using a ten-eleven translocation (TET) enzyme, or using a chemical reagent, e.g., in a manner such as described in greater detail below.
  • TET ten-eleven translocation
  • the 5-carboxyl group of the 5-caC is coupled to the quencher (Q), e.g., in a manner such as described in greater detail below.
  • the amplicons are illustrated as having a single methylated nucleotide coupled to a single quencher, it will be appreciated that the amplicons may include several methylated nucleotides which respectively may be coupled to quenchers. Additionally, in some examples, the amplicons may include different types of nucleotides that optionally may be coupled to different types of quenchers in a manner such as described with reference to FIGS.3A-3E.
  • a sequencing-by-synthesis read 2 procedure is performed to determine the sequence of the amplicons.
  • another set of primers may be hybridized to the amplicons, and a polymerase used to add FFNs to the primer.
  • the sequence of the polynucleotide is complementary to the sequence of fluorescently labeled nucleotides that are added.
  • the quenchers inhibit fluorescence from fluorophores that are added at locations that are sufficiently close to the quenchers.
  • the fluorescence intensity during read 2 may include a local minimum corresponding to the location of the methylated nucleotide, e.g., in a manner such as described with reference to FIGS.2A-2C or 4.
  • the methylated nucleotide may be identified using the wavelength of fluorescence from the complementary, fluorescently labeled nucleotide added during read 1 and/or read 2. Note that if there was sufficient fluorescence during read 2 to sequence the amplicons with desired accuracy and also to identify the methylated nucleotides, then read 1 may not be necessary and optionally may be omitted.
  • the quencher is coupled to 5-caC, and the 5-carboxyl group of the 5-caC may be reacted with a molecule to form a product.
  • the 5-caC may be naturally occurring in the polynucleotide, or may be generated by oxidizing 5-mC, 5-hmC, or 5-fC in the polynucleotide to 5-caC. The oxidation may be performed using any suitable combination of chemical and/or enzymatic reagents.
  • a ten-eleven translocation (TET) dioxygenase is used to oxidize the 5-mC, 5-hmC, or 33 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO 5-fC to 5-caC.
  • 5-mC may be oxidized to 5-caC using menadione, ultraviolet (UV) radiation at 365 nm, under oxygen, followed by 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO)/bis(acetoxyiodobenzene) (BAIB) in a manner such as described in Kore et al., “Concise synthesis of 5-methyl, 5-formyl, and 5-carboxy analogues of 2 ⁇ -deoxycytidine-5 ⁇ - triphosphate,” Tetrahedron letters 54(39): 5325-5327 (2013), the entire contents of which are incorporated by reference herein.
  • UV ultraviolet
  • BAIB bis(acetoxyiodobenzene)
  • 5-hmC or 5-fC may be oxidized to 5-caC using TEMPO/BAIB in a manner such as described in Sun et al., “Efficient synthesis of 5-hydroxymethyl-, 5-formyl-, and 5-carboxyl- 2 ⁇ -deoxycytidine and their triphosphates,” RSC Advances 4(68): 36036-36039 (2014), the entire contents of which are incorporated by reference herein.
  • an iron(IV)-oxo complex is used to oxidize 5- mC to 5-caC in a manner such as described in Schmidl et al., “Biomimetic iron complex achieves TET enzyme reactivity,” Angewandte Chemie Int’l Ed.60(39): 21457-21463 (2021), the entire contents of which are incorporated by reference herein.
  • the 5-carboxyl group of the 5-caC may be reacted with the molecule in any suitable manner.
  • the reaction may include activating the 5-carboxyl group of the 5-caC before reacting the 5-carboxyl group with the molecule.
  • the 5-carboxyl group of the 5-caC may be activated using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- morpholinium chloride (DMTMM) or 1-ethyl-3-(3'- (dimethylamino)propyl)carbodiimide (EDC).
  • DTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- morpholinium chloride
  • EDC 1-ethyl-3-(3'- (dimethylamino)propyl)carbodiimide
  • the molecule with which the activated carboxylic acid is reacted may include a nucleophile.
  • the product of reaction between the carboxyl group of the 5-caC and the molecule includes the quencher, while in other examples the quencher is subsequently coupled to the product.
  • the molecule also may include the quencher or a functional group to which a quencher subsequently may be coupled (such as biotin or N 3 ).
  • a quencher or a functional group to which a quencher subsequently may be coupled
  • 4-(2H-azyrin-3-yl)-phenol (AZ-9) and 4-(2H-azyrin-3-yl)-phenyl derivatives are intended to encompass different functionalizations that are compatible with this chemistry, e.g., the identities of the black sphere illustrated above (e.g., quencher, biotin, or N 3 ).
  • 4-(2H-azyrin-3-yl)-phenol alternatively may be functionalized with an alkyne, but it should be appreciated that any substituted 3-phenyl-2H-azirine may be used, wherein the substitution includes a quencher or may be coupled to a quencher.
  • an azirine such as 4-(2H-azyrin-3-yl)-phenol
  • 5-mC methylated nucleotide that can be coupled to a quencher
  • 5-mC may be oxidized enzymatically or chemically to 5-caC and then reacted with a molecule that includes, or may be coupled to, a quencher.
  • other reaction schemes may be used to couple the molecule to 5-mC without the need for oxidizing the 5-mC to 5-caC, e.g., such as now will be described.
  • the quencher may be coupled to 5-mC using operations that include reacting the 5-methyl group of the 5-mC with a first molecule to form a product; and reacting the product with a second molecule to couple the first quencher to the first product.
  • reacting the methyl group of the 5-mC with the first molecule may include 37 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO using CMD1 to couple the first molecule (e.g., a cis-diol) to the 5-methyl group.
  • CMD1 is a homolog of TET, and uses 5-mC and vitamin C as substrates.
  • the product of such reaction may include a diol to which a suitable second molecule may be coupled.
  • the second molecule may include a boronate (-B(OH) 2 ) that coordinates to the diol: are a , can be removed if desired because their reaction with the diol is reversible.
  • the boronate may be a phenyl boric acid derivative.
  • the quencher may be coupled to the 5-mC using operations that include oxidizing the 5-mC to 5-hmC; reacting the 5-hydroxymethyl group of the 5-hmC with a first molecule to form a first product; and reacting the first product with a second molecule to couple the first quencher to the first product.
  • the 5-mC may be oxidized to 5- 38 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO hmC using any suitable combination of enzymatic and/or chemical reagents.
  • a TET dioxygenase is used to oxidize the 5-mC to the 5-hmC, illustratively ccTET, which is a TET homolog from a fungus.
  • the 5-hydroxymethyl group of the 5-hmC then may be reacted with the first molecule.
  • the reaction of the hydroxymethyl group of the 5- hmC with the first molecule may include using Mha.I to couple the first molecule to the hydroxymethyl group.
  • Mha.I is an evolved methylase (Msss.I) that may be used to functionalize 5-hmC. More specifically, Mha.I is a methyltransferase that, when lacking the SAM cofactor, can transfer a thiol onto 5-hmC.
  • MhA.I is used to couple an aminothiol to the 5-hmC.
  • the aminothiol then is reacted with any suitable second molecule, such as an N-hydroxysuccinimide (NHS) ester, an isocyanate, or an isothiocyanate: 39 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO .
  • the second molecule may include a quencher, or may include a functional group (such as biotin or N 3 ) to which a quencher subsequently may be coupled.
  • a methylated nucleotide that can be coupled to a quencher is 5-hmC.
  • 5-hmC may be oxidized enzymatically or chemically to 5-caC and then reacted with a molecule that includes, or may be coupled to, a quencher.
  • a quencher may be coupled to the 5-hmC using operations that include reacting the 5-hydroxymethyl group of the 5-hmC with a first molecule to form a first product; and reacting the first product with a 40 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO second molecule to couple the first quencher to the first product.
  • Mha.I may be used to couple the first molecule (e.g., aminothiol) to the hydroxymethyl group of 5-hmC in a similar manner as described above with reference to 5- mC.
  • the first product then may be coupled to a second molecule (e.g., NHS ester, isocyanate, or isothiocyanate) in a similar manner as described above with reference to 5-mC.
  • a similar reaction scheme may be used to couple a quencher to naturally occurring 5-mC that is oxidized to 5-hmC (e.g., using ccTET), or may be used to couple a quencher to naturally occurring 5-hmC.
  • a methylated nucleotide that can be coupled to a quencher is 6-mA.
  • the quencher may be coupled to 6-mA using an FTO-assisted chemical labeling method termed m 6 A-SEAL.
  • m6A-SEAL FTO-assisted chemical labeling method
  • the quencher may be attached with the same strategy used in Wang (but by replacing a quencher with the biotin) or with any of the commercially available thiol specific chemistries, for example such as described in Ochtrop et al., “Recent advances of thiol-selective bioconjugation reactions,” Current Opinion in Chemical Biology 58: 28-36 (2020), the entire contents of which are incorporated by reference herein. [0106] Furthermore, it should be appreciated that the reaction schemes provided herein respectively are selective for a particular type of methylated nucleotide, and thus may be expected to couple a quencher selectively to that type of methylated nucleotide.
  • carbonyl activation and nucleophilic attack may be expected to substantially react with the 5- carboxyl group of 5-caC, and substantially may not be expected to react with 5-mC, 5-hmC, or 5-fC.
  • azirines may be expected to substantially react with the 5-carboxyl group of 5-caC, and substantially may not be expected to react with 5-mC, 5-hmC, or 5-fC.
  • CMD1 may be expected to react selectively with 5-mC and substantially may not be expected to react with 5-hmC, 5-fC, or 5-caC.
  • ccTET may be expected to react selectively with 5-mC and substantially may not be expected to react with 5-hmC, 5-fC, or 5- caC.
  • reaction product may include any suitable functional group that may be coupled to a quencher.
  • the functional group may include a SNAP protein and the quencher may include O-benzylguanine.
  • the functional group may include a CLIP protein and the quencher may include an O- benzylcytosine.
  • the functional group may include SpyTag and the quencher may include SpyCatcher.
  • the functional group may include SpyCatcher and the quencher may include SpyTag.
  • the functional group may include biotin and the quencher may include streptavidin.
  • the functional group may include streptavidin and the quencher may include biotin.
  • the functional group may include NTA and the quencher may include His-Tag.
  • the functional group may include His-Tag and the quencher may include NTA.
  • the functional group may include N 3 and the quencher may include an alkyne.
  • the functional group may include an alkyne and the functional group may include N 3 .
  • Other example binding partners that may be used include, but are not limited to: SnoopTagJr and Dogtag the coupling of which may be catalyzed using SnoopLigase in a manner such as described in Buldun et al., “SnoopLigase catalyzes peptide-peptide locking and enables solid-phase conjugate isolation,” J. Am. Chem.
  • FIG.6 illustrates a flow of operations in an example method for detecting a methylated nucleotide using a quencher coupled to the methylated nucleotide.
  • Method 600 illustrated in FIG.6 may include coupling a quencher to a methylated nucleotide in a polynucleotide (operation 610), for example in a manner such as described with reference to FIG.1B, FIG.3A, or operation 4 of FIG.5.
  • a quencher for example in a manner such as described with reference to FIG.1B, FIG.3A, or operation 4 of FIG.5.
  • Method 600 also may include adding fluorescently labeled nucleotides to a primer hybridized to the polynucleotide (operation 620), for example in a manner such as described with reference to FIGS.1D-1H, FIGS.3B- 3E, or operation 5 of FIG.5.
  • Method 600 also may include using the quencher to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides (operation 630), for example in a manner such as described with reference to FIGS.1E-1H, FIGS.3D-3E, or operation 5 of FIG.5.
  • Method 600 also may include using the reduced fluorescence caused by the quencher to detect the methylated nucleotide (operation 640), for example in a manner such as described with reference to FIGS.2A-2C, 4, or the plot in FIG.5.
  • the polynucleotide that is sequenced using method 600 includes a second methylated nucleotide which it is also desired to detect.
  • the method may be modified to include coupling a second quencher to the second methylated nucleotide, for example using a different set of reactions than was used to couple the quencher to the other nucleotide.
  • the method may include using the second quencher to reduce fluorescence from at least one of the added, fluorescently labeled nucleotides. Such operation may be performed concurrently with operation 630 described above.
  • the method also may include using the reduced fluorescence caused by the second quencher to detect the second methylated nucleotide. Such operation may be performed concurrently with operation 640 above.
  • a cluster of amplicons of a polynucleotide may be generated, wherein at least some of the amplicons include the methylated nucleotide. In some examples, substantially all of the amplicons include the methylated nucleotide.
  • FIGS.7A-7F schematically illustrate example compositions and operations in a process flow for producing clonal clusters that preserve the CpG methylation state of a target polynucleotide.
  • amplification site 10 of an array and a limited number of amplicons of a target polynucleotide are shown.
  • the figures use the following convention when numbering polynucleotides: strands including a methylated cytosine are numbered (e.g., strand 13 of FIG.7C); strands including a non-methylated cytosine are also numbered but the number is modified with the prime symbol " ' " (e.g., strand 13' of FIG. 7B).
  • the method is performed using an array that includes a plurality of amplification sites 10 such as illustrated in FIG.7A.
  • Each amplification site 10 includes a plurality of orthogonal capture primers 11, 15 attached to the amplification site.
  • individual amplification sites can include a single-stranded (ss) polynucleotide 12 which is attached to the amplification site surface by hybridization between an adapter of the polynucleotide (installed during library preparation) and one of the capture primers.
  • polynucleotide 12 is shown annealed to a capture primer 11.
  • Polynucleotide 12 may be referred to as a template, a target polynucleotide, and/or a seed polynucleotide, and in some examples may be a member of a sequencing library that has been exposed to conditions that preserve an epigenetic marker present, such as the methylation state of one or more nucleotides in the polynucleotide.
  • the sequencing library has been produced using methods that do not include amplification. For example, in FIG.7A the polynucleotide 12 is shown with a CpG dinucleotide where the C is methylated.
  • the method further includes extending the 3' end of the capture primer 11 with a polymerase (not specifically shown), using the methylated single-strand polynucleotide 12 as template strand, to produce a complementary strand which is unmethylated.
  • such extension generates a double-stranded (ds) polynucleotide 14, where one strand includes polynucleotide 12, and the other strand includes the capture primer 11 and the newly synthesized amplicon 13', which is the unmethylated complement of the 44 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO template strand 12.
  • the complement of methylated CpG dinucleotide of strand 12 is present on the complementary strand 13', but the methylation status is not preserved, resulting in a hemi-methylated state for that dinucleotide present on the double-stranded polynucleotide 14.
  • the method includes exposing the array to conditions that transfer the hemi- methylated CpG dinucleotides of the original single-strand polynucleotide 12 to the complementary strand 13 ⁇ .
  • the conditions include exposing the amplification sites to an enzyme, such as DNMT1, described elsewhere herein and for which an example sequence is shown in FIG.8.
  • the enzyme transfers the methylation state of the methylated CpG dinucleotide of the template strand 12 to complementary strand 13 ⁇ , to generate the now methylated complementary strand 13, converting the hemi-methylated site to methylated CpG dinucleotides on both strands 12, 13. It should be apparent to one skilled in the art that such a treatment would not result in methylation at CpG sites that were not originally hemimethylated, e.g., a nonmethylated CpG site would remain unmethylated after treatment.
  • strand 13 is covalently coupled to the amplification site 10 via capture primer 11, while strand 12 is noncovalently coupled to the amplification site 10 and may be removed by dehybridization.
  • the double-stranded polynucleotide 14 at each amplification site 10 is amplified to include a clonal population of immobilized polynucleotides.
  • the clonal population includes a first sub- population of single-strand polynucleotides 13 ⁇ having the same nucleotide sequence as strand 13 generated in a manner such as described with reference to FIGS.7A-7C, but the CpG dinucleotides in strands 13 ⁇ are not methylated.
  • Strand 13 retains its methylated CpG dinucleotides produced using operations including the above-described transfer using, for example, the enzyme DNMT1.
  • the clonal population at amplification site 10 also includes a second sub-population of single-stranded polynucleotides 12 ⁇ that include the nucleotide sequence of the template strand 12, but do not contain any methylated CpG dinucleotides.
  • an amplification site includes one methylated strand 13 and multiple copies of the same unmethylated nucleotide sequence 13', coupled to the 45 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO amplification site surface via respective capture primers 11.
  • amplification sites also include multiple copies of the unmethylated nucleotide sequence 12', which includes the same nucleotide sequence as 12 in FIG.7A-C but is now attached to the amplification site surface via respective capture primers 15.
  • amplification methods include, but are not limited to, solid- phase amplification.
  • solid-phase amplification refers to any polynucleotide amplification reaction carried out on or in association with a solid support such that all or a portion of the amplified products are immobilized on the solid support as they are formed.
  • Solid phase amplification includes, but is not limited to, systems such as arrays, where one primer is anchored to the surface of the array and the other is in free solution; emulsions, where one primer is anchored to a bead and the other is in free solution; and colony formation in solid phase gel matrices, where one primer is anchored to the surface and one is in free solution.
  • methods that rely on bridge amplification, where both primers are attached to a surface are used.
  • methods are used that rely on kinetic exclusion, where recombinase-facilitated amplification and isothermal conditions amplify the library (U.S. Pat. No.9,309,502, U.S. Pat.
  • a composition for amplifying polynucleotides at amplification sites referred to herein as an "amplification reagent,” is typically capable of rapidly making copies of polynucleotides at amplification sites.
  • An amplification reagent used in a method of the present disclosure will generally include a polymerase and nucleotide triphosphates (NTPs).
  • polymerases 46 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO suitable for use in embodiments of the present disclosure include, but are not limited to, DNA polymerase (such as Klenow fragment, T4 DNA polymerase, Bst (Bacillus stearothermophilus) polymerase), thermostable DNA polymerases (such as Taq, Vent, Deep Vent, Pfu, Tfl, and 9°N DNA polymerases) as well as their genetically modified derivatives (see, for example, U.S.
  • an amplification reagent can also include recombinase, accessory protein, and single-stranded DNA binding (SSB) protein for recombinase-facilitated amplification (see, for example, U.S. Pat. No.8,071,308, the entire contents of which are incorporated by reference herein).
  • SSB single-stranded DNA binding
  • DMSO dimethyl sulfoxide
  • BSA Bovine Serum Albumin
  • PEG poly-ethylene glycol
  • Betaine Triton X-100
  • denaturant e.g., formamide
  • MgCl2 MgCl2
  • the method further includes propagating, at each amplification site, the methylated CpG dinucleotide present on one strand 13 of the clonal population to other members 12 ⁇ , 13 ⁇ of the clonal population.
  • an isothermal amplification reaction can be performed by incubating the amplification sites with a reaction mixture under conditions that transfer the methylated CpG dinucleotides of strand 13 to other strands 12 ⁇ , 13 ⁇ - that is, to unmethylated strands 12 ⁇ otherwise having the sequence of the original template strand 12 or to unmethylated strands 13 ⁇ having the sequence of the original complementary strand 13 ⁇ .
  • the conditions include exposing the amplification sites to an enzyme such as DNMT1 and a DNA helicase or recombinase.
  • an enzyme such as DNMT1 and a DNA helicase or recombinase.
  • recombinase is intended to be consistent with its use in the art and include, for example, RecA protein, the T4 UvsX protein, the RB69 bacteriophage UvsX protein, and the like. Examples of these proteins are readily available to the skilled person (U.S. Pat. No.8,071,308, the entire contents of which are incorporated by reference herein). Examples of formulations that 47 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO include a helicase protein are described in U.S. Pat.
  • Propagation of the CpG methylation occurs by many cycles of steps that include (i) hybridization of the complementary single-strand polynucleotides within each amplification site, forming a bridged double-stranded fragment, (ii) transfer of any methylated C of methylated CpG dinucleotides from one strand to the other paired strand by an enzyme such as DNMT1, and (iii) unwinding of the now fully-methylated duplex by either helicase- mediated unwinding or strand invasion by another fragment in the cluster mediated by a recombinase.
  • complementary strands 12' and 13 shown in operation I anneal to form the double-stranded hemi-methylated structure shown in operation II.
  • the methylation status of CpG dinucleotides on strand 13 is transferred to stand 12' to convert strand 12' to 12 as shown in operation III.
  • the double-stranded structure shown in operation III is unwound and the process is repeated until the methylation status of the one methylated single-stranded polynucleotide 13 is propagated through both sub-populations of polynucleotides 12 ⁇ and 13 ⁇ at the amplification sites, resulting in substantially fully methylated clusters as shown at operation IV.
  • the capture primers coupling the other one of the sub-populations to the surface may be cleaved to remove that other sub- population from the surface.
  • the cleaving of a nucleotide sequence to permit the optional removal of a specific strand is referred to herein as "linearization.”
  • linearization cleaving of a nucleotide sequence to permit the optional removal of a specific strand.
  • FIG.7F the sub-population of amplicons 12 have been removed using linearization, leaving the amplicons 13 ready for further analysis in a manner such as described elsewhere herein.
  • suitable methods for linearization are described in application number WO 2007/010251, U.S. Pat. No.8,431,348, U.S. Pat.
  • Any suitable cleavage reaction can be used for linearization.
  • cleavage reactions include, but are not limited to, enzymatic, chemical, and photochemical.
  • Cleavage can be achieved by, for example, RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavage site can include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., TCEP), in which case the cleavage site should include an appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, 48 SMRH:4879-1594-7704.2 IP-2313-PCT 47CX-386091-WO in which case the cleavage site should include a diol linkage; and generation of an abasic site and subsequent hydrolysis.
  • a reducing agent e.g., TCEP
  • Suitable cleavage techniques for use in the method of the disclosure include, but are not limited to, chemical cleavage, cleavage of an abasic site, cleavage of a ribonucleotide, photochemical cleavage, PCR stoppers, cleavage of a peptide linker, enzymatic digestion with nicking endonuclease.
  • the person of ordinary skill in the art will recognize that use of some conditions described herein, for example heat or alkali, may be undesirable in view of the potential for denaturation of the complementary strand from the shortened capture primer.
  • an abasic site is generated and cleaved.
  • abasic site is defined as a position in a polynucleotide from which the base component has been removed.
  • Abasic sites can occur naturally in DNA under physiological conditions by hydrolysis of nucleoside residues, but can also be formed chemically under artificial conditions or by the action of enzymes. Once formed, abasic sites can be cleaved (e.g., by treatment with an endonuclease or other single-stranded cleaving enzyme, exposure to heat or alkali), providing a means for site-specific cleavage the capture primer.
  • an abasic site can be created at a pre-determined position of the capture primer and then cleaved by first incorporating deoxyuridine (U) at the pre-determined cleavage site.
  • the enzyme uracil DNA glycosylase (UDG) can then be used to remove the uracil base, generating an abasic site.
  • the strand including the abasic site may then be cleaved at the abasic site by treatment with endonuclease (e.g. EndoIV endonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali.
  • endonuclease e.g. EndoIV endonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIII glycosylase/AP lyase
  • Abasic sites may also be generated at non- natural/modified deoxyribonucleotides other than deoxyuridine and cleaved in an analogous manner by treatment with endonuclease, heat or alkali.
  • endonuclease heat or alkali.
  • 8-oxo-guanine can be converted to an abasic site by exposure to FPG glycosylase.
  • Deoxyinosine can be converted to an abasic site by exposure to AlkA glycosylase.
  • the abasic sites generated may then be cleaved, typically by treatment with a suitable endonuclease (e.g., EndoIV, AP lyase).
  • the molecules to be cleaved may be exposed to a mixture containing the appropriate glycosylase and one or more suitable endonucleases.
  • the glycosylase and the endonuclease will typically be present in an activity ratio of at least about 2:1.
  • the USER reagent available from New England Biolabs (NEB #M5505S) is used for the creation of a single nucleotide gap at a uracil base in a capture primer.

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Abstract

Des exemples de la présente invention concernent la détection d'un nucléotide méthylé à l'aide d'un extincteur couplé au nucléotide méthylé. Dans certains exemples, un nucléotide méthylé dans un polynucléotide peut être détecté à l'aide d'un procédé qui comprend le couplage d'un extincteur au nucléotide méthylé. Le procédé peut inclure l'ajout de nucléotides marqués par fluorescence à une amorce hybridée au polynucléotide. Le procédé peut inclure l'utilisation de l'extincteur pour réduire la fluorescence provenant d'au moins l'un des nucléotides marqués par fluorescence ajoutés. Le procédé peut inclure l'utilisation de la fluorescence réduite provoquée par le premier extincteur pour détecter le nucléotide méthylé.
PCT/US2024/029126 2023-05-31 2024-05-13 Détection d'un nucléotide méthylé à l'aide d'un extincteur Pending WO2024249061A1 (fr)

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