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WO2024249200A1 - Procédés de conservation de l'état de méthylation pendant le regroupement - Google Patents

Procédés de conservation de l'état de méthylation pendant le regroupement Download PDF

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Publication number
WO2024249200A1
WO2024249200A1 PCT/US2024/030506 US2024030506W WO2024249200A1 WO 2024249200 A1 WO2024249200 A1 WO 2024249200A1 US 2024030506 W US2024030506 W US 2024030506W WO 2024249200 A1 WO2024249200 A1 WO 2024249200A1
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strand
amplification
nucleic acid
array
population
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PCT/US2024/030506
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English (en)
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Colin Brown
Sarah E. SHULTZABERGER
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Illumina, Inc.
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Publication of WO2024249200A1 publication Critical patent/WO2024249200A1/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • Embodiments of the present disclosure relate to preparing nucleic acids for sequencing.
  • embodiments of the methods, compositions, systems, and kits provided herein relate to using sequencing libraries to obtain sequence data that include methylation status.
  • Methylation of cytosine residues at the 5 position of the pyrimidine ring is proposed to have diverse roles in regulation of gene expression, parental imprinting, and molecular etiology of human diseases including cancer.
  • the standard detection method for 5mC is whole-genome bisulfite sequencing (WGBS). This method has proven useful in obtaining whole genome methylation status; however, this method is known to cause significant degradation of sample DNA resulting in GC-bias, overestimation of 5mC abundance, and poor performance with low-input samples.
  • WGBS sodium bisulfite is used to induce deamination of unmodified cytosine residues to produce deoxyuracil, while leaving 5mC residues unaffected.
  • C ⁇ T e.g., cytosine to thymine
  • 5mC residues are still read as C.
  • SNPs single nucleotide polymorphisms
  • Sensitive detection of methylation signals by whole-genome bisulfite sequencing is aided by amplification of bisulfite-converted sequences by standard 4-base PCR following conversion. Many problems with degradation and dropout could in principle be mitigated by performing an amplification step prior to bisulfite conversion, but standard PCR chemistry does not allow for propagation of the methylation signal during amplification. Prior work has attempted to create "methylation-aware" PCR by including the human maintenance methyltransferase in the reaction.
  • a templatedependent DNA methyltransferase such as the enzyme DNA (cytosine-5)- methyltransferase 1 (DNMT1), preferentially identifies hemi-m ethylated CpG dinucleotides sites.
  • a hemi-methylated CpG dinucleotide also referred to as a hemimethylated 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.
  • a template-dependent DNA methyltransferase such as DNMT1 allows propagation of the methylation signal at CpG sites following the extension step of PCR.
  • wildtype DNMT1 is unable to survive the high temperatures encountered during PCR cycling. For this reason, DNMT1 -based methyl-CpG amplification requires either the use of an engineered thermostable DNMT1 or the addition of fresh DNMT1 following each PCR cycle.
  • CpG amplification strategy that extends existing methods for creating clonal clusters in an array, such as flow cell nanowells, to allow preservation of the CpG methylation state of a seed molecule across the entire cluster. This enables an on- array workflow for bisulfite sequencing that incorporates a CpG amplification step prior to bisulfite treatment, with the potential to avoid issues with sequence bias and DNA damage as well as allow for 'pseudo-four-base' bisulfite sequencing.
  • the term "array” refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array.
  • An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and/or complementary sequence thereof).
  • the sites of an array can be different features located on the same substrate. Exemplary features include without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate.
  • the sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.
  • the term "amplification site" refers to a site in or on an array where one or more amplicons can be generated.
  • An amplification site can be further configured to provide for immobilizing or attaching a nucleic acid at the site and to contain, hold, or attach at least one amplicon that is generated at the site.
  • amplicon when used in reference to a nucleic acid, means the product of copying the nucleic acid, wherein the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid.
  • An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, e.g., a target nucleic acid or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, isothermal amplification (e g., kinetic exclusion amplification), or ligation chain reaction.
  • An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a polymerase extension product) or multiple copies of the nucleotide sequence (e.g., a concatameric product of RCA).
  • a first amplicon of a target nucleic acid is typically a complementary copy.
  • Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon.
  • a subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.
  • the first amplicon is produced using a template strand, where the template strand is obtained from a sample and is not subject to amplification prior to annealing the template strand to the amplification site (see “seed nucleic acid” herein).
  • seed nucleic acid seed nucleic acid
  • the addition of template strands to amplification sites permits the retention of methylated nucleotides, including methylated cytosines of CpG dinucleotides.
  • an interstitial region refers to an area in a substrate or on a surface that separates other areas of the substrate or surface.
  • an interstitial region can separate one feature of an array from another feature of the array.
  • the two regions that are separated from each other can be discrete, lacking contact with each other.
  • an interstitial region can separate a first portion of a feature from a second portion of a feature.
  • the separation provided by an interstitial region can be partial or full separation.
  • Interstitial regions will typically have a surface material that differs from the surface material of the features on the surface.
  • features of an array can have an amount or concentration of capture agents that exceeds the amount or concentration present at the interstitial regions. In some embodiments the capture agents may not be present at the interstitial regions.
  • capture agent refers to a material, chemical, molecule, or moiety thereof that is capable of attaching, retaining, or binding to a target molecule (e.g., a target nucleic acid).
  • Exemplary capture agents include, without limitation, a capture nucleic acid that is complementary to at least a portion of a modified target nucleic acid (e.g., a universal capture binding sequence), a member of a receptor-ligand binding pair (e g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) capable of binding to a modified target nucleic acid (or linking moiety attached thereto), or a chemical reagent capable of forming a covalent bond with a modified target nucleic acid (or linking moiety attached thereto).
  • a capture agent is a nucleic acid.
  • a nucleic acid capture agent can also be used as an amplification primer.
  • P5 and P7 may be used when referring to a nucleic acid capture agent.
  • P5 1 P5 prime
  • P7 1 P7 prime
  • any suitable nucleic acid capture agent can be used in the methods presented herein, and that the use of P5 and P7 are exemplary embodiments only.
  • Uses of nucleic acid capture agents such as P5 and P7 on flow cells is known in the art, as exemplified by the disclosures of WO 2007/010251, WO 2006/064199, WO 2005/065814, WO 2015/106941, WO 1998/044151, and WO 2000/018957.
  • nucleic acid capture agent can also function as an amplification primer.
  • any suitable nucleic acid capture agent can act as a forward amplification primer, whether immobilized or in solution, and can be useful in the methods presented herein for hybridization to a sequence (e.g., a universal capture binding sequence) and amplification of a sequence.
  • any suitable nucleic acid capture agent can act as a reverse amplification primer, whether immobilized or in solution, and can be useful in the methods presented herein for hybridization to a sequence (e.g., a universal capture binding sequence) and amplification of a sequence.
  • a forward amplification primer whether immobilized or in solution
  • reverse amplification primer whether immobilized or in solution
  • the term "universal sequence” refers to a region of sequence that is common to two or more target nucleic acids, where the molecules also have regions of sequence that differ from each other.
  • a universal sequence that is present in different members of a collection of molecules can allow capture of multiple different nucleic acids using a population of capture nucleic acids that are complementary to a portion of the universal sequence, e.g., a universal capture binding sequence.
  • Non-limiting examples of universal capture binding sequences include sequences that are identical to or complementary to P5 and P7 primers.
  • a universal sequence present in different members of a collection of molecules can allow the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, e.g., a universal primer binding site.
  • Target nucleic acid molecules may be modified to attach universal adapters (also referred to herein as adapters), for example, at one or both ends of the different target sequences, as described herein.
  • the term "adapter” and its derivatives refers generally to any linear oligonucleotide which can be attached to a target nucleic acid.
  • the adapter is substantially non-complementary to the 3' end or the 5' end of any target sequence present in a sample.
  • suitable adapter lengths are in the range of about 10-100 nucleotides, about 12-60 nucleotides and about 15- 50 nucleotides in length.
  • the adapter can include any combination of nucleotides and/or nucleic acids.
  • the adapter can include one or more cleavable groups at one or more locations.
  • the adapter can include a sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a capture nucleic acid.
  • the adapter can include a barcode, also referred to as an index or tag, to assist with downstream error correction, identification, or sequencing.
  • the terms “adaptor” and “adapter” are used interchangeably.
  • nucleic acid is intended to be consistent with its use in the art and includes naturally occurring nucleic acids and functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence.
  • Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds.
  • An analog structure can have an alternate backbone linkage including any of a variety of those known in the art.
  • Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).
  • a nucleic acid can contain any of a variety of analogs of these sugar moieties that are known in the art.
  • a nucleic acid can include native or non-native bases.
  • a native deoxyribonucleic acid can have one or more bases selected from adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from uracil, adenine, cytosine or guanine.
  • Useful non-native bases that can be included in a nucleic acid are known in the art.
  • a "seed nucleic acid” is a member of a sequencing library that has been exposed to conditions that preserve an epigenetic marker present, such as the methylation state of nucleotides.
  • a sequencing library is seed nucleic acids if the sequencing library is not exposed to conditions resulting in linear or exponential amplification.
  • a seed nucleic acid can be the single-stranded nucleic acid that is annealed to a capture nucleic acid at the surface of an amplification site and used as a template strand for production of an amplicon.
  • a seed nucleic acid can be the single-stranded nucleic acid that is immobilized to an amplification site at its 5’ end and has been exposed to conditions to propagate an epigenetic marker present, such as the methylation state of nucleotides.
  • a target nucleic acid having a universal sequence at each end, for instance a universal adapter at each end, can be referred to as a "modified target nucleic acid.”
  • the terms “sequencing library” and “library” refer to the collection of target nucleic acids or modified target nucleic acids.
  • clonal cluster As used herein, the terms “clonal cluster,” “clonal population” and “monoclonal population” are used interchangeably and refer to a population of nucleic acids that is homogeneous with respect to a particular nucleotide sequence.
  • the homogenous sequence is typically at least 10 nucleotides long, but can be even longer including for example, at least 50, at least 100, at least 250, at least 500, or at least 1000 nucleotides long.
  • a clonal population can be derived from a single target nucleic acid. Typically, all of the nucleic acids in a clonal population will have the same nucleotide sequence.
  • Conditions that are "suitable” for an event to occur or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.
  • providing in the context of an item described herein, such as a composition, an article, or a nucleic acid means making the composition, article, or nucleic acid, purchasing the composition, article, or nucleic acid, or otherwise obtaining the compound, composition, article, or nucleic acid.
  • steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • FIG. 1A-1I shows schematic drawings of an embodiment of producing clonal clusters that preserve the CpG methylation state of a seed nucleic acid. For simplicity, only one amplification site of an array and a limited number of target nucleic acids are shown. The figures use the following convention when numbering single strands of nucleic acids: strands showing a methylated cytosine are numbered (e.g., strand 13 of FIG. 1C); strands showing a non-methylated cytosine are also numbered but the number is modified with the symbol (e.g., strand 13' of FIG.
  • FTG. 2 shows a schematic drawing of an alternate embodiment of producing clonal clusters that preserve the CpG methylation state of a seed nucleic acid. For simplicity, only one amplification site of an array and a limited number of target nucleic acids are shown. In this embodiment, treatment with DNMT1 occurs during bridge amplification. This embodiment of bridge amplification utilizes thermocycling amplification.
  • FIG. 3A-3G shows a schematic drawing of an alternate embodiment of producing clonal clusters that preserve the CpG methylation state of a seed nucleic acid. For simplicity, only one amplification site of an array and a limited number of target nucleic acids are shown. In this embodiment, treatment with DNMT1 occurs simultaneously with kinetic exclusion bridge amplification.
  • FIG. 4 shows the sequence of the human template-dependent DNA (cytosine-5)- methyltransferase 1 (DNMT1, SEQ ID NO: 1).
  • the present disclosure provides methods for determining the methylation status of genomic DNA by producing clonal clusters that preserve epigenetic markers, such as the methylation state of a seed nucleic acid.
  • the method includes providing an array that includes a plurality of amplification sites.
  • Each amplification site includes a plurality of capture nucleic acids attached by the 5’ end to the amplification site.
  • Individual amplification sites can include one single-stranded (ss) nucleic acid attached to the amplification site surface by hybridization between nucleotides at the 3' end of the single-strand nucleic acid and nucleotides at the 3' end of one capture nucleic acid. For instance, as shown in FIG. 1 A the single-strand nucleic acid 12 is shown annealed to a capture nucleic acid 11.
  • the single-strand nucleic acid is a seed nucleic acid; it is a member of a sequencing library that has been exposed to conditions that preserve an epigenetic marker present, such as the methylation state of nucleotides.
  • the sequencing library has been produced using methods that do not include amplification.
  • FIG. 1A the single-strand nucleic acid 12 is shown with a CpG dinucleotide where the C is methylated.
  • a single-strand nucleic acid could include any number of methylated nucleotides, including multiple distinct CpG dinucleotides where the C is methylated.
  • the method further includes extension of the 3' end of the capture nucleic acid with a DNA polymerase using the single-strand nucleic acid as template strand to produce the complementary strand.
  • a double-stranded (ds) nucleic acid 14 results after the extension, where one strand made up of the capture nucleic acid 11 and the newly synthesized amplicon 13', which is the unmethylated complement of the template strand 12.
  • the complementary 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 nucleic acid 14.
  • the method includes exposing the array to conditions that transfer the hemi-methylated CpG dinucleotides of the original single-strand nucleic acid to the complementary strand.
  • the conditions include exposing the amplification sites to an enzyme, such as DNMT1. For instance, as shown in FIG.
  • the methylation state of the methylated CpG dinucleotide of the template strand 12 is transferred to the now methylated complementary strand 13, converting the hemi- methylated site to methylated CpG dinucleotides on both strands. 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.
  • the single double-stranded nucleic acid at each amplification site is amplified to include a clonal population of immobilized nucleic acids.
  • the clonal population includes a first sub-population of single-strand nucleic acids having the same nucleotide sequence as the complement described above (e.g., complementary strand 13') , but the CpG dinucleotides are not methylated.
  • One of the strands of this subpopulation includes the methylated CpG dinucleotides produced by the transfer using, for instance, the enzyme DNMT1.
  • the clonal population also includes a second subpopulation of single-stranded nucleic acids which includes the nucleotide sequence of the template strand, 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'.
  • the amplification sites also include multiple copies of the unmethylated nucleotide sequence 12', which includes the same nucleotide sequence as 12 in FIG. 1A-C but is now attached to the amplification site surface by a different capture nucleic acid 15.
  • the method further includes propagating, at each amplification site, the methylated CpG dinucleotide present on one strand of the clonal population to other members 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 one strand of the clonal population to other strands.
  • the conditions propagate the methylation status to both template and complementary strands.
  • the conditions include exposing the amplification sites to an enzyme such as DNMT1 and a DNA helicase or recombinase.
  • Propagation of the CpG signal occurs by many cycles of steps that include (i) hybridization of the complementary single-strand nucleic acids 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.
  • steps that include (i) hybridization of the complementary single-strand nucleic acids 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
  • the methylation status of CpG dinucleotides on strand 13 is transferred to strand 12' to convert strand 12' to 12 as shown in III.
  • the double-stranded structure shown in III is unwound and the process is repeated until the methylation status of the one methylated single-stranded nucleic acid 13 is propagated through both sub-populations of nucleic acids at the amplification sites, resulting in fully methylated clusters IV.
  • one of the capture nucleic acids that is attached to the surface is cleaved to remove one sub-population of single-stranded nucleic acids from the amplification sites.
  • the cleaving of a nucleotide sequence to permit the optional removal of a specific strand is referred to herein as "linearization.”
  • the sub-population of amplicons 12 have been removed, leaving the amplicons 13 ready for sequencing.
  • the method further includes hybridizing a sequencing primer to the single-stranded amplicon and using standard sequencing methods, such as sequencing-by-synthesis (SBS), to generate a reference read representing the full 4- base genomic sequence of the target nucleic acid.
  • SBS sequencing-by-synthesis
  • the amplification sites of the array are exposed to conditions for on-array chemical treatment to allow detection of methylated residues. For instance, as shown in FIG. 1H, treatment of the array with sodium bisulfite converts all non-methylated cytosines to uracils to result in methylated and chemically treated strand 13"
  • the converted amplicons can be used in a standard paired-end resynthesis and linearization to generate amplification sites complementary to the reference read, where the G ⁇ -A mutations show the locations of the non-methylated cytosines and the guanosine residues show the locations of methylated cytosines.
  • the sub-population of amplicons 13" have been removed after being used to synthesize amplicons 12".
  • FIG. II also shows a sequencing primer 17 annealed to the single-stranded amplicon 12".
  • An array of amplification sites used in a method set forth herein can be present as one or more substrates.
  • Exemplary types of substrate materials that can be used for an array include glass, modified glass, functionalized glass, inorganic glasses, microspheres (e.g., inert and/or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell (e g., microtiter) plates.
  • Exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and TeflonTM.
  • Exemplary silica-based materials include silicon and various forms of modified silicon.
  • a substrate can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, bottle, or the like.
  • a particularly useful vessel is a flow-cell, for example, as described in US Pat. No. 8,241,573 or Bentley et al., Nature 456:53-59 (2008). Exemplary flow-cells are those that are commercially available from Illumina, Inc. (San Diego, Calif.).
  • Another particularly useful vessel is a well in a multi-well plate or microtiter plate.
  • the amplification sites of an array can be configured as features on a surface.
  • the features can be present in any of a variety of desired formats.
  • the sites can be wells, pits, channels, ridges, raised regions, pegs, posts or the like.
  • the amplification sites can contain beads. However, in particular embodiments the sites need not contain a bead or particle.
  • Exemplary sites include wells that are present in substrates used for commercially available sequencing platforms (e.g., Ion Torrent, (a subsidiary of Thermo Fisher Scientific).
  • Other substrates having wells include, for example, etched fiber optics and other substrates described in U.S. Pat. No. 6,266,459; U.S. Pat. No.
  • the amplification sites of an array can be metal features on a non-metallic surface such as glass, plastic or other materials exemplified herein.
  • a metal layer can be deposited on a surface using methods known in the art such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering, or the like. Any of a variety of commercial instruments can be used as appropriate including, for example, the FlexAL®, Op AL®, lonfab 300Plus®, or Optofab 3000® systems (Oxford Instruments, UK).
  • a metal layer can also be deposited by e-beam evaporation or sputtering as set forth in Thornton, Ann. Rev.
  • Metal layer deposition techniques can be combined with photolithography techniques to create metal regions or patches on a surface. Exemplary methods for combining metal layer deposition techniques and photolithography techniques are provided in U.S. Pat. No. 8,778,848 and U.S. Pat. No. 8,895,249.
  • An array of features can appear as a grid of spots or patches.
  • the features can be located in a repeating pattern or in an irregular non-repeating pattern.
  • Particularly useful patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like.
  • Asymmetric patterns can also be useful.
  • the pitch can be the same between different pairs of nearest neighbor features or the pitch can vary between different pairs of nearest neighbor features.
  • features of an array can each have an area that is larger than about 100 nm 2 , 250 nm 2 , 500 nm 2 , 1 pm 2 , 2.5 pm 2 , 5 pm 2 , 10 pm 2 , 100 pm 2 , or 500 pm 2 .
  • features of an array can each have an area that is smaller than about 1 mm 2 , 500 pm 2 , 100 pm 2 , 25 pm 2 , 10 pm 2 , 5 pm 2 , 1 pm 2 , 500 nm 2 , or 100 nm 2 .
  • a region can have a size that is in a range between an upper and lower limit selected from those exemplified above.
  • the features can be discrete, being separated by interstitial regions.
  • the size of the features and/or spacing between the regions can vary such that arrays can be high density, medium density, or lower density. High density arrays are characterized as having regions separated by less than about 15 pm. Medium density arrays have regions separated by about 15 to 30 pm, while low density arrays have regions separated by greater than 30 pm.
  • An array useful in the disclosure can have regions that are separated by less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, or 0.5 pm.
  • an array can include a collection of beads or other particles.
  • the particles can be suspended in a solution or they can be located on the surface of a substrate.
  • bead arrays in solution are those commercialized by Luminex (Austin, TX, USA).
  • arrays having beads located on a surface include those wherein beads are located in wells such as a BeadChip array (Illumina Inc., San Diego, CA, USA) or substrates used in sequencing platforms from Ion Torrent (a subsidiary of Life Technologies, Carlsbad, CA USA).
  • BeadChip array Illumina Inc., San Diego, CA, USA
  • substrates used in sequencing platforms from Ion Torrent a subsidiary of Life Technologies, Carlsbad, CA USA.
  • Other arrays having beads located on a surface are described in U.S. Pat. No. 6,266,459; U.S. Pat. No. 6,355,431; U.S. Pat. No.
  • the beads can be made to include amplification primers and the beads can then be used to load an array, thereby forming amplification sites for use in a method set forth herein.
  • the substrates can be used without beads.
  • amplification primers can be attached directly to the wells or to gel material in wells.
  • Amplification sites of an array can include a plurality of capture agents capable of binding to target nucleic acids.
  • a capture agent includes a capture nucleic acid.
  • the nucleotide sequence of the capture nucleic acid is complementary to a universal sequence of the target nucleic acids.
  • the capture nucleic acid can also function as a primer for amplification of the target nucleic acid.
  • one population of capture nucleic acid includes a P5 primer or the complement thereof.
  • the amplification sites also include a plurality of a second capture nucleic acid, and this second capture nucleic acid can include a P7 primer or the complement thereof.
  • a capture nucleic acid can include a cleavage site. Cleavage sites in a capture nucleic acid are described in greater detail herein.
  • a capture agent such as a capture nucleic acid
  • the capture agent can be attached to the surface of a feature of an array.
  • the attachment can be via an intermediate structure such as a bead, particle or gel.
  • An example of attachment of capture nucleic acids to an array via a gel is described in U.S. Pat. No. 8,895,249 and further exemplified by flow cells available commercially from Illumina Inc. (San Diego, CA, USA) or described in WO 2008/093098.
  • Exemplary gels that can be used in the methods and apparatus set forth herein include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, SFA (see, for example, US Pat. App. Pub. No. 2011/0059865 Al) or PAZAM (see, for example, U.S. Prov. Pat. App. Ser. No. 61/753,833 and U.S. Pat. No. 9,012,022). Attachment via a bead can be achieved as exemplified in the description and cited references set forth previously herein.
  • a colloidal structure such as agarose
  • polymer mesh structure such as gelatin
  • cross-linked polymer structure such as polyacrylamide, SFA (see, for example, US Pat. App. Pub. No. 2011/0059865 Al) or PAZAM (see, for example, U.S. Prov. Pat. App. Ser. No. 61/753
  • the features on the surface of an array substrate are non-contiguous, being separated by interstitial regions of the surface.
  • Interstitial regions that have a substantially lower quantity or concentration of capture agents, compared to the features of the array, are advantageous.
  • Interstitial regions that lack capture agents are particularly advantageous.
  • a relatively small amount or absence of capture moieties at the interstitial regions favors localization of target nucleic acids, and subsequently generated clusters, to desired features.
  • the features can be concave features in a surface (e g., wells) and the features can contain a gel material.
  • the gelcontaining features can be separated from each other by interstitial regions on the surface where the gel is substantially absent or, if present the gel is substantially incapable of supporting localization of nucleic acids.
  • An array used in a method described herein includes modified target nucleic acids.
  • the target nucleic acid may be essentially any nucleic acid of known or unknown sequence. It may be, for example, a fragment of genomic DNA. Sequencing may result in determination of the sequence of the whole or a part of the target molecule.
  • the targets can be processed into templates suitable for amplification by the placement of universal amplification sequences, e.g., sequences present in a universal adaptor, at the ends of each target fragment.
  • the primary nucleic acid sample may originate in double-stranded DNA (dsDNA) form (e.g., genomic DNA or genomic DNA fragments).
  • dsDNA double-stranded DNA
  • the precise sequence of the polynucleotide molecules from a primary nucleic acid sample is generally not material to the disclosure and may be known or unknown.
  • the primary polynucleotide molecules from a primary nucleic acid sample are DNA molecules. More particularly, the primary polynucleotide molecules represent the entire genetic complement of an organism and are genomic DNA molecules which include both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. In one embodiment, particular subsets of polynucleotide sequences or genomic DNA can be used, such as, for example, particular chromosomes. Yet more particularly, the sequence of the primary polynucleotide molecules is not known. Still yet more particularly, the primary polynucleotide molecules are human genomic DNA molecules.
  • the nucleic acid sample can include high molecular weight material such as genomic DNA (gDNA).
  • the sample can include low molecular weight material such as nucleic acid molecules obtained from formalin-fixed paraffin-embedded or archived DNA samples.
  • low molecular weight material includes enzymatically or mechanically fragmented DNA.
  • the sample can include cell-free circulating DNA.
  • the sample can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissections, surgical resections, and other clinical or laboratory obtained samples.
  • the sample can be an epidemiological, agricultural, forensic or pathogenic sample.
  • the sample can include nucleic acid molecules obtained from an animal such as a human or mammalian source.
  • the sample can include nucleic acid molecules obtained from a non-mammalian source such as a plant, a bacterium, a virus, or a fungus.
  • the source of the nucleic acid molecules may be an archived or extinct sample or species.
  • Additional non-limiting examples of sources of biological samples can include whole organisms as well as a sample obtained from a patient.
  • the biological sample can be obtained from any biological fluid or tissue and can be in a variety of forms, including fluid, e.g., liquid or gas, tissue, e.g., solid tissue, and preserved forms of such a fluid or tissue, such as dried, frozen, and fixed forms.
  • the sample may be of any biological tissue, cells, or fluid.
  • Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), ascitic fluid, urine, saliva, tears, sputum, vaginal fluid (discharge), washings obtained during a medical procedure (e.g., pelvic or other washings obtained during biopsy, endoscopy or surgery), tissue, nipple aspirate, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or micro-dissected cells or extracellular parts thereof.
  • the sample can be a blood sample, such as, for example, a whole blood sample.
  • the sample is an unprocessed dried blood spot sample.
  • the sample is a formalin- fixed paraffin-embedded sample.
  • the sample is a saliva sample.
  • the sample is a dried saliva spot sample.
  • Exemplary biological samples from which target nucleic acids can be derived include, for example, those from a eukaryote, for instance a mammal, such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or nonhuman primate; a plant, such as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or soybean; an algae, such as Chlamydomonas reinhardtii, a nematode such as Caenorhabditis elegans, an insect, such as Drosophila melanogaster, mosquito, fruit fly, honey bee or spider; a fish, such as zebrafish; a reptile; an amphibian, such as a frog or Xenopus laevis,' a Dictyostelium discoideum a fungi, such
  • Target nucleic acids can also be derived from a prokaryote such as a bacterium, Escherichia coll, staphylococci or Mycoplasma pneumoniae, an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid.
  • a prokaryote such as a bacterium, Escherichia coll, staphylococci or Mycoplasma pneumoniae, an archaeon
  • a virus such as Hepatitis C virus or human immunodeficiency virus
  • a viroid can be derived from a homogeneous culture or population of organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
  • the target nucleic acids used in the methods and compositions of the present disclosure can be derived by fragmentation, such as random fragmentation.
  • Random fragmentation refers to the fragmentation of a polynucleotide molecule from a primary nucleic acid sample in a non-ordered fashion by enzymatic, chemical, or mechanical methods. Such fragmentation methods are known in the art and use standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). Moreover, random fragmentation is designed to produce fragments irrespective of the sequence identity or position of nucleotides comprising and/or surrounding the break.
  • the random fragmentation is by mechanical means such as nebulization or sonication to produce fragments of about 50 base pairs in length to about 1500 base pairs in length, still more particularly 50-700 base pairs in length, yet more particularly 50-400 base pairs in length. Most particularly, the method is used to generate smaller fragments of from 50-150 base pairs in length.
  • fragmentation of polynucleotide molecules by mechanical means results in fragments with a heterogeneous mix of blunt and 3'- and 5'-overhanging ends. It is therefore desirable to repair the fragment ends using methods or kits (such as the LUCIGENTM DNAterminatorTM End Repair Kit) known in the art to generate ends that are optimal for insertion, for example, into blunt sites of cloning vectors.
  • the fragment ends of the population of nucleic acids are blunt ended. More particularly, the fragment ends are blunt ended and phosphorylated.
  • the phosphate moiety can be introduced via enzymatic treatment, for example, using polynucleotide kinase.
  • the target fragment sequences are prepared with single overhanging nucleotides by, for example, activity of certain types of DNA polymerase such as Taq polymerase or Klenow exo minus polymerase which has a non-template-dependent terminal transferase activity that adds a single deoxynucleotide, for example, deoxyadenosine (A) to the 3' ends of a DNA molecule, for example, a PCR product.
  • DNA polymerase such as Taq polymerase or Klenow exo minus polymerase which has a non-template-dependent terminal transferase activity that adds a single deoxynucleotide, for example, deoxyadenosine (A) to the 3' ends of a DNA molecule, for example, a PCR product.
  • A deoxyadenosine
  • an ‘A’ could be added to the 3' terminus of each end repaired strand of the double-stranded target fragments by reaction with Taq or Klenow exo minus polymerase, while the universal adapter polynucleotide construct could be a T-construct with a compatible ‘T’ overhang present on the 3' terminus of each region of double-stranded nucleic acid of the universal adapter.
  • This end modification also prevents self-ligation of both vector and target such that there is a bias towards formation of target nucleic acids having a universal adapter at each end.
  • fragmentation can be accomplished using a process often referred to as tagmentation.
  • Tagmentation uses a transposome complex and combines into a single step fragmentation and ligation to add universal adapters (Gunderson et al., WO 2016/130704).
  • a transposome complex is a transposase bound to a transposase recognition site and can insert the transposase recognition site into a target nucleic acid in a process sometimes termed "tagmentation.” In some such insertion events, one strand of the transposase recognition site may be transferred into the target nucleic acid.
  • a transposome complex includes a dimeric transposase having two subunits, and two non-contiguous transposon sequences.
  • a transposase includes a dimeric transposase having two subunits, and a contiguous transposon sequence.
  • Some embodiments can include the use of a hyperactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol.
  • MuA transposase and a Mu transposase recognition site comprising R1 and R2 end sequences
  • R1 and R2 end sequences can also be used by a skilled artisan.
  • transposon sequences useful with the methods and compositions described herein are provided in U.S. Patent Application Pub. No. 2012/0208705, U.S. Patent Application Pub. No. 2012/0208724 and Int. Patent Application Pub. No. WO 2012/061832.
  • a transposon sequence includes a first transposase recognition site and a second transposase recognition site.
  • transposome complexes useful herein include a transposase having two transposon sequences.
  • the two transposon sequences are not linked to one another, in other words, the transposon sequences are non-contiguous with one another. Examples of such transposomes are known in the art (see, for instance, U.S. Patent Application Pub. No. 2010/0120098).
  • tagmentation is used to produce target nucleic acids that include different universal sequences at each end. This can be accomplished by using two types of transposome complexes, where each transposome complex includes a different nucleotide sequence that is part of the transferred strand.
  • a population of target nucleic acids can have an average strand length that is desired or appropriate for a particular application of the methods or compositions set forth herein.
  • the average strand length can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides.
  • the average strand length can be greater than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides.
  • the average strand length for a population of target nucleic acids can be in a range between a maximum and minimum value set forth herein. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have an average strand length that is in a range between an upper and lower limit selected from those exemplified above.
  • a population of target nucleic acids can be produced under conditions or otherwise configured to have a maximum length for its members.
  • the maximum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be less than 100,000 nucleotides, less than 50,000 nucleotides, less than 10,000 nucleotides, less than 5,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, less than 100 nucleotides, or less than 50 nucleotides.
  • a population of target nucleic acids can be produced under conditions or otherwise configured to have a minimum length for its members.
  • the minimum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be more than 10 nucleotides, more than 50 nucleotides, more than 100 nucleotides, more than 500 nucleotides, more than 1,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, more than 50,000 nucleotides, or more than 100,000 nucleotides.
  • the maximum and minimum strand length for target nucleic acids in a population can be in a range between a maximum and minimum value set forth above. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have maximum and/or minimum strand lengths in a range between the upper and lower limits exemplified above.
  • a target nucleic acid used in a method or composition described herein includes a universal adapter attached to each end.
  • a target nucleic acid having a universal adapter at each end can be referred to as a "modified target nucleic acid.”
  • Methods for attaching a universal adapter to each end of a target nucleic acid used in a method described herein are known to the person skilled in the art. The attachment can be through tagmentation using transposase complexes (WO 2016/130704), or through standard library preparation techniques using ligation (U.S. Pat. Pub. No. 2018/0305753).
  • double-stranded target nucleic acids from a sample are treated by first ligating identical universal adaptor molecules to the 5' and 3' ends of the double-stranded target nucleic acids.
  • the universal adapters are "matched" adapters because the two strands of the adaptors are formed by annealing complementary polynucleotide strands.
  • the universal adapters used in the method of the disclosure are referred to as "mismatched" adaptors because the adaptors include a region of sequence mismatch, i.e., they are not formed by annealing fully complementary polynucleotide strands.
  • the universal adaptor typically includes universal capture binding sequences that aid in immobilizing the target nucleic acids on an array for subsequent sequencing, and universal primer binding sites useful for the sequencing.
  • a universal adapter can optionally include at least one index.
  • An index can be used as a marker characteristic of the source of particular target nucleic acids on an array (U.S. Pat. No. 8,053,192).
  • the index is a synthetic sequence of nucleotides that is part of the universal adapter which is added to the target nucleic acids as part of the library preparation step.
  • an index is a nucleic acid sequence which is attached to each of the target molecules of a particular sample, the presence of which is indicative of, or is used to identify, the sample or source from which the target molecules were isolated.
  • an index may be up to 20 nucleotides in length, more preferably 1-10 nucleotides, and most preferably 4-6 nucleotides in length.
  • a four nucleotide index gives a possibility of multiplexing 256 samples on the same array, a six base index enables 4096 samples to be processed on the same array.
  • the precise nucleotide sequence of the universal adapters is generally not material to the disclosure and may be selected by the user such that the desired sequence elements are ultimately included in the common sequences of the plurality of different modified target nucleic acids, for example, to provide for the universal capture binding sequences for immobilizing the target nucleic acids on an array for subsequent sequencing, and binding sites for particular sets of universal amplification primers and/or sequencing primers. Additional sequence elements may be included, for example, to provide binding sites for sequencing primers which will ultimately be used in sequencing of target nucleic acids in the library, sequencing of an index, or products derived from amplification of the target nucleic acids in the library, for example on a solid support.
  • preparation of an array that includes target nucleic acids at amplification sites includes seeding the amplification sites with single-stranded nucleic acids and then amplifying the seeded target nucleic acids.
  • the method described herein includes additional steps between the annealing and the amplifying. In some other embodiments, the method described herein includes additional steps after the amplifying.
  • Generating clonal clusters of the present disclosure also includes extension of annealed single-stranded nucleic acids to produce a complementary strand that is not methylated, resulting in a hemi-methylated double-stranded nucleic acid, and then transferring the methylated status to the complementary strand to result in both strands of each duplex being fully methylated.
  • the seeding of amplification sites of an array includes the use of single-stranded members of a sequencing library.
  • the members of a sequencing library are exposed to conditions that preserve epigenetic markers, such as methylated CpG sites.
  • Monoclonal amplification sites (amplification sites occupied with just one single-stranded member of a sequencing library) are most desirable, as sequencing monoclonal populations of amplicons yields much higher signal -to-noise ratios, increased intensity, and increased percentage of amplification sites that pass filter, all of which contribute to increased data output and data quality.
  • the method includes adding single-stranded members of a sequencing library to the array to result in hybridization of universal sequences on the modified target nucleic acids to capture nucleic acids present on the amplification sites.
  • the single-stranded nucleic acids have fluidic access to the amplification sites of an array, and the single-stranded nucleic acids can be transported, for example by passive diffusion or other processes, to amplification sites.
  • the concentration of single-stranded nucleic acids and hybridization conditions can be selected to obtain a maximum number of amplification sites occupied with one single-stranded nucleic acid. Typically, the number of the single-stranded nucleic acids added to the array exceeds the number of amplification sites in the array.
  • a concentration of single-stranded nucleic acids is added to result in a maximum of about 37% occupancy .
  • the conditions for annealing can be isothermal or use varying temperatures. For instance, a constant temperature can be used. Alternatively, a temperature ramp can be used, typically starting at a higher temperature and reducing over time to a lower temperature. Any time period can be used, and shorter hybridization times are generally preferred. For instance, as shown in FIG. 1A, the single-strand nucleic acid 12 is shown annealed to a capture nucleic acid 11.
  • Hybridization of target nucleic acids to amplification sites is followed by extension of the capture nucleic acids using the hybridized seed nucleic acids as a template. Extension occurs in the presence of components that, when combined with the hybridized seed nucleic acids and the capture nucleic acids, cause the synthesis of a strand complementary to the seed nucleic acid and immobilized to the amplification site by the capture nucleic acid.
  • the result is a plurality of amplification sites that each include one double-stranded nucleic acid. If the seed nucleic acid includes a methylated CpG site, the resulting doublestranded nucleic acid is hemi-methylated at each methylated CpG site.
  • the extension includes a DNA polymerase, free nucleotides, and components and conditions suitable for the extension to occur.
  • Conditions for extension of a nucleic acid at an amplification site using DNA polymerase are known to the skilled person. For instance, as shown in FIG. IB, a double-stranded (ds) nucleic acid 14 results after the extension, where one strand made up of the capture nucleic acid 11 and the newly synthesized amplicon 13', which is the unmethylated complement of the template strand 12.
  • the methylated CpG dinucleotide is present on strand 12, and the unmethylated complement CpG is present on the complementary strand 13', resulting in a hemi-methylated state for that dinucleotide present on the double-stranded nucleic acid 14.
  • the amplification sites are further exposed to conditions that transfer the methylation status of the seed nucleic acid to the newly synthesized complementary strand.
  • a templatedependent DNA methyltransferase is used to transfer the methylation status.
  • the template-dependent DNA methyltransferase is the enzyme DNA (cytosine-5)-methyltransferase 1 (DNMT1).
  • DNMT1 preferentially identifies hemimethylated CpG sites and methylates the cytosine of the complementary unmethylated CpG dinucleotide, resulting in a CpG dinucleotide on both strands.
  • DNA methyltransferase enzymes are commercially available, for example from Sigma AldrichTM (catalog no.
  • the methylation state of the methylated CpG dinucleotide of the template strand 12 is transferred to the now methylated complementary strand 13.
  • the amplification sites of an array can be exposed to denaturing conditions after transfer of the methylation status from the methylated seed strand to the unmethylated complementary strand.
  • the denaturing results in amplification sites having the now methylated complementary strand covalently attached to the site by the capture nucleic acid.
  • Denaturing conditions include, but are not limited to, formamide, heat, or alkali.
  • human DNMT1 SEQ ID NO: 1 may be used with the methods, arrays, and compositions described herein.
  • the rate at which methylation occurs can be increased by increasing the concentration or amount of one or more of the active components of the reaction, for example, the concentration of enzyme.
  • Methylation rates can also be increased in a method set forth herein by adjusting the temperature.
  • the rate of methylation of the unmethylated complementary strand can be increased by increasing the temperature up to a maximum temperature where reaction rate declines due to denaturation or other adverse events.
  • Optimal or desired temperatures can be determined from known properties of the enzyme in use or empirically determined for a given mixture.
  • the nucleic acid is amplified to generate a clonal population (also referred to as a cluster) of amplicons.
  • the amplification reaction proceeds until a sufficient number of amplicons are generated to fill the capacity of the respective amplification site.
  • the amplification is under conditions that do not preserve the methylated state of the covalently attached single-stranded nucleic acid present at an amplification site. The result is an amplification site with a plurality of amplicons.
  • One amplicon includes the methylated CpG sites methylated by the action of the template-dependent DNA methyltransferase, such as DNMT1.
  • the remaining amplicons include an unmethylated nucleotide sequence that is identical to, or the complement of, the original seed nucleic acid.
  • an amplification site includes one strand 13 having methylated CpG dinucleotides and multiple copies of the same unmethylated nucleotide sequence 13' having unmethylated CpG dinucleotides.
  • the amplification sites also include multiple copies of the unmethylated nucleotide sequence 12' having unmethylated CpG dinucleotides, which includes the same nucleotide sequence as strand 12 in FIGs. 1A-C but is now attached to the amplification site surface by a different capture nucleic acid 15.
  • the amplification is under conditions that preserve the methylated states of the covalently attached single-stranded methylation state of the covalently attached single-stranded nucleic acid present at an amplification site.
  • a template-dependent DNA methyltransferase may be added to an amplification reaction mixture.
  • Some template-dependent DNA methyltransferases, such as DNMT1 are not stable at high temperatures for extended periods of time. Thus, when a template-dependent DNA methyltransferase is added to an amplification mixture, it may be added following any steps performed at an elevated temperature.
  • DNMT1 may be added to an amplification reaction after denaturation of a template nucleic acid and primer annealing, such as during or after polymerase extension.
  • an amplification reaction includes multiple steps performed at an elevated temperature
  • the template-dependent DNA methyltransferase may be added after each of these steps.
  • FIG. 2 depicts an exemplary embodiment of a bridge amplification workflow wherein the bridged product is treated with a templatedependent DNA methyltransferase following extension.
  • a single-stranded nucleic acid 13 is immobilized to an amplification site at its 5' end.
  • the single-stranded nucleic acid 13 is a seed nucleic acid.
  • the single-stranded nucleic acid 13 may be produced using, for example, a method consistent with the method depicted in FIG. 1A-C.
  • the amplification site additionally includes immobilized capture nucleic acids 11, 15.
  • the seed nucleic acid has at its 3' end a sequence that is complementary to at least part of the first immobilized capture nucleic acid 15.
  • the seed nucleic acid has at its 5' end a sequence that is substantially identical to at least part of the second immobilized capture nucleic acid 11.
  • the single-stranded nucleic acid 13 binds to the first immobilized capture nucleic acid 15 via the sequence at its 5' end.
  • the first immobilized capture nucleic acid 15 is extended using the single-stranded nucleic acid 13 as a template.
  • the methylation state of the single-stranded nucleic acid 13 is propagated to the newly synthesized strand 12’.
  • the methylation state is propagated by contacting the amplification site with a template-directed DNA methyltransferase.
  • the single-stranded nucleic acid 13 and the newly synthesized and methylated strand 12” are denatured.
  • Both the single-stranded nucleic acid 13 and the newly synthesized strand 12” are attached to the amplification site at their 5' ends.
  • both the single-stranded nucleic acid 13 and the newly synthesized strand 12” include complementary methylated nucleic acids, e.g., the methylation status of the single-stranded nucleic acid 13 has been propagated to the newly synthesized strand 12”.
  • the single-stranded nucleic acid 13 may bind a new first immobilized capture sequence 15.
  • the newly synthesized strand 12” may bind the second immobilized capture sequence 11.
  • Each strand is extended to produce a new complementary strand.
  • the methylation state of each strand is propagated by contacting the amplification site with a template-directed DNA methyltransferase. As many DNA methyltransferases are not thermostable, this step may include adding a fresh aliquot of template-directed DNA methyltransferase to the amplification site. The process may be repeated for as many cycles as are necessary to populate the amplification site.
  • amplification methods include, but are not limited to, solid-phase amplification.
  • solid-phase amplification refers to any nucleic acid 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.
  • the term encompasses solid-phase polymerase chain reaction (solidphase PCR) and solid phase isothermal amplification which are reactions analogous to standard solution phase amplification, except that one or both of the forward and reverse amplification primers are immobilized on the solid support.
  • 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 see, e.g., WO 2000/018957, U.S. Pat. No. 7,972,820; U.S. Pat. No.
  • Kinetic exclusion can exploit a relatively slow rate for making a first copy of a target nucleic acid vs. a relatively rapid rate for making subsequent copies of the target nucleic acid or of the first copy.
  • kinetic exclusion occurs due to the relatively slow rate of target nucleic acid seeding (e.g., relatively slow diffusion or transport) vs. the relatively rapid rate at which amplification occurs to fill the site with copies of the nucleic acid seed.
  • kinetic exclusion can occur due to a delay in the formation of a first copy of a target nucleic acid that has seeded a site (e.g., delayed or slow activation) vs. the relatively rapid rate at which subsequent copies are made to fill the site.
  • an individual site may have been seeded with several different target nucleic acids (e.g., several target nucleic acids can be present at each site prior to amplification).
  • first copy formation for any given target nucleic acid can be activated randomly such that the average rate of first copy formation is relatively slow compared to the rate at which subsequent copies are generated.
  • kinetic exclusion will allow only one of those target nucleic acids to be amplified. More specifically, once a first target nucleic acid has been activated for amplification, the site will rapidly fill to capacity with its copies, thereby preventing copies of a second target nucleic acid from being made at the site.
  • An amplification reagent can include further components that facilitate amplicon formation and in some cases, increase the rate of amplicon formation.
  • Recombinase such as for example UvsX, can facilitate amplicon formation by allowing repeated invasion/extension. More specifically, recombinase can facilitate invasion of a double stranded target nucleic acid by an immobilized single- stranded capture sequence and extension of the capture sequence by the polymerase using the target nucleic acid as a template for amplicon formation. This process can be repeated as a chain reaction where amplicons produced from each round of invasion/extension serve as templates in a subsequent round.
  • the process can occur more rapidly than standard PCR since a denaturation cycle (e.g., via heating or chemical denaturation) is not required.
  • recombinase-facilitated amplification can be carried out isothermally. It is generally desirable to include ATP, or other nucleotides (or in some cases non-hydrolyzable analogs thereof) in a recombinase- facilitated amplification reagent to facilitate amplification.
  • a mixture of recombinase and single-stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification.
  • Exemplary formulations for recombinase-facilitated amplification include those sold commercially as TwistAmp kits by TwistDx (Cambridge, UK).
  • Useful components of recombinase-facilitated amplification reagent and reaction conditions are set forth in U.S. Pat. No. 5,223,414 and U.S. Pat. No. 7,399,590.
  • FIG. 3 depicts an exemplary embodiment of a workflow including propagation of methylation state during a protocol such as ExAmp.
  • ExAmp may be advantageous in that it does not require a heat or chemical denaturation step.
  • template-dependent DNA methyltransferases are often sensitive to heat denaturation. However, such methyltransferases would likely not be denatured during an isothermal ExAmp workflow.
  • a single initial addition of a template-dependent DNA methyltransferase may enable propagation of the methylation status of a template to an extended strand simultaneously during template amplification.
  • FIG. 3 depicts a method wherein a singlestranded nucleic acid 12 is hybridized to a first capture sequence 11 on an amplification site 10 (FIG. 3A).
  • the amplification site 10 includes SSB proteins 18 that bind to the singlestranded nucleic acid and each of the capture sequences.
  • the single-stranded nucleic acid may include at least one methylated nucleotide, such as a methylated CpG site.
  • the first capture sequence 11 is extended by a polymerase using the single-stranded nucleic acid 12 as a template. The polymerase is able to remove the SSB proteins 18 as it processes along the single-stranded nucleic acid 12.
  • the resulting complementary strand 13’ is immobilized to the amplification site 10 at its 5' end (FIG. 3B).
  • the methylation status of the single-stranded nucleic acid 12 is propagated to the complementary strand 13’ by a template-dependent DNA methyltransferase.
  • the templatedependent DNA methyltransferase may be added to the amplification site at this point.
  • the template-dependent DNA methyltransferase may already be present at the amplification site.
  • the template-dependent DNA methyltransferase may be added simultaneously with the polymerase. The resulting product is depicted in FIG.
  • the amplification site is treated with a recombinase 19, which can bind the second capture sequence 15 to form a nucleoprotein filament (FIG. 3D).
  • the second capture sequence 15 can then invade the single-stranded nucleic acid 12 and complementary strand 13” to form the structure depicted in FIG. 3E.
  • the second capture sequence 15 is extended using a polymerase.
  • the polymerase displaces the single-stranded nucleic acid 12 as it processes along the complementary strand 13” (FIG. 3F)
  • the template-dependent DNA methyltransferase is able to act on the newly synthesized strand 20” at any point following extension of a region complementary to a methylated nucleotide of the complementary strand 13.
  • propagation of the methylation status to the newly synthesized strand 20” may occur simultaneously with its extension.
  • the single-stranded nucleic acid 12 is no longer bound to the complementary strand 13” (FIG. 3G).
  • the single-stranded nucleic acid 12 may be removed, leaving the resulting doublestranded, bridged structure depicted in FIG. 3G.
  • New strand synthesis may proceed by treating the amplification site 10 with a recombinase to facilitate invasion of the 3' end of the newly synthesized strand 20” with a new first capture sequence 11. Extension and propagation of methylation status may proceed to populate the amplification site 10 with a monoclonal population of methylated nucleic acids.
  • compositions that include a RB69 bacteriophage UvsX, a RB69 bacteriophage single-stranded DNA binding protein (gp32), and a DNA polymerase can result.
  • the DNA polymerase is B. subtilis polymerase I (Bsu).
  • the composition can also include a RB69 bacteriophage UvsY.
  • the composition does not include a crowding agent, such as, but not limited to, a polyethylene glycol (PEG), dextran or Ficoll.
  • the composition can include other components as described herein, including, but not limited to, ATP, an ATP analog, or a combination thereof.
  • the ATP or ATP analog can be ATP, ATP-y-S, ATP-0-S, ddATP, or a combination thereof.
  • ATP can present at a concentration of 2.3 mM to 2.8 mM and ATP-y-S can be present at a concentration of 0.1 mM to 0.5 mM.
  • the composition can further include one or more dNTPs, such as dATP, dGTP, dCTP, dTTP, or a combination thereof.
  • composition that can result includes ATP, ATP-y-S, and a DNA polymerase.
  • the ATP can be present at a concentration of 2.3 mM to 2.8 mM, and the ATP-y-S can be present at a concentration of 0.1 mM to 0.5 mM.
  • the DNA polymerase is B. subtilis polymerase I (Bsu), E. coli DNA polymerase I Klenow fragment, B. stearothermophilus polymerase (Bst), or B. subtilis Phi-29 polymerase.
  • the composition can further include an accessory protein such as RB69 bacteriophage UvsY, a singlestranded DNA binding protein such as RB69 bacteriophage gp32, a recombinase loading protein such as RB69 bacteriophage UvsX, or a combination thereof.
  • the composition can optionally include a helicase such as RuvA, RuvB, or a combination thereof, and in one embodiment does not include a helicase.
  • the composition can include an ATP analog ATP-y-S, ddATP, or a combination thereof.
  • the composition can further include one or more dNTPs, such as dATP, dGTP, dCTP, dTTP, or a combination thereof.
  • a composition for amplifying nucleic acids at amplification sites is typically capable of rapidly making copies of nucleic acids at amplification sites.
  • An amplification reagent used in a method of the present disclosure will generally include a polymerase and nucleotide triphosphates (NTPs). Any of a variety of polymerases known in the art can be used, but in some embodiments, it may be preferable to use a polymerase that is exonuclease negative.
  • nucleic acid polymerases 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 instance, U.S. Pat. No. 9,677,057, U.S. Pat. No. , 11,001,816, and U.S. Published Pat. Application U.S. Pat. Pub. No. 2020/0131484A1).
  • 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
  • an amplification reagent can also include recombinase, accessory protein, and singlestranded DNA binding (SSB) protein for recombinase-facilitated amplification (see, for instance, U.S. Pat. No. 8,071,308).
  • SSB singlestranded DNA binding
  • the NTPs can be deoxyribonucleotide triphosphates (dNTPs) for embodiments where DNA copies are made.
  • dNTPs deoxyribonucleotide triphosphates
  • the NTPs can be ribonucleotide triphosphates (rNTPs) for embodiments where RNA copies are made.
  • rNTPs ribonucleotide triphosphates
  • NTPs can be modified with a fluorescent or radioactive group.
  • a large variety of synthetically modified nucleic acids have been developed for chemical and biological methods in order to increase the detectability and/or the functional diversity of nucleic acids. These functionalized/modified molecules (e.g., nucleotide analogs) can be fully compatible with natural polymerizing enzymes, maintaining the base pairing and replication properties of the natural counterparts.
  • amplification solution is added consequently to the choice of the polymerase, and they are essentially corresponding to compounds known in the art as being effective to support the activity of each polymerase.
  • concentration of compounds like dimethyl sulfoxide (DMSO), Bovine Serum Albumin (BSA), poly-ethylene glycol (PEG), Betaine, Triton X-100, denaturant (e g., formamide), or MgCh is well known in the prior art as being important to have an optimal amplification, and therefore the operator can easily adjust such concentrations for the methods of the present disclosure on the basis of the examples presented hereafter and the knowledge generally available.
  • the rate at which an amplification reaction occurs can be increased by increasing the concentration or amount of one or more of the active components of an amplification reaction, for example, the amount or concentration of polymerase, nucleotide triphosphates, or primers.
  • the one or more active components of an amplification reaction that are increased in amount or concentration are non-nucleic acid components of the amplification reaction.
  • Amplification rate can also be increased in a method set forth herein by adjusting the temperature.
  • the rate of amplification at one or more amplification sites can be increased by increasing the temperature at the site(s) up to a maximum temperature where reaction rate declines due to denaturation or other adverse events.
  • Optimal or desired temperatures can be determined from known properties of the amplification components in use or empirically for a given amplification reaction mixture. Such adjustments can be made based on a priori predictions of primer melting temperature (Tm) or empirically.
  • Tm primer melting temperature
  • the temperature of an amplification reaction is at least 35°C to no greater than 70°C.
  • an amplification reaction can be at least 35°C to no greater than 48°C.
  • nucleic acids sequenced according to the present disclosure are attached to the surface by hybridization to a nucleic acid that is anchored to the surface. Accordingly, lower temperatures are often preferred.
  • denaturing conditions include, but are not limited to, formamide, heat, or alkali.
  • the method further includes propagating, at each amplification site, the methylated CpG dinucleotide present on one strand of the clonal population of amplicons to other unmethylated members 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 one strand of the clonal population to the other strands, thereby propagating the methylation status to both strands present at an amplification site.
  • the conditions include exposing the amplification sites to a template-dependent DNA methyltransferase, such as the enzyme DNMT1, and a DNA helicase or recombinase.
  • Amplification of the methylated CpG signal occurs by many cycles of steps that include hybridizing complementary strands, transferring methylation state from one methylated CpG to the complementary unmethylated CpG, and unwinding the hybridized complementary strands.
  • the hybridization of the complementary single-stranded nucleic acids within each amplification site results in the formation of a bridged double-stranded fragment that is hemi-methylated at each CpG.
  • DNMT1 transfers the methylation status from a methylated CpG of one strand to the complementary unmethylated CpG of the other paired strand.
  • the unwinding of the now fully methylated duplex can be accomplished by either strand invasion by another fragment in the cluster mediated by a recombinase or by helicase- mediated unwinding.
  • the term "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).
  • Complementary strands 12' and 13 shown at amplification site I anneal to form the doublestranded hemi-methylated structure shown in II.
  • the methylation status of methylated CpG dinucleotides on strand 13 is transferred to stand 12' to convert strand 12' to 12 as shown in III.
  • the double- stranded fully methylated structure shown at amplification site III is unwound and the process is repeated until the methylation status of the one methylated single-stranded nucleic acid 13 is propagated through both sub-populations of nucleic acids at the amplification sites, resulting in fully methylated clusters (amplification site IV).
  • any given methylated CpG-containing strand can only undergo bridging and DNMT1 methylation with neighboring strands, sufficient density within the cluster is expected to allow the methylated CpG signal to propagate across the cluster as strands dissociate and re-anneal to different neighboring strands.
  • the result of amplification and propagation of methylation state is a set of clonal singlestranded methylated amplicons at the amplification sites.
  • the single-stranded amplicons are immobilized on the surface of an amplification site at the 5' ends and include two populations, one population that includes a sequence that is identical to the seed nucleic acid originally used to seed the amplification site and a second population that includes a sequence complementary to the seed nucleic acid. Both populations include methylated CpG dinucleotides. For instance, see FIG. IE, where a fully methylated cluster IV includes a population of single-stranded amplicons 12 and single-stranded amplicons 13 attached to an amplification site by capture nucleic acids 15 and 11, respectively.
  • Production of clusters for sequencing can include removal of one of the two populations of amplicons.
  • one of the capture nucleic acids that is attached to the surface is cleaved to allow the optional removal one of the two populations of amplicons.
  • the cleaving of a nucleotide sequence to permit the optional removal of a specific strand is referred to herein as "linearization.” Examples of suitable methods for linearization are described herein and are described in application number WO 2007/010251, U.S. Pat. No. 8,431,348, and U.S. Pat. No. 8,017,335.
  • the cleavage site is typically present in the capture nucleic acid and is typically at a predetermined position that results in a substantial portion of the original capture nucleic acid to be retained and permit hybridization in a later step during resynthesis of the second strand as described herein.
  • the single-stranded amplicon 12 in the cluster IV includes a cleavage site X within the capture nucleic acid 15. Cleavage results in a cleaved capture nucleic acid 15* and one population of methylated target nucleic acids, 13, as shown in FIG. IF.
  • Any suitable cleavage reaction can be used to cleave at site X.
  • 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, 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 instance heat or alkali, may be undesirable in view of the potential for denaturation of the complementary strand from the shortened capture nucleic acid. Examples of cleavage sites and methods for cleaving the sites can be found in U.S. Pat. No. 8,765,381, and U.S. Pat. Pub. No. 2019/0309360.
  • an abasic site is generated and cleaved.
  • An "abasic site” is defined as a position in a nucleic acid 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 nucleic acid.
  • Bisulfite treatment can give results with single-base resolution but can create DNA damage that necessitates large amounts of sample input and results in systematic biases that make exact quantitation of methylation stoichiometry difficult. Side reactions during whole genome bisulfate chemistry steps can also result in DNA backbone cleavage, leading to dropout of regions of the genome with a high proportion of nonmethylated C residues, as well as reducing overall library conversion. Because multiple copies of the template are present and DNA damage is expected to occur stochastically, even if a large proportion of templates in the cluster are cleaved some intact templates are expected to survive bisulfite treatment.
  • bisulfite conversion is one of any number of different conversion methods that can be conducted on-flow cell after propagation of methylation with a methyltransferase enzyme.
  • an enzymatic conversion method can be used after DNMT1 propagation.
  • One such enzymatic method is the enzymatic methyl-seq (EM- seq) method, as known to those of ordinary skill in the art, and available commercially from New England BiolabsTM.
  • Suitable APOBEC mutants for use in the embodiments presented herein include those described in U.S. Patent Application No. 63/328,444 filed on 7 April 2022 and titled “ALTERED CYTIDINE DEAMINASES AND METHODS OF USE”, the contents of which are incorporated by reference in their entirety.
  • sequence of the immobilized modified target nucleic acids is determined using any suitable sequencing technique, and methods for determining the sequence of immobilized modified target nucleic acids, including strand re-synthesis, are known in the art and are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al.
  • nucleic acid sequencing techniques can be used in conjunction with a variety of nucleic acid sequencing techniques. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Embodiments in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable.
  • the process to determine the nucleotide sequence of an target nucleic acid can be an automated process. Preferred embodiments include sequencing-by-synthesis ("SBS") techniques.
  • SBS sequencing-by-synthesis
  • SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand.
  • a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery.
  • more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.
  • a nucleotide monomer includes locked nucleic acids (LNAs) or bridged nucleic acids (BNAs).
  • LNAs locked nucleic acids
  • BNAs bridged nucleic acids
  • SBS can use nucleotide monomers that have a terminator moiety or those that lack any terminator moieties.
  • Methods using nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth in further detail herein.
  • the number of nucleotides added in each cycle is generally variable and dependent upon the template sequence and the mode of nucleotide delivery.
  • the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which uses dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.).
  • SBS techniques can use nucleotide monomers that have a label moiety or that lack a label moiety.
  • incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like.
  • a characteristic of the label such as fluorescence of the label
  • a characteristic of the nucleotide monomer such as molecular weight or charge
  • a byproduct of incorporation of the nucleotide such as release of pyrophosphate
  • the different nucleotides can be distinguishable from each other, or alternatively the two or more different labels can be the indistinguishable under the detection techniques being used.
  • the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.).
  • Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P.
  • PPi inorganic pyrophosphate
  • PPi adenosine triphosphate
  • ATP adenosine triphosphate
  • the nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array.
  • An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images.
  • the images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.
  • cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026.
  • reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026.
  • Solexa now Illumina Inc.
  • WO 07/123,744 The availability of fluorescently- labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing.
  • Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.
  • the labels do not substantially inhibit extension under SBS reaction conditions.
  • the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features.
  • each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels.
  • different nucleotide types can be added sequentially and an image of the array can be obtained between each addition step. In such embodiments, each image will show nucleic acid features that have incorporated nucleotides of a particular type.
  • nucleotide monomers can include reversible terminators.
  • reversible terminators/cleavable fluorophores can include fluorophores linked to the ribose moiety via a 3' ester linkage (Metzker, Genome Res. 15: 1767-1776 (2005)).
  • Other approaches have separated the terminator chemistry from the cleavage of the fluorescence label (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005)). Ruparel et al. described the development of reversible terminators that used a small 3' allyl group to block extension, but could easily be deblocked by a short treatment with a palladium catalyst.
  • the fluorophore was attached to the base via a photocl eavable linker that could easily be cleaved by a 30 second exposure to long wavelength UV light.
  • disulfide reduction or photocleavage can be used as a cleavable linker.
  • Another approach to reversible termination is the use of natural termination that ensues after placement of a bulky dye on a dNTP.
  • the presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and/or electrostatic hindrance.
  • the presence of one incorporation event prevents further incorporations unless the dye is removed.
  • Cleavage of the dye removes the fluorophore and effectively reverses the termination. Examples of modified nucleotides are also described in U.S. Pat. Nos. 7,427,673, and 7,057,026.
  • Some embodiments can use detection of four different nucleotides using fewer than four different labels.
  • SBS can be performed using methods and systems described in the incorporated materials of U.S. Pub. No. 2013/0079232.
  • a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g., via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair.
  • nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal.
  • one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels.
  • An exemplary embodiment that combines all three examples is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g., dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g., dGTP having no label).
  • a first nucleotide type that is detected in a first channel e g., dATP having a label that is detected in the first channel when excited by
  • sequencing data can be obtained using a single channel.
  • the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated.
  • the third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.
  • Some embodiments can use sequencing by ligation techniques. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.
  • the oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize.
  • images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images.
  • Some embodiments can use nanopore sequencing (Deamer, D. W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis", Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope” Nat. Mater. 2:611-615 (2003)).
  • the target nucleic acid passes through a nanopore.
  • the nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin.
  • each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore.
  • a single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130, 818-820 (2008)). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herein.
  • Some embodiments can use methods involving the real-time monitoring of DNA polymerase activity.
  • Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019, and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Pub. No.
  • FRET fluorescence resonance energy transfer
  • Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product.
  • sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pub. Nos. 2009/0026082;
  • Methods set forth herein for amplifying target nucleic acids using kinetic exclusion amplification can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.
  • an advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of cm 2 , in parallel. Accordingly, the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified herein.
  • an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized target nucleic acids, the system including components such as pumps, valves, reservoirs, fluidic lines and the like.
  • a flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in US Pat. No. 8,241,573 and US Pat. No.
  • one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method.
  • one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above.
  • an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods.
  • compositions and/or articles of manufacture can result.
  • a composition can result that includes a template-dependent DNA methyltransferase, such as DNMT1 enzyme and an array with a plurality of amplification sites having one double-stranded nucleic acid.
  • DNMT1 enzyme a template-dependent DNA methyltransferase
  • an array with a plurality of amplification sites having one double-stranded nucleic acid.
  • One strand of the double-stranded nucleic acid is attached to the amplification site by the 5' end and the other complementary strand is not attached to the amplification site.
  • the doublestranded nucleic acid includes at least one hemi-methylated site; a methylated CpG dinucleotide on the strand that is not attached to the amplification site, and a complementary unmethylated CpG dinucleotide with an unmethylated CpG dinucleotide on the strand that is attached to the amplification site.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the one double-stranded nucleic acid.
  • an array can result that includes a plurality of amplification sites having one double-stranded nucleic acid.
  • One strand of the double-stranded nucleic acid is attached to the amplification site by the 5' end and the other complementary strand is not attached to the amplification site.
  • the double-stranded nucleic acid includes at least one at least one methylated CpG dinucleotide on one strand and the other strand includes the complement to the at least one methylated CpG, and the cytosine of the CpG dinucleotide of each strand is methylated.
  • the array can be part of a composition that also includes a template-dependent DNA methyltransferase, such as DNMT1 enzyme.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the one double-stranded nucleic acid.
  • an array can result that includes a plurality of amplification sites having a plurality of clonal single-stranded nucleic acids attached thereto by the 5' end.
  • the plurality of single-stranded nucleic acids at each amplification site includes two populations.
  • the first population of single-strand nucleic acids have the same nucleotide sequence and the CpG dinucleotides are not methylated.
  • One of the strands of this population includes the methylated CpG dinucleotides.
  • the second population of singlestranded nucleic acids are the complement of the first population, and the CpG dinucleotides are not methylated.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the plurality of single-stranded nucleic acids attached thereto by the 5' end.
  • an array can result that includes a plurality of amplification sites where each amplification site includes a plurality of clonal single-stranded nucleic acids attached thereto by the 5' end.
  • the plurality of single-stranded nucleic acids at each amplification site can include two populations, a first population that is a single-stranded nucleic acid and a second population that is the complement of the single-stranded nucleic acid.
  • the first population of the plurality of single-stranded nucleic acids includes methylated CpG dinucleotides
  • the second population of the plurality of singlestranded nucleic acids includes the complement of the at least one methylated CpG dinucleotide, and the cytosine of the CpG dinucleotides of both strands is methylated.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the plurality of clonal single-stranded nucleic acids immobilized thereto by the 5' end.
  • an array can result that includes a plurality of amplification sites, and the amplification sites each include a plurality of clonal single-stranded nucleic acids immobilized thereto by the 5' end.
  • the plurality of clonal single-stranded nucleic acids at each amplification site include either (i) a population that includes a template strand or (ii) a second population that includes the complement of the template strand.
  • the plurality of clonal single-stranded nucleic acids of at least one amplification site includes methylated CpG dinucleotides.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the plurality of clonal single-stranded nucleic acids immobilized thereto by the 5' end.
  • the array can also include a sequencing primer annealed to complementary nucleotides of the clonal singlestranded nucleic acids.
  • the array can be part of a composition that also includes components suitable for sequencing the clonal single- stranded nucleic acids.
  • an array can result that includes a plurality of amplification sites that each include a plurality of clonal single-stranded nucleic acids immobilized thereto by the 5' end.
  • the plurality of clonal single-stranded nucleic acids at each amplification site include either (i) a population that includes a template strand, or (ii) a second population that includes the complement of the template strand, and the plurality of clonal singlestranded nucleic acids of the amplification sites includes at least one methylated CpG dinucleotide.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the plurality of amplification sites can have the plurality of clonal single-stranded nucleic acids immobilized thereto by the 5' end.
  • the nucleotides of the clonal single-stranded nucleic acids are adenine, thymine, guanine, uracil, and methylated cytosine nucleotides.
  • kits for practicing one more aspects the methods provided herein can be used for producing clonal clusters.
  • the kit can be used for on-array chemical treatment to allow detection of methylated residues.
  • the kit can include in separate containers a template-dependent DNA methyltransferase, such as DNMT1, and a DNA helicase and/or recombinase.
  • the kit can include components useful for methylation detection, such as a chemical or enzymatic treatment to allow detection of methylated residues.
  • the components of a kit can be present in a suitable packaging material in an amount sufficient for producing at least one library.
  • reagents such as a buffer solution (either prepared or present in its constituent components, where one or more of the components may be premixed or all of the components may be separate), and the like, are also included. Instructions for use of the packaged components are also typically included.
  • the phrase "packaging material” refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging material has a label which indicates that the components can be used for on-array chemical treatment to allow detection of methylated residues.
  • the packaging material contains instructions indicating how the materials within the kit are employed for practicing one more aspects of the methods provided herein.
  • the term "package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits one or more components of the kit.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • Aspect 1 is an array comprising a plurality of amplification sites, wherein at least one amplification site comprises one double-stranded target nucleic acid immobilized thereto, wherein a first strand of the double-stranded target nucleic acid is attached to the amplification site at its 5' end, wherein a second strand of the double-stranded target nucleic acid is complementary to and annealed to a region of the first strand comprising the 3' end of the first strand, and the second strand is not attached to the amplification site, and wherein the first strand of the double-stranded target nucleic acid comprises at least one first strand CpG dinucleotide and the second strand comprises at least one complementary second strand CpG dinucleotide complementary to the at least one first strand CpG dinucleotide, wherein each cytosine of the first strand CpG dinucleotide and the complementary second strand CpG is methylated
  • Aspect 2 is the composition array of aspect 1, wherein at least 10% of the plurality of amplification sites comprise a double-stranded target nucleic acid, wherein the first strand of the each double-stranded target nucleic acid comprises at least one first strand CpG dinucleotide and the second strand comprises the complementary at least one second strand CpG dinucleotide, wherein the each cytosine of the first strand CpG dinucleotide and the second strand CpG dinucleotide of each strand is methylated, and wherein the second strand of the double-stranded target nucleic acids are members of a sequencing library.
  • Aspect 3 is the array composition of aspect 1 or 2, further comprising a template-dependent DNA methyltransferase.
  • Aspect 4 is the array composition of aspect 3, wherein the template-dependent DNA methyl transferase is DNMT1 enzyme.
  • Aspect 5 is the array composition of any preceding aspect, further comprising components suitable for kinetic amplification or bridge amplification.
  • Aspect 6 is a composition comprising an array and a template-dependent DNA methyltransferase, wherein the array comprises a plurality of amplification sites comprising one double-stranded target nucleic acid immobilized to each amplification site, wherein a first strand of the double-stranded target nucleic acid is attached to the amplification site at its 5' end, wherein a second strand of the double- stranded nucleic acid is not attached to the amplification site, is complementary to and annealed to a region of the first strand, the region comprising the 3' end of the first strand, wherein the second strand of the doublestranded nucleic acid is a member of a sequencing library, and wherein the double-stranded target nucleic acid comprises at least one hemi-methylated CpG dinucleotide, the at least one hemi-methylated CpG having a methylated cytosine on the second strand of the double-strande
  • Aspect 7 is the composition of any preceding aspect wherein the template-dependent DNA methyl transferase is DNMT1 enzyme.
  • Aspect 8 is an array comprising a plurality of amplification sites comprising a plurality of single-stranded nucleic acids attached thereto by the 5' end, wherein the plurality of singlestranded nucleic acids at each amplification site comprises two populations, a first population comprising a template strand and a second population comprising the complement of the template strand, wherein one of the complements of the template strand at the amplification sites comprises at least one methylated CpG dinucleotide and the other complements of the template strand at the amplification sites do not comprise the at least one methylated CpG dinucleotide.
  • Aspect 9 is the array of any preceding aspect, wherein at least 10% of the plurality of amplification sites comprise the plurality of single-stranded nucleic acids at each amplification site comprising two populations.

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Abstract

La présente invention concerne des compositions, des articles, des kits et des procédés de production d'une matrice qui comprend des agrégats clonaux. Dans un mode de réalisation, les procédés comprennent la production de groupes clonaux sur une matrice qui préserve l'état de méthylation, tel que l'état de méthylation de dinucléotides CpG méthylés des molécules d'ADN germes.
PCT/US2024/030506 2023-05-26 2024-05-22 Procédés de conservation de l'état de méthylation pendant le regroupement WO2024249200A1 (fr)

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Citations (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
US5223414A (en) 1990-05-07 1993-06-29 Sri International Process for nucleic acid hybridization and amplification
WO1998044151A1 (fr) 1997-04-01 1998-10-08 Glaxo Group Limited Methode d'amplification d'acide nucleique
WO2000018957A1 (fr) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Procedes d'amplification et de sequençage d'acide nucleique
WO2000063437A2 (fr) 1999-04-20 2000-10-26 Illumina, Inc. Detection de reactions d'acide nucleique sur microsupports de billes en reseau
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6266459B1 (en) 1997-03-14 2001-07-24 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
WO2003080862A1 (fr) * 2002-03-25 2003-10-02 Epigenomics Ag Procede et dispositifs pour analyser la methylation de l'adn
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
US6859570B2 (en) 1997-03-14 2005-02-22 Trustees Of Tufts College, Tufts University Target analyte sensors utilizing microspheres
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
WO2006064199A1 (fr) 2004-12-13 2006-06-22 Solexa Limited Procede ameliore de detection de nucleotides
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
WO2007010251A2 (fr) 2005-07-20 2007-01-25 Solexa Limited Preparation de matrices pour sequencage d'acides nucleiques
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
WO2007107710A1 (fr) * 2006-03-17 2007-09-27 Solexa Limited Procédés isothermiques pour créer des réseaux moléculaires clonales simples
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
WO2008093098A2 (fr) 2007-02-02 2008-08-07 Illumina Cambridge Limited Procedes pour indexer des echantillons et sequencer de multiples matrices nucleotidiques
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US7790418B2 (en) 2000-12-08 2010-09-07 Illumina Cambridge Limited Isothermal amplification of nucleic acids on a solid support
US7829284B2 (en) 2002-09-20 2010-11-09 New England Biolabs, Inc. Helicase-dependent amplification of nucleic acids
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US8017335B2 (en) 2005-07-20 2011-09-13 Illumina Cambridge Limited Method for sequencing a polynucleotide template
US8071308B2 (en) 2006-05-04 2011-12-06 Alere San Diego, Inc. Recombinase polymerase amplification
WO2012061832A1 (fr) 2010-11-05 2012-05-10 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120208724A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120270305A1 (en) 2011-01-10 2012-10-25 Illumina Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US8431348B2 (en) 2006-10-06 2013-04-30 Illumina Cambridge Limited Method for pairwise sequencing of target polynucleotides
US20130260372A1 (en) 2012-04-03 2013-10-03 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing
WO2013188582A1 (fr) * 2012-06-15 2013-12-19 Illumina, Inc. Amplification par exclusion cinétique de banques d'acides nucléiques
US8778848B2 (en) 2011-06-09 2014-07-15 Illumina, Inc. Patterned flow-cells useful for nucleic acid analysis
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
WO2015106941A1 (fr) 2014-01-16 2015-07-23 Illumina Cambridge Limited Modification de polynucléotides sur support solide
US9309502B2 (en) 2002-02-21 2016-04-12 Alere San Diego Inc. Recombinase polymerase amplification
WO2016130704A2 (fr) 2015-02-10 2016-08-18 Illumina, Inc. Procédés et compositions pour analyser des composants cellulaires
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
US9677057B2 (en) 2014-09-30 2017-06-13 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
WO2018165366A1 (fr) * 2017-03-08 2018-09-13 President And Fellows Of Harvard College Procédés d'amplification d'adn pour maintenir un état de méthylation
US20180305753A1 (en) 2017-04-23 2018-10-25 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
US20190309360A1 (en) 2013-12-20 2019-10-10 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples
US20200131484A1 (en) 2018-10-31 2020-04-30 Illumina, Inc. Polymerases, compositions, and methods of use
US11001816B2 (en) 2018-12-05 2021-05-11 Illumina, Inc. Polymerases, compositions, and methods of use
WO2021178893A2 (fr) * 2020-03-06 2021-09-10 Singular Genomics Systems, Inc. Séquençage de brin apparié lié
WO2023154897A1 (fr) * 2022-02-11 2023-08-17 Singular Genomics Systems, Inc. Amplification d'acide nucléique et rétention de motif de méthylation

Patent Citations (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
US5223414A (en) 1990-05-07 1993-06-29 Sri International Process for nucleic acid hybridization and amplification
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6266459B1 (en) 1997-03-14 2001-07-24 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
US6859570B2 (en) 1997-03-14 2005-02-22 Trustees Of Tufts College, Tufts University Target analyte sensors utilizing microspheres
US20050100900A1 (en) 1997-04-01 2005-05-12 Manteia Sa Method of nucleic acid amplification
WO1998044151A1 (fr) 1997-04-01 1998-10-08 Glaxo Group Limited Methode d'amplification d'acide nucleique
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
WO2000018957A1 (fr) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Procedes d'amplification et de sequençage d'acide nucleique
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
WO2000063437A2 (fr) 1999-04-20 2000-10-26 Illumina, Inc. Detection de reactions d'acide nucleique sur microsupports de billes en reseau
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6770441B2 (en) 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US7972820B2 (en) 2000-12-08 2011-07-05 Illumina Cambridge Limited Isothermal amplification of nucleic acids on a solid support
US7790418B2 (en) 2000-12-08 2010-09-07 Illumina Cambridge Limited Isothermal amplification of nucleic acids on a solid support
US20060188901A1 (en) 2001-12-04 2006-08-24 Solexa Limited Labelled nucleotides
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US9309502B2 (en) 2002-02-21 2016-04-12 Alere San Diego Inc. Recombinase polymerase amplification
WO2003080862A1 (fr) * 2002-03-25 2003-10-02 Epigenomics Ag Procede et dispositifs pour analyser la methylation de l'adn
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
US20070166705A1 (en) 2002-08-23 2007-07-19 John Milton Modified nucleotides
US7829284B2 (en) 2002-09-20 2010-11-09 New England Biolabs, Inc. Helicase-dependent amplification of nucleic acids
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
US20110059865A1 (en) 2004-01-07 2011-03-10 Mark Edward Brennan Smith Modified Molecular Arrays
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
WO2006064199A1 (fr) 2004-12-13 2006-06-22 Solexa Limited Procede ameliore de detection de nucleotides
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
US8017335B2 (en) 2005-07-20 2011-09-13 Illumina Cambridge Limited Method for sequencing a polynucleotide template
WO2007010251A2 (fr) 2005-07-20 2007-01-25 Solexa Limited Preparation de matrices pour sequencage d'acides nucleiques
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
WO2007107710A1 (fr) * 2006-03-17 2007-09-27 Solexa Limited Procédés isothermiques pour créer des réseaux moléculaires clonales simples
US8241573B2 (en) 2006-03-31 2012-08-14 Illumina, Inc. Systems and devices for sequence by synthesis analysis
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US8071308B2 (en) 2006-05-04 2011-12-06 Alere San Diego, Inc. Recombinase polymerase amplification
US8431348B2 (en) 2006-10-06 2013-04-30 Illumina Cambridge Limited Method for pairwise sequencing of target polynucleotides
US8765381B2 (en) 2006-10-06 2014-07-01 Illumina Cambridge Limited Method for pairwise sequencing of target polynucleotides
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US7948015B2 (en) 2006-12-14 2011-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
WO2008093098A2 (fr) 2007-02-02 2008-08-07 Illumina Cambridge Limited Procedes pour indexer des echantillons et sequencer de multiples matrices nucleotidiques
US8053192B2 (en) 2007-02-02 2011-11-08 Illumina Cambridge Ltd. Methods for indexing samples and sequencing multiple polynucleotide templates
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2012061832A1 (fr) 2010-11-05 2012-05-10 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
US8951781B2 (en) 2011-01-10 2015-02-10 Illumina, Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
US20120270305A1 (en) 2011-01-10 2012-10-25 Illumina Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120208724A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US8778848B2 (en) 2011-06-09 2014-07-15 Illumina, Inc. Patterned flow-cells useful for nucleic acid analysis
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US20130260372A1 (en) 2012-04-03 2013-10-03 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
WO2013188582A1 (fr) * 2012-06-15 2013-12-19 Illumina, Inc. Amplification par exclusion cinétique de banques d'acides nucléiques
US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
US20190309360A1 (en) 2013-12-20 2019-10-10 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic dna samples
WO2015106941A1 (fr) 2014-01-16 2015-07-23 Illumina Cambridge Limited Modification de polynucléotides sur support solide
US9677057B2 (en) 2014-09-30 2017-06-13 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
WO2016130704A2 (fr) 2015-02-10 2016-08-18 Illumina, Inc. Procédés et compositions pour analyser des composants cellulaires
WO2018165366A1 (fr) * 2017-03-08 2018-09-13 President And Fellows Of Harvard College Procédés d'amplification d'adn pour maintenir un état de méthylation
US20180305753A1 (en) 2017-04-23 2018-10-25 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
US20200131484A1 (en) 2018-10-31 2020-04-30 Illumina, Inc. Polymerases, compositions, and methods of use
US11001816B2 (en) 2018-12-05 2021-05-11 Illumina, Inc. Polymerases, compositions, and methods of use
WO2021178893A2 (fr) * 2020-03-06 2021-09-10 Singular Genomics Systems, Inc. Séquençage de brin apparié lié
WO2023154897A1 (fr) * 2022-02-11 2023-08-17 Singular Genomics Systems, Inc. Amplification d'acide nucléique et rétention de motif de méthylation

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
ADESSI ET AL., NUCLEIC ACIDS RESEARCH, vol. 28, no. 20, 2000, pages E87
BENTLEY ET AL., NATURE, vol. 456, 2008, pages 53 - 59
COCKROFT, S. L.CHU, J.AMORIN, M.GHADIRI, M. R.: "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution", J. AM. CHEM. SOC., vol. 130, 2008, pages 818 - 820, XP055097434, DOI: 10.1021/ja077082c
DEAMER, D. W.AKESON, M.: "Nanopores and nucleic acids: prospects for ultrarapid sequencing", TRENDS BIOTECHNOL, vol. 18, 2000, pages 147 - 151, XP004194002, DOI: 10.1016/S0167-7799(00)01426-8
DEAMER, D.D. BRANTON: "Characterization of nucleic acids by nanopore analysis", ACC. CHEM. RES., vol. 35, 2002, pages 817 - 825, XP002226144, DOI: 10.1021/ar000138m
GORY SHINREZNIKOFF, J. BIOL. CHEM., vol. 273, 1998, pages 7367
HEALY, K: "Nanopore-based single-molecule DNA analysis", NANOMED, vol. 2, 2007, pages 459 - 481, XP009111262, DOI: 10.2217/17435889.2.4.459
KORLACH, J ET AL.: "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures", PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 1176 - 1181
LEVENE, M. J. ET AL.: "Zero-mode waveguides for single-molecule analysis at high concentrations", SCIENCE, vol. 299, 2003, pages 682 - 686, XP002341055, DOI: 10.1126/science.1079700
LI, J.M. GERSHOWD. STEINE. BRANDINJ. A. GOLOVCHENKO: "DNA molecules and configurations in a solid-state nanopore microscope", NAT. MATER., vol. 2, 2003, pages 611 - 615, XP009039572, DOI: 10.1038/nmat965
LUNDQUIST, P. M. ET AL.: "Parallel confocal detection of single molecules in real time", OPT. LETT., vol. 33, 2008, pages 1026 - 1028, XP001522593, DOI: 10.1364/OL.33.001026
METZKER, GENOME RES, vol. 15, 2005, pages 1767 - 1776
MIZUUCHI, K., CELL, vol. 35, 1983, pages 785
RONAGHI, M., KARAMOHAMED, S., PETTERSSON, B., UHLEN, M., NYREN, P.: "Real-time DNA sequencing using detection of pyrophosphate release", ANALYTICAL BIOCHEMISTRY, vol. 242, no. 1, 1996, pages 84 - 9, XP002388725, DOI: 10.1006/abio.1996.0432
RONAGHI, M.UHLEN, M.NYREN, P.: "A sequencing method based on real-time pyrophosphate", SCIENCE, vol. 281, no. 5375, 1998, pages 363, XP002135869, DOI: 10.1126/science.281.5375.363
RONAGHI, M: "Pyrosequencing sheds light on DNA sequencing", GENOME RES, vol. 11, no. 1, 2001, pages 3 - 11, XP000980886, DOI: 10.1101/gr.11.1.3
RUPAREL ET AL., PROC NATL ACAD SCI USA, vol. 102, 2005, pages 5932 - 7
SAMBROOKRUSSELL: "A Laboratory Manual", article "Molecular Cloning"
SAVILAHTI, H ET AL., EMBO J., vol. 14, 1995, pages 4893
SONI, G. V.MELLER: "A. Progress toward ultrafast DNA sequencing using solid-state nanopores", CLIN. CHEM., vol. 53, 2007, pages 1996 - 2001, XP055076185, DOI: 10.1373/clinchem.2007.091231
THORNTON, ANN. REV. MATER. SCI., vol. 7, 1977, pages 239 - 60

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