REAGENTS, METHODS, AND LIBRARIES FOR GEL-FREE BEAD-BASED SEQUENCING
First Claim
1. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
- (a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe is attached to a microparticle, wherein the microparticle is attached to a substrate, and wherein the microparticle is not immobilized in a semi-solid support;
(b) identifying one or more nucleotides of the polynucleotide; and
(c) repeating steps (a) and (b) until the sequence of nucleotides is determined.
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Accused Products
Abstract
The present disclosure provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles typically comprise steps of extension, ligation, and cleavage. In certain embodiments, the methods make use of extension probes containing phosphorothiolate linkages and agents capable of cleaving such linkages. Methods of determining information about a sequence using at least two distinguishably labeled probe families are provided, as are methods of performing multiple sequencing reactions on a single template. Automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions and/or sequence information that can be used in accordance with such methods are also provided. In certain embodiments, blocking oligonucleotides are provided to facilitate sequencing using disclosed methods.
235 Citations
118 Claims
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1. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe is attached to a microparticle, wherein the microparticle is attached to a substrate, and wherein the microparticle is not immobilized in a semi-solid support; (b) identifying one or more nucleotides of the polynucleotide; and (c) repeating steps (a) and (b) until the sequence of nucleotides is determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the probe having an extendable terminus; (b) ligating an extension oligonucleotide probe to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe, wherein the oligonucleotide probe is attached to a microparticle, wherein the microparticle is attached to a substrate, and wherein the microparticle is not immobilized in a semi-solid support; (c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe; (d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and (e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) providing a template polynucleotide attached to a microparticle that is immobilized in or on a semi-solid support or is attached to a substantially planar, rigid substrate; (b) contacting the template polynucleotide with a blocking oligonucleotide; (c) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe optionally comprises a scissile linkage; (d) identifying one or more nucleotides of the polynucleotide; and (e) repeating steps (c) and (d) until the sequence of nucleotides is determined. - View Dependent Claims (32, 33, 34, 35, 36, 37)
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38. A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the probe having an extendable terminus and the template having a blocking oligonucleotide hybridized thereto, the probe-template duplex being attached to a microparticle that is embedded in or on a semi-solid support or attached to a substrate; (b) ligating an extension oligonucleotide probe to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe, wherein the extension probe comprises a phosphorothiolate linkage; (c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe; (d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and (e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined. - View Dependent Claims (39, 40, 41, 42, 43, 44)
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45. A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:
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(a) amplifying a template polynucleotide molecule in a compartment of an emulsion in the presence of a microparticle so that a microparticle having a clonal population of template polynucleotides attached thereto is produced; (b) recovering the microparticle from the emulsion; (c) embedding the microparticle in or on a semi-solid support or attaching the microparticle to a substrate; (d) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a scissile linkage; (e) identifying one or more nucleotides of the polynucleotide; and (f) repeating steps (d) and (e) until the sequence of nucleotides is determined. - View Dependent Claims (46, 47, 48, 49, 50, 78, 79, 80, 81, 82)
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51. A method for determining information about a sequence of nucleotides in a template polynucleotide using a first collection of at least 2 distinguishably labeled oligonucleotide probe families, the method comprising the steps of:
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(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe is a member of the collection of distinguishably labeled oligonucleotide probe families and has a blocking oligonucleotide hybridized thereto; (c) detecting a label associated with the oligonucleotide probe; and (d) repeating steps (b) and (c) until an ordered list of probe family names is obtained; and (d) using the ordered list of probe family names to eliminate one or more possibilities for the sequence of nucleotides. - View Dependent Claims (52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 83, 84)
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85. A method for determining information about a sequence of nucleotides in a template polynucleotide using a first collection of at least 2 distinguishably labeled oligonucleotide probe families, the method comprising the steps of:
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(a) contacting a probe-template complex comprising a duplex portion with an extendable terminus and a single-stranded portion of interest to be sequenced with at least two distinguishably labeled oligonucleotide probe families, so that hybridization occurs between an oligonucleotide probe that comprises a portion complementary to the portion of the template immediately adjacent to the duplex portion, wherein the template has a blocking oligonucleotide hybridized thereto; (b) ligating the hybridized oligonucleotide probe to the extendable terminus, thereby producing a probe-template complex comprising an extended duplex; (c) detecting a label associated with the ligated probe; (d) generating an extendable probe terminus on the extended duplex if one is not already present; and (e) repeating steps (a) to (d) until an ordered list of probe family names is obtained. - View Dependent Claims (86, 87)
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88. A method for determining information about a sequence of nucleotides in a template polynucleotide using a first collection of oligonucleotide probe families, the method comprising the steps of:
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(a) performing sequential cycles of extension, ligation, detection, and cleavage, wherein the detection step comprises acquiring on average 2 bits of information simultaneously from each of at least 2 nucleotides in the template without acquiring two bits of information from any individual nucleotide, wherein the template has a blocking oligonucleotide hybridized thereto; and (b) combining the information obtained in step (a) with at least one bit of additional information to determine the sequence. - View Dependent Claims (89)
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90. A method of preparing a plurality of template polynucleotides comprising steps of:
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(a) contacting a plurality of microparticles with a semi-solid support, wherein at least some of the microparticles have a template attached thereto and wherein the semi-solid support comprises primers attached thereto or embedded therein, so that the templates hybridize to the primers; and (b) extending the primers to form templates complementary to the templates attached to the microparticles. - View Dependent Claims (91, 92, 93, 94)
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95. A collection of components for preparing a population of microparticles, the collection comprising:
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(a) a population of microparticles wherein individual microparticles have at least first and second populations of primers attached thereto, wherein the primers of the first population have a different sequence from that of the primers of the second population; (b) a library of nucleic acid fragments, wherein each nucleic acid fragment contains first and second nucleic acid segments of interest, and wherein the first and second primers correspond to universal sequences located external to the first and second nucleic acid segments of interest; and (c) a blocking oligonucleotide that binds to a common region of the nucleic acid fragments. - View Dependent Claims (96, 97, 98)
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- 99. A template comprising a population of substantially identical template molecules, wherein the template molecules comprise at least one common region and at least one segment of interest, and wherein at least some of the template molecules have a blocking oligonucleotide hybridized to the common region.
- 110. A microparticle attached to the substrate, wherein the microparticle has a template attached thereto.
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116. A method for preparing an array comprising steps of:
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providing a population of microparticles having templates attached thereto, wherein the templates comprise biotin; and contacting the microparticles with a substrate comprising a biotin-binding protein under conditions in which biotin binds to the biotin-binding protein, thereby producing an array of microparticles. - View Dependent Claims (117, 118)
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Specification