Method for preparing single-stranded DNA libraries
First Claim
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1. A method for clonally isolating a library comprising a plurality of single stranded DNA molecules comprising:
- (a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules;
(b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to form a mixture of adaptor ligated DNA molecules;
(c) isolating a plurality of single stranded DNA molecules each comprising a first single stranded universal adaptor and a second single stranded universal adaptor to obtain a library; and
(d) delivering the single stranded DNA molecules into reactors such that a plurality of the reactors include one DNA molecule, thereby clonally isolating the library.
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Abstract
This invention relates to methods of generating single stranded DNA libraries for use in amplification and sequencing reactions. In various aspects, the disclosed methods include: fragmenting DNA; polishing the fragments'"'"' ends; ligating the fragments to universal adaptors; performing strand displacement and extension of the nicked fragments; purifying the double-stranded ligation products; capturing the double-stranded ligation products onto a solid support; and isolating single stranded DNA library fragments, and binding these fragments to another solid support.
67 Citations
55 Claims
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1. A method for clonally isolating a library comprising a plurality of single stranded DNA molecules comprising:
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(a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules; (b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to form a mixture of adaptor ligated DNA molecules; (c) isolating a plurality of single stranded DNA molecules each comprising a first single stranded universal adaptor and a second single stranded universal adaptor to obtain a library; and (d) delivering the single stranded DNA molecules into reactors such that a plurality of the reactors include one DNA molecule, thereby clonally isolating the library. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 55)
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2. A method for generating a library comprising a plurality of single stranded DNA molecules, comprising:
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(a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules; (b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules, wherein the first universal adaptor contains a moiety that binds to a solid support; (c) attaching to a solid support those DNA molecules comprising a first double stranded universal adaptor; (d) removing adaptor ligated DNA molecules which have not attached to a solid support; (e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules having a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and (f) isolating a library of the released single stranded DNA molecules of step (e) away from those DNA molecules that remain attached to the solid support.
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36. A method for generating a single stranded DNA library attached to solid supports comprising:
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(a) generating a plurality of single stranded DNA templates; (b) attaching each of the plurality of ssDNA templates to a solid support; and (c) isolating the solid supports on which the single stranded DNA templates are attached.
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37. A method for generating a single stranded DNA library attached to solid supports comprising:
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(a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules; (b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to make a mixture of adaptor ligated DNA molecules; (c) isolating those single stranded DNA molecules which comprise a first single stranded universal adaptor and a second single stranded universal adaptor; and (d) attaching the isolated single stranded molecules from (c) to a solid support. - View Dependent Claims (38, 39, 40, 41, 42)
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- 43. A nucleic acid molecule comprising a first adaptor, a fragment of template DNA, and a second adaptor, wherein the first adaptor and second adaptor each comprise a sequencing primer, a PCR primer, and a discriminating key sequence, and wherein the first adaptor and second adaptor, when dissociated, do not cross-hybridize to each other under stringent hybridization conditions.
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49. A method for preparing single stranded DNA molecules, comprising:
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(a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules; (b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules; (c) attaching adaptor ligated DNA molecules comprising a first double stranded universal adaptor and a second double stranded adaptor to a solid support via one strand of the first double stranded universal adaptor; (d) washing away adaptor ligated DNA molecules which have not attached to a solid support; (e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules comprising a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and
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50. A method for delivering nucleic acid templates to a plurality of reaction centers comprising the steps of:
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(a) providing a population of nucleic acid templates; (b) isolating each nucleic acid template from said population to a sequestering agent to form a population of sequestered nucleic acid templates; (c) delivering said population of sequestered nucleic acid templates to said plurality of reaction centers wherein each reaction center receives one sequestered nucleic acid. - View Dependent Claims (51, 52, 53, 54)
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Specification