METHODS OF SAMPLE PREPARATION
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Accused Products
Abstract
The present disclosure provides methods, compositions, and kits for methods that can improve techniques nucleic acid analysis, and can allow for more reliable and accurate targeted, multiplexed, high throughput sequencing. The methods, compositions, and kits can be used for sequencing target loci of nucleic acid. The methods, compositions, and kits disclosed herein can be used for assisted de novo targeted sequencing. The methods, compositions, and kits disclosed herein can also be used for library labeling for de novo sequencing and phasing.
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Citations
172 Claims
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1-152. -152. (canceled)
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153. A tagged nucleic acid library, comprising at least 100 library nucleic acids, each tagged library nucleic acid comprising:
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a) a first marker region comprising a first marker sequence identical to a first sequence in a marker sequence oligonucleotide population; b) a sample insert region having an independently determined length and a sample insert sequence corresponding to a contiguous subset of a sample nucleic acid sequence; c) a second marker region comprising a second marker sequence identical to a second sequence in a marker sequence oligonucleotide population; wherein the first marker sequence, the sample insert region length, and the second marker sequence independently vary among each library nucleic acid of said library. - View Dependent Claims (154, 155, 156)
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157. A composition comprising:
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a first nucleic acid strand comprising; a 5′
sequence comprising at least 6 bases of indeterminate sequence,a 3′
sequence comprising a fragment of a nucleic acid sample sequence,a 3′
terminal end that cannot support strand extension; andat least one affinity tag; a second nucleic acid strand comprising a second stand oligo of indeterminate sequence, wherein the second nucleic acid strand is annealed to the first nucleic acid strand. - View Dependent Claims (158, 159, 160, 161)
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162. A method of generating a tagged nucleic acid library comprising the steps of:
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annealing a first oligo population to a library template; performing library template-directed nucleic acid extension from the annealed first oligo population; affinity tagging the first extension products; terminating the library template-directed nucleic acid extension to produce a population of first extension products of indeterminate length; and adding a second oligo sequence near the 3′
end of the first extension product;such that a tagged library of nucleic acid molecules is generated comprising nucleic acids each independently comprising a first oligo sequence, a template derived nucleic acid sequence of indeterminate length, and a second oligo sequence. - View Dependent Claims (163, 164, 165, 166, 167)
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168. A method of subdividing a nucleic acid sample into library constituents suitable for sequencing, said method comprising the steps of:
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contacting the nucleic acid sample to a population of oligonucleotides, a DNA polymerase, dNTPs, a buffer suitable for nucleic acid extension, an affinity tag and a nucleic acid chain extension terminating moiety, providing conditions suitable for annealing and nucleic acid extension, contacting the nucleic acid sample to an affinity-tag binding moiety, and separating bound from unbound components; wherein the bound components comprise library constituents suitable for sequencing. - View Dependent Claims (169, 170, 171)
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172. A method of generating a data set on a computer comprising at least 1,000 non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a nucleic acid sample, the method comprising:
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obtaining a first nucleic acid molecule sequence comprising a first 5′
molecular tag sequence, a first insertion sequence from said nucleic acid sample having a first length, and a first 3′
molecular tag sequence;obtaining a second nucleic acid molecule sequence comprising a second 5′
molecular tag sequence, a second insertion sequence having a second length, and a second 3′
molecular tag sequence; anddiscarding said second double-stranded nucleic acid molecule sequence if; said first 5′
molecular tag sequence is identical to said second 5′
molecular tag sequence;said first 3′
molecular tag sequence is identical to said second 3′
molecular tag sequence;second insertion sequence is identical to said first insertion sequence; and said second target sequence length is identical to said first target sequence length.
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Specification