Methods and compositions for rapid nucleic acid library preparation
First Claim
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1. A method of generating a data set representative of a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:
- a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample,wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to said target nucleic acid sample in the presence of a nucleic acid polymerase and a nucleotide triphosphate, wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates;
b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule;
c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence;
d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence;
e) sequencing said first double-stranded nucleic acid molecule and said second double-stranded nucleic acid molecule to generate a data set;
f) observing that;
said third molecular tag sequence is identical to said first molecular tag sequence;
said fourth molecular tag sequence is identical to said second molecular tag sequence;
said second target sequence is identical to said first target sequence; and
said second target sequence length is identical to said first target sequence length; and
g) removing a sequence of said second double-stranded nucleic acid molecule from said data set.
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Abstract
Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.
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Citations
20 Claims
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1. A method of generating a data set representative of a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:
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a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample, wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to said target nucleic acid sample in the presence of a nucleic acid polymerase and a nucleotide triphosphate, wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates; b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule; c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence; d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence; e) sequencing said first double-stranded nucleic acid molecule and said second double-stranded nucleic acid molecule to generate a data set; f) observing that; said third molecular tag sequence is identical to said first molecular tag sequence; said fourth molecular tag sequence is identical to said second molecular tag sequence; said second target sequence is identical to said first target sequence; and said second target sequence length is identical to said first target sequence length; and g) removing a sequence of said second double-stranded nucleic acid molecule from said data set. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of generating a data set representative of a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:
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a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample, wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to said target nucleic acid sample in the presence of a nucleic acid polymerase and a nucleotide triphosphate wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates; b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule; c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence; d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence; e) sequencing said first double-stranded nucleic acid molecule and said second double-stranded nucleic acid molecule to generate a data set; and f) observing that at least one of; said third molecular tag sequence is different from said first molecular tag sequence; said fourth molecular tag sequence is different from said second molecular tag sequence; said second target sequence is different from said first target sequence;
orsaid second target sequence length is different from said first target sequence length; and g) retaining a sequence of said second double-stranded nucleic acid molecule in said data set thereby generating a population of non-identical, tagged nucleic acid molecules each comprising a subset of sequence from said target nucleic acid sample.
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Specification