METHODS AND COMPOSITIONS FOR LIBRARY NORMALIZATION
First Claim
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1. A method of removing high abundance species from a plurality of nucleic acid molecules, comprising:
- hybridizing a plurality of first oligonucleotides comprising a binding moiety with a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules comprises at least one high abundance species;
extending the plurality of first oligonucleotides to generate a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the binding moiety;
denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the first plurality of nucleic acid molecules;
partially reannealing the plurality of complementary strands of the first plurality of nucleic acid molecules; and
removing the reannealed complementary strands of the first plurality of nucleic acid molecules by a capture molecule immobilized on one or more solid support to generate a second plurality of nucleic acid molecules, wherein the capture molecules specifically bind to the binding moiety,whereby the content of the at least one high abundance species in the second plurality of nucleic acid molecules is reduced in comparison to the content of the at least one high abundance species in the first plurality of nucleic acid molecules.
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Abstract
The disclosure provides for methods, compositions, and kits for normalizing nucleic acid libraries, for example sequencing libraries.
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Citations
30 Claims
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1. A method of removing high abundance species from a plurality of nucleic acid molecules, comprising:
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hybridizing a plurality of first oligonucleotides comprising a binding moiety with a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules comprises at least one high abundance species; extending the plurality of first oligonucleotides to generate a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the binding moiety; denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the first plurality of nucleic acid molecules; partially reannealing the plurality of complementary strands of the first plurality of nucleic acid molecules; and removing the reannealed complementary strands of the first plurality of nucleic acid molecules by a capture molecule immobilized on one or more solid support to generate a second plurality of nucleic acid molecules, wherein the capture molecules specifically bind to the binding moiety, whereby the content of the at least one high abundance species in the second plurality of nucleic acid molecules is reduced in comparison to the content of the at least one high abundance species in the first plurality of nucleic acid molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of generating a normalized nucleic acid library, comprising:
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hybridizing a plurality of first oligonucleotides comprising a binding moiety with a plurality of nucleic acid targets; extending the plurality of first oligonucleotides to generate a plurality of complementary strands of the plurality of nucleic acid targets comprising the binding moiety; denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the plurality of nucleic acid targets; partially reannealing the plurality of complementary strands of the plurality of nucleic acid targets; and removing the reannealed complementary strands of the plurality of nucleic acid targets by a capture molecule immobilized on one or more solid support, wherein the capture molecules specifically bind to the binding moiety, whereby a normalized nucleic acid library of the plurality of nucleic acid targets is generated. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of generating a normalized nucleic acid library, comprising:
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hybridizing a plurality of first oligonucleotides comprising a binding moiety with a plurality of nucleic acid targets in an unnormalized nucleic acid library; extending the plurality of first oligonucleotides to generate a plurality of complementary strands of the plurality of nucleic acid targets comprising the binding moiety; denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the plurality of nucleic acid targets; partially reannealing the plurality of complementary strands of the plurality of nucleic acid targets; and removing the reannealed complementary strands of the plurality of nucleic acid targets, whereby a normalized nucleic acid library of the plurality of nucleic acid targets is generated. - View Dependent Claims (27, 28, 29, 30)
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Specification