Methods and compositions for library normalization
First Claim
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1. A method of reducing the content of high abundance species from a first plurality of nucleic acid molecules, comprising:
- hybridizing a plurality of first oligonucleotides comprising a binding moiety with a first plurality of nucleic acid molecules, thereby forming hybridization complexes comprising the plurality of first oligonucleotides, wherein the first plurality of nucleic acid molecules comprises at least one high abundance species and at least one low abundance species, wherein the binding moiety comprises a functional group attached to the first oligonucleotides, wherein the first plurality of nucleic acid molecules are mRNAs;
extending the plurality of first oligonucleotides of the hybridization complexes, thereby generating a plurality of double-stranded nucleic acid molecules comprising (i) a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the binding moiety and (ii) the first plurality of nucleic acid molecules;
denaturing the plurality of double-stranded nucleic acid molecules, thereby forming a denatured mixture;
forming an annealing mixture by partially reannealing the denatured mixture in a condition such that reannealed double stranded nucleic acids are generated in the annealing mixture, wherein the reannealed double stranded nucleic acids comprise the plurality of complementary strands of the first plurality of nucleic acid molecules, the at least one high abundance species, the at least one low abundance species, and the binding moiety, and the reannealed double stranded nucleic acids contain more amounts of the at least one high abundance species than the amounts of the at least one low abundance species; and
removing the reannealed double stranded nucleic acids from the annealing mixture by a capture molecule immobilized on one or more solid support, thereby generating a second plurality of nucleic acid molecules and reducing the content of the high abundance species from the first plurality of nucleic acid molecules, wherein the capture molecules specifically bind to the binding moiety and 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
27 Claims
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1. A method of reducing the content of high abundance species from a first 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, thereby forming hybridization complexes comprising the plurality of first oligonucleotides, wherein the first plurality of nucleic acid molecules comprises at least one high abundance species and at least one low abundance species, wherein the binding moiety comprises a functional group attached to the first oligonucleotides, wherein the first plurality of nucleic acid molecules are mRNAs; extending the plurality of first oligonucleotides of the hybridization complexes, thereby generating a plurality of double-stranded nucleic acid molecules comprising (i) a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the binding moiety and (ii) the first plurality of nucleic acid molecules; denaturing the plurality of double-stranded nucleic acid molecules, thereby forming a denatured mixture; forming an annealing mixture by partially reannealing the denatured mixture in a condition such that reannealed double stranded nucleic acids are generated in the annealing mixture, wherein the reannealed double stranded nucleic acids comprise the plurality of complementary strands of the first plurality of nucleic acid molecules, the at least one high abundance species, the at least one low abundance species, and the binding moiety, and the reannealed double stranded nucleic acids contain more amounts of the at least one high abundance species than the amounts of the at least one low abundance species; and removing the reannealed double stranded nucleic acids from the annealing mixture by a capture molecule immobilized on one or more solid support, thereby generating a second plurality of nucleic acid molecules and reducing the content of the high abundance species from the first plurality of nucleic acid molecules, wherein the capture molecules specifically bind to the binding moiety and 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, 12, 13)
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14. 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, thereby forming hybridization complexes comprising the plurality of first oligonucleotides, wherein the binding moiety comprises a functional group attached to the plurality of first oligonucleotides, wherein the plurality of nucleic acid targets comprise one or more high abundance nucleic acid targets and one or more low abundance nucleic acid targets, and wherein the plurality of nucleic acid targets are mRNAs; extending the plurality of first oligonucleotides of the hybridization complexes, thereby generating a plurality of double-stranded nucleic acid molecules comprising (i) a plurality of complementary strands of the plurality of nucleic acid targets comprising the binding moiety and (ii) the plurality of nucleic acid targets; denaturing the plurality of double-stranded nucleic acid molecules, thereby forming a denatured mixture; forming an annealing mixture by partially reannealing the denatured mixture in a condition such that reannealed double stranded nucleic acids are generated in the annealed mixture, wherein the reannealed double stranded nucleic acids comprise the plurality of complementary strands of the plurality of nucleic acid targets, the one or more high abundance nucleic acid targets, the one or more low abundance nucleic acid targets, and the binding moiety, and the reannealed double stranded nucleic acids contain more amounts of the one or more high abundance nucleic acid targets than the amounts of the one or more low abundance nucleic acid targets; and removing the reannealed double stranded nucleic acids from annealing mixture, thereby generating the normalized nucleic acid library. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification