Normalization of nucleic acid libraries
This disclosure provides methods and compositions for removing one or more high abundance species from a plurality of nucleic acid molecules. In some embodiments, the methods and compositions can be used for normalizing nucleic acid libraries. In some embodiments, molecular labels are used in conjunction with the methods and compositions disclosed herein to improve sequencing efficiency.
1. A composition, comprising:
a plurality of first oligonucleotides comprising an affinity moiety, wherein each of the plurality of first oligonucleotides is a non-target-specific primer comprising a universal primer binding site;
a plurality of selective depletion oligonucleotides comprising an affinity moiety, wherein the plurality of selective depletion oligonucleotides specifically bind nucleic acid molecules that encode:
(i) ribosomal proteins, and
(ii) mitochondrial proteins encoded by mitochondrial mRNA; and
a plurality of capture molecules immobilized on one or more solid supports, wherein the capture molecules are configured to specifically bind to the affinity moiety of the plurality of first oligonucleotides and the plurality of selective depletion oligonucleotides, wherein the plurality of first oligonucleotides, the plurality of selective depletion oligonucleotides, and the plurality of capture molecules immobilized on one or more solid supports are provided separately.
2. The composition of claim 1 , wherein the affinity moiety of the plurality of first oligonucleotides and the plurality of selective depletion oligonucleotides is a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click-chemistry moiety, digoxigenin, primary amine(s), carboxyl(s), hydroxyl(s), aldehyde(s), ketone(s), and any combination thereof.
3. The composition of claim 1 , wherein the affinity moiety of the plurality of first oligonucleotides and the plurality of selective depletion oligonucleotides is biotin and the capture molecule is streptavidin.
4. The composition of claim 1 , wherein the affinity moiety of the plurality of first oligonucleotides and the plurality of selective depletion oligonucleotides is streptavidin and the capture molecule is biotin.
5. The composition of claim 1 , wherein the solid support is a bead.
6. The composition of claim 1 , wherein each of the plurality of first oligonucleotides comprises a stochastic barcode.
7. The composition of claim 1 , comprising one or more reagents for nucleic acid extension reactions.
8. The composition of claim 7 , comprising a reverse transcriptase.
9. The composition of claim 1 , comprising one or more reagents for PCR reactions.
10. The composition of claim 1 , comprising:
instructions for performing:
(i) hybridizing the plurality of first oligonucleotides and the plurality of selective depletion oligonucleotides with a first plurality of nucleic acid molecules comprising at least one nucleic acid molecule that encodes:
(i) a ribosomal protein, and/or
(ii) a mitochondrial protein encoded by mitochondrial mRNA;
(ii) extending the first plurality of oligonucleotides and the plurality of selective depletion oligonucleotides to generate a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the affinity moiety;
(iii) denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the first plurality of nucleic acid molecules;
(iv) partially reannealing the plurality of complementary strands of the first plurality of nucleic acid molecules; and
(v) removing the partially reannealed complementary strands of the first plurality of nucleic acid molecules by the plurality of capture molecules immobilized on one or more solid supports to generate a second plurality of nucleic acid molecules.
11. The composition of claim 10 , further comprising a plurality of second oligonucleotides capable of hybridizing to the plurality of complementary strands of the first plurality of nucleic acid molecules.
12. The composition of claim 11 , wherein each of the plurality of second oligonucleotides comprises a universal primer binding site.
13. The composition of claim 11 , wherein each of the plurality of second oligonucleotides comprises a stochastic barcode.
14. The composition of claim 1 , further comprising Cas9 protein.
15. The composition of claim 14 , wherein the Cas9 protein is complexed with a guide oligonucleotide that specifically binds a nucleic acid molecule encoding a ribosomal protein or a mitochondrial protein.