METHODS AND COMPOSITIONS FOR LIBRARY NORMALIZATION

• US 20170073730A1
• Filed: 09/08/2016
• Published: 03/16/2017
• Est. Priority Date: 09/11/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Betts, Craig, Fu, Glenn
• Granted Patent: US 10,619,186 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/305 Denaturation or renaturation by physical action
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

The disclosure provides for methods, compositions, and kits for normalizing nucleic acid libraries, for example sequencing libraries.

Claims

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.

2. The method of claim 1, wherein the binding moiety 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 method of claim 1, further comprising synthesizing a second strand for at least one of the plurality of complementary strands of the first plurality of nucleic acid molecules to generate one or more of the plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the first plurality of nucleic acid molecules.

4. The method of claim 3, wherein the synthesizing comprises hybridizing a plurality of second oligonucleotides to the plurality of complementary strands of the first plurality of nucleic acid molecules and extending the plurality of second oligonucleotide.

5. The method of claim 4, wherein the plurality of first oligonucleotides or the plurality of second oligonucleotides comprises a universal primer binding site.

6. The method of claim 1, further comprising amplifying the plurality of double-stranded nucleic acid molecules.

7. The method of claim 1, wherein the at least one high abundance species represents at least 50% of the first plurality of nucleic acid molecules.

8. The method of claim 1, further comprising sequencing the second plurality of nucleic acid molecules to generate a plurality of sequencing reads.

9. The method of claim 1, further comprising adding a plurality of blockers during the partially reannealing step.

10. The method of claim 9, wherein the plurality of blockers hybrids to the universal primer binding site of the first oligonucleotide or the universal primer binding site of the second oligonucleotide.

11. The method of claim 10, wherein the plurality of blockers prevents hybridization between the universal primer binding site of the first oligonucleotide or the universal primer binding site of the second oligonucleotide and its complementary sequence.

12. A method of generating a normalized nucleic acid library, comprising:

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.

13. The method of claim 12, wherein the binding moiety 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.

14. The method of claim 12, further comprising synthesizing a second strand for one or more of the plurality of complementary strands of the plurality of nucleic acid targets to generate one or more of the plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the plurality of nucleic acid targets.

15. The method of claim 14, wherein the synthesizing comprises hybridizing a plurality of second oligonucleotides to the plurality of complementary strands of the plurality of nucleic acid targets and extending the plurality of second oligonucleotide.

16. The method of claim 15, wherein the plurality of first oligonucleotides or the plurality of second oligonucleotides comprises a universal primer binding site.

17. The method of claim 14, further comprising amplifying the plurality of double-stranded nucleic acid molecules.

18. The method of claim 12, further comprising sequencing the normalized nucleic acid library to generate a plurality of sequencing reads.

19. The method of claim 12, further comprising adding a plurality of blockers during the partially reannealing step.

20. The method of claim 19, wherein the plurality of blockers hybrids to the universal primer binding site of the first oligonucleotide or the universal primer binding site of the second oligonucleotide.

21. The method of claim 20, wherein the plurality of blockers prevents hybridization between the universal primer binding site of the first oligonucleotide or the universal primer binding site of the second oligonucleotide and its complementary sequence.

22. The method of claim 12, wherein the plurality of nucleic acid targets comprises mRNA.

23. The method of claim 12, wherein the plurality of first oligonucleotides comprises target-specific primers.

24. The method of claim 12, wherein the plurality of first oligonucleotides comprises non-target-specific primers.

25. The method of claim 12, wherein the plurality of nucleic acid targets is from a single cell.

26. A method of generating a normalized nucleic acid library, comprising:

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.

27. The method of claim 26, wherein the unnormalized nucleic acid library comprises one or more high abundance nucleic acid targets and one or more low abundance nucleic acid targets.

28. The method of claim 26, wherein the unnormalized nucleic acid library is a cDNA library.

29. The method of claim 26, wherein the unnormalized nucleic acid library is a genomic library.

30. The method of claim 26, wherein the unnormalized nucleic acid library is a single-cell nucleic acid library.