Methods and compositions for library normalization

• US 10,619,186 B2
• Filed: 09/08/2016
• Issued: 04/14/2020
• 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
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US15/260,106
• Publication Number:
  US 20170073730A1
• Time in Patent Office: 1,314 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• 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 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.

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 complementary strand of at least one of the plurality of complementary strands of the first plurality of nucleic acid molecules.

4. The method of claim 3, wherein the synthesizing step 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 oligonucleotides.

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 first oligonucleotides comprises a universal primer binding site and the plurality of blockers hybridizes to the universal primer binding site of the plurality of first oligonucleotides.

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

12. The method of claim 1, wherein the first plurality of nucleic acid molecules is a part of a genomic library.

13. The method of claim 1, wherein the first plurality of nucleic acid molecules is a part of a single-cell nucleic acid library.

14. 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, 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.

15. The method of claim 14, 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.

16. The method of claim 14, further comprising synthesizing a complementary strand of one or more of the plurality of complementary strands of the plurality of nucleic acid targets.

17. The method of claim 16, wherein the synthesizing step 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 oligonucleotides.

18. The method of claim 17, wherein the plurality of first oligonucleotides or the plurality of second oligonucleotides comprises a universal primer binding site.

19. The method of claim 16, further comprising amplifying the plurality of double-stranded nucleic acid molecules.

20. The method of claim 14, further comprising sequencing the normalized nucleic acid library to generate a plurality of sequencing reads.

21. The method of claim 14, further comprising adding a plurality of blockers during the partially reannealing step.

22. The method of claim 21, wherein the plurality of first oligonucleotides comprises a universal primer binding site and the plurality of blockers hybridizes to the universal primer binding site of the plurality of first oligonucleotides.

23. The method of claim 22, wherein the plurality of blockers prevents hybridization between the universal primer binding site of the plurality of first oligonucleotides and its complementary sequence.

24. The method of claim 14, wherein the reannealed double stranded nucleic acids are removed by a capture molecule immobilized on one or more solid support, wherein the capture molecules specifically bind to the binding moiety.

25. The method of claim 14, wherein the plurality of first oligonucleotides comprises target-specific primers.

26. The method of claim 14, wherein the plurality of first oligonucleotides comprises non-target-specific primers.

27. The method of claim 14, wherein the plurality of nucleic acid targets is from a single cell.