Normalization of nucleic acid libraries

• US 10,301,677 B2
• Filed: 05/23/2017
• Issued: 05/28/2019
• Est. Priority Date: 05/25/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Shum, Eleen, Fu, Glenn, Betts, Craig
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/603,239
• Publication Number:
  US 20170342484A1
• Time in Patent Office: 735 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

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.

Claims

1. A method of removing high abundance species from a plurality of nucleic acid molecules, comprising:

hybridizing a plurality of first oligonucleotides comprising an affinity moiety with a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules comprises at least one high abundance species, wherein the at least one high abundance species represents at least 1% of the first plurality of nucleic acid molecules;

extending the plurality of oligonucleotides to generate a plurality of complementary strands of the first plurality of nucleic acid molecules comprising the affinity 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 partially 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 affinity moiety,

whereby the at least one high abundance species is reduced in the second plurality of nucleic acid molecules.

2. The method of claim 1, wherein the affinity 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 each of the plurality of complementary strands of the first plurality of nucleic acid molecules to generate 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 first plurality of nucleic acid molecules comprises a plurality of high abundance species, wherein the plurality of high abundance species represents at least 10% of the first plurality of nucleic acid molecules.

8. The method of claim 7, wherein the second plurality of nucleic acid molecules comprises the plurality of high abundance species.

9. The method of claim 1, wherein the first plurality of nucleic acid molecules comprises a plurality of low abundance species, wherein a low abundance species of the plurality of low abundance species represents less than 0.1% of the first plurality of nucleic acid molecules.

10. The method of claim 9, wherein the second plurality of nucleic acid molecules comprises the plurality of low abundance species.

11. The method of claim 1, wherein each of the first plurality of nucleic acid molecules or each of the second plurality of nucleic acid molecules comprises a stochastic barcode.

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

13. The method of claim 12, wherein each of the plurality of sequencing reads comprises a molecular label.

14. The method of claim 1, further comprising selectively depleting a high abundance species, wherein the high abundance species represents at least 10% of the first plurality of nucleic acid molecules.

15. The method of claim 14, wherein the selectively depleting comprises hybridizing a target-specific oligonucleotide that specifically binds to the high abundance species.

16. The method of claim 14, wherein the selectively depleting comprises treating the first plurality of nucleic acid molecules with a Cas9 protein complexed with a guide oligonucleotide that specifically binds to the high abundance species.

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

hybridizing a plurality of first oligonucleotides comprising an affinity moiety with a plurality of nucleic acid targets in a sample;

extending the plurality of oligonucleotides to generate a plurality of complementary strands of the plurality of nucleic acid targets comprising the affinity 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 partially 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 affinity moiety,

whereby a normalized nucleic acid library of the plurality of nucleic acid targets is generated.

18. The method of claim 17, wherein the plurality of nucleic acid targets comprises mRNA.

19. The method of claim 18, wherein the plurality of nucleic acid targets comprises a plurality of low abundance species, wherein the plurality of low abundance species comprises 7,000 genes with the lowest number of transcripts within the plurality of nucleic acid targets.

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

hybridizing a plurality of first oligonucleotides comprising an affinity moiety with a plurality of nucleic acid targets in a sample nucleic acid library;

extending the plurality of oligonucleotides to generate a plurality of complementary strands of the plurality of nucleic acid targets comprising the affinity 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 partially 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.