Methods of analyzing nucleic acids

• US 10,640,809 B2
• Filed: 05/27/2016
• Issued: 05/05/2020
• Est. Priority Date: 05/29/2015
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Epicentre Technologies Corporation (Illumina Incorporated)
• Inventors: Kuersten, Scott, Radek, Agnes, Vaidyanathan, Ramesh, Grunenwald, Haiying Li
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/576,382
• Publication Number:
  US 20180148767A1
• Time in Patent Office: 1,439 Days
• Field of Search: 506 16
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Abstract

Presented herein are methods and compositions for analyzing rare nucleic acid species. Some methods presented herein use DNA reassociation kinetics following thermal denaturation to define populations of nucleic acid sequences, e.g., highly abundant (e.g., cDNA from rRNA), moderately abundant, and less abundant or rare sequences (e.g., cDNA from mRNA).

Claims

1. A method of analyzing rare nucleic acid species comprising:

(a) providing a library of double-stranded nucleic acids;

(b) denaturing the library;

(c) renaturing the library under conditions sufficient to renature abundant nucleic acid species, wherein a portion of the library comprising less abundant nucleic acid species does not renature;

(d) contacting the library with a transposase that preferentially binds to double-stranded nucleic acids; and

(e) separating renatured abundant nucleic acid species from the less abundant nucleic acid species.

2. The method of claim 1, wherein separating comprises immobilizing the transposase to a solid support.

3. The method of claim 1, wherein the transposase comprises Tn5 or TS-Tn5 transposase.

4. The method of claim 1, wherein the transposase comprises adaptor sequences which comprise a binding moiety capable of binding to a solid support.

5. The method of claim 4, wherein the binding moiety comprises biotin and the solid support comprises streptavidin.

6. The method of claim 1, wherein the transposase comprises adaptor sequences which are not compatible with downstream amplification and sequencing.

7. The method of claim 1, wherein denaturing comprises heat denaturing.

8. The method of claim 1, wherein renaturing comprises lowering the denaturation temperature to a temperature of about 40°

C. to about 65°

C. for a sufficient period of time to renature abundant nucleic acid species.

9. The method of claim 1, wherein denaturing comprises chemical denaturing.

10. The method of claim 1, further comprising sequencing the separated less abundant nucleic acid species.

11. The method of claim 1, wherein the library comprises genomic DNA.

12. The method of claim 1, wherein the library comprises randomly sheared DNA.

13. The method of claim 1, wherein the library comprises double-stranded cDNA.

14. The method of claim 1, wherein the nucleic acids in the library comprise adaptor sequences ligated to the ends of the nucleic acids.

15. The method of claim 14, wherein the adaptor sequences comprise one or more of:

amplification priming binding region, sequencing primer binding region, and promotor sequences for in vitro transcription.

16. The method of claim 1, wherein the abundant nucleic acid species comprise highly repetitive sequences.

17. The method of claim 1, wherein the library comprises nucleic acid from a single cell or a small group of cells.

18. The method of claim 1, wherein the abundant nucleic acid species comprise host sequences and the less abundant nucleic acid species comprise pathogen content.

19. The method of claim 1, wherein the library comprises genomic RNA.