Compositions and methods for enrichment of nucleic acids

• US 10,435,685 B2
• Filed: 01/14/2016
• Issued: 10/08/2019
• Est. Priority Date: 08/19/2014
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Tsai, Yu-Chih, Vilfan, Igor Drasko, Luong, Khai
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/995,646
• Publication Number:
  US 20160208241A1
• Time in Patent Office: 1,363 Days
• Field of Search: None
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a target region, which is typically located within one or more target fragments. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations.

Claims

1. A method for enrichment of a target region in a DNA library comprising:

a) providing a DNA library of double-stranded fragments with hairpin adapters on both ends, wherein the double-stranded fragments in the DNA library are not amplified nucleic acids, and wherein one or more of the double-stranded fragments are target fragments that comprise the target region;

b) subjecting the DNA library to endonuclease cleavage with an engineered endonuclease from a gene editing system that cleaves the target fragments at a first location to produce double-stranded ends, wherein the first location is present only once within at least one of the target fragments and is not within the target region;

c) linking stem-loop adapters to the double-stranded ends produced by the endonuclease cleavage, thereby forming asymmetric-adapter-ligated fragments, wherein the stem-loop adapters have a different sequence than the hairpin adapters; and

d) isolating the asymmetric-adapter-ligated fragments from other fragments in the reaction mixture that are not linked to the stem-loop adapters.

2. The method of claim 1, wherein each of the hairpin adapters comprises a primer binding site complementary to a sequencing primer.

3. The method of claim 1, wherein each of the stem-loop adapters comprises an oligonucleotide binding site complementary to an oligonucleotide linked to a solid surface.

4. The method of claim 3, wherein the solid surface is a bead.

5. The method of claim 1, further comprising subjecting the asymmetric-adapter-ligated fragments isolated in d) to a single-molecule sequencing reaction.

6. The method of claim 5, wherein the single-molecule sequencing reaction is a sequencing-by-synthesis reaction.

7. The method of claim 5, wherein the single-molecule sequencing reaction is a nanopore sequencing reaction.

8. The method of claim 5, wherein the single-molecule sequencing reaction generates redundant sequence information from single molecules of the asymmetric-adapter-ligated fragments isolated in d).

9. The method of claim 1, further comprising amplifying the asymmetric-adapter-ligated fragments isolated in d).

10. The method of claim 1, wherein the DNA library is a whole-genome DNA library.

11. The method of claim 1, wherein the target region is a repeat region comprising at least 50 repeats.

12. The method of claim 1, wherein the target region is a repeat region that is a diagnostic marker.

13. The method of claim 1, wherein the target region comprises epigenetic modifications.

14. The method of claim 13, wherein the target region comprises an imprinted gene.

15. The method of claim 1, wherein the target region is a repeat region comprising sequence interruptions, and further wherein the asymmetric-adapter-ligated fragments isolated in d) are sequenced using a technology that can both determine how many repeats are in the repeat region and can identify each of the sequence interruptions in the repeat region.

16. The method of claim 1, wherein the target region is a repeat region comprising epigenetic modifications, and further wherein the asymmetric-adapter-ligated fragments isolated in d) are sequenced using a single-molecule sequencing technology that can detect both a nucleotide sequence and the epigenetic modifications during a single sequencing reaction.

17. The method of claim 1, wherein the target region is a full-length gene.

18. The method of claim 1, wherein the first location is at least 100 base pairs away from the target region.

19. The method of claim 1, wherein the first location is at least 150 base pairs away from the target region.

20. The method of claim 1, wherein the first location is at least 200 base pairs away from the target region.

21. The method of claim 1, wherein, in step b), an RNA-endonuclease complex associates with the target fragments such that the 3′

end of a targeting RNA is nearer to the target region.

22. The method of claim 1, wherein no end repair is performed following the endonuclease cleavage and prior to the linking of said stem-loop adapters.

23. The method of claim 1, where the engineered endonuclease from a gene editing system is selected from the group consisting of:

a TAL Effector Nuclease and a zinc-finger nuclease.

24. The method of claim 1, wherein the gene editing system is a CRISPR-Cas system.

25. The method of claim 24, wherein the engineered endonuclease is an RNA-Cas 9 complex, wherein at least one targeting RNA in the RNA-Cas 9 complex comprises a sequence complementary to the first location, wherein subjecting step b) comprises combining the RNA-Cas 9 complex with the DNA library in a reaction mixture under conditions that promote binding of the RNA-endonuclease complex to the first location in the target fragments.

26. The method of claim 25, wherein the RNA-Cas 9 complex comprises a single targeting RNA.

27. The method of claim 25, wherein the RNA-Cas 9 complex comprises two targeting RNAs.