Compositions and methods for enrichment of nucleic acids

Patent metadata

Field	Value
Publication number	US-10858651-B2
Application number	US-201916553915-A
Country	US
Kind code	B2
Filing date	Aug 28, 2019
Priority date	Aug 19, 2014
Publication date	Dec 8, 2020
Grant date	Dec 8, 2020

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.

First claim

The invention claimed is:

1. A method for sequencing a target region in a DNA library comprising:
a) generating a reaction mixture by combining (i) a DNA library comprising double-stranded DNA fragments with exonuclease-resistant adapters on both ends, wherein one or more of the double-stranded DNA fragments are target DNA fragments comprising a target region, and (ii) an engineered endonuclease specific for a first DNA sequence within the one or more target DNA fragments;
b) placing the reaction mixture under conditions that promote cleavage of the first DNA sequence by the engineered endonuclease to produce double-stranded ends;
c) linking sequencing adapters that are resistant to exonuclease digestion to the double-stranded ends in the reaction mixture, wherein the sequencing adapters have a different sequence than the exonuclease-resistant adapters, thereby forming asymmetric-adapter-ligated fragments;
c) treating the reaction mixture with (i) an endonuclease specific for a second DNA sequence within one or more non-target DNA fragments and (ii) one or more exonucleases that degrade DNA fragments that have at least one end that does not have an adapter;
d) placing the reaction mixture under conditions that promote cleavage of the second DNA sequence by the endonuclease and exonuclease activity; and
e) sequencing the asymmetric-adapter-ligated fragments.

2. The method of claim 1 , wherein the first DNA sequence is flanking the target region.

3. The method of claim 1 , wherein the one or more exonucleases comprises ExoIII and ExoVII.

4. The method of claim 1 , wherein the adapters that are resistant to exonuclease digestion are hairpin adapters.

5. The method of claim 1 , wherein the sequencing adapter is a stem-loop adapter.

6. The method of claim 1 , wherein the sequencing adapter is a linear adapter comprising modified nucleotides.

7. The method of claim 1 , wherein the sequencing reaction is a single-molecule sequencing reaction.

8. The method of claim 7 , wherein the single-molecule sequencing reaction is one that generates nucleotide sequence and epigenetic modification data.

9. The method of claim 7 , wherein the sequencing adapter comprises a primer binding site complementary to a sequencing primer and the single-molecule sequencing reaction is a sequencing-by-synthesis reaction.

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

11. The method of claim 7 , wherein the single-molecule sequencing reaction generates redundant sequence information from single molecules of the asymmetric-adapter-ligated fragments.

12. The method of claim 1 , wherein the target region is a repeat region comprising at least 50 repeats, is a repeat region that is a diagnostic marker, comprises epigenetic modifications, or comprises an imprinted gene.

13. The method of claim 1 , wherein the engineered nuclease is an RNA-directed Cas9 endonuclease, a TAL Effector Nuclease, a zinc-finger nuclease, or a bacteriophage P1 recombination enhancement function protein.

14. The method of claim 1 , wherein the double-stranded DNA fragments in the DNA library are not amplified nucleic acids.

15. The method of claim 1 , wherein the DNA library is a whole-genome DNA library, wherein the whole-genome DNA library is generated by fragmenting genomic DNA and ligating the adapters that are resistant to exonuclease digestion to the ends of the resultant DNA fragments.

16. The method of claim 15 , wherein the fragmenting is done using one or more restriction endonucleases that do not cleave within the target region.

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

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

19. The method of claim 1 , wherein no end repair is performed prior to the linking step.

20. The method of claim 1 , wherein the DNA library comprises one or more second target DNA fragments comprising a second target region, wherein a second engineered endonuclease specific for a DNA sequence within the one or more second target DNA fragments is combined in the reaction mixture in step (a).

Assignees

• Pacific Biosciences California Inc

Inventors

• Tsai Yu-Chih
• Vilfan Igor
• Luong Khai

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title