Transportation of native chromatin for personal epigenomics

• US 10,738,357 B2
• Filed: 05/21/2019
• Issued: 08/11/2020
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Giresi, Paul, Buenrostro, Jason D., Chang, Howard Y., Greenleaf, William J.
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US16/418,796
• Publication Number:
  US 20190316196A1
• Time in Patent Office: 448 Days
• Field of Search: 435 912
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

Claims

1. A composition comprising:

a permeabilized cell nucleus comprising;

(a) a plurality of polynucleotide molecules; and

(b) an insertional enzyme complex comprising an insertional enzyme and

an insert element comprising a predetermined sequence.

2. A composition comprising:

a permeabilized cell nucleus comprising;

(a) chromatin comprising genomic DNA and a plurality of DNA binding proteins, wherein the chromatin comprises a plurality of open chromatin regions; and

(b) an insertional enzyme complex comprising an insertional enzyme and

an insert element comprising a predetermined sequence, wherein the insertional enzyme complex is bound to one of said open chromatin regions.

3. The composition of claim 2, wherein the insertional enzyme comprises one or more enzymatic moieties.

4. The composition of claim 3, wherein the insertional enzyme comprises two or more enzymatic moieties.

5. The composition of claim 4, wherein the two or more enzymatic moieties are linked together.

6. The composition of claim 2, wherein the insertional enzyme comprises a nuclear localization signal.

7. The composition of claim 2, wherein the insertional enzyme complex is configured to insert the insert element into the genomic DNA at an open chromatin region.

8. The composition of claim 7, wherein the insertional enzyme complex is configured to insert the insert element into the genomic DNA in a substantially sequence independent manner.

9. The composition of claim 2, wherein the insertional enzyme complex comprises an enzyme selected from the group consisting of:

transposases, Hermes transposase, and human immunodeficiency virus (HIV) integrases.

10. The composition of claim 9, wherein the insertional enzyme complex comprises a transposase.

11. The composition of claim 10, wherein the transposase is a Tn transposase.

12. The composition of claim 11, wherein the Tn transposase is a Tn5 transposase.

13. The composition of claim 2, wherein the insert element comprises one or more molecular tags.

14. The composition of claim 13, wherein one or more of the molecular tags comprises a sequencing adaptor.

15. The composition of claim 14, wherein one or more of the sequencing adaptors comprises a barcode label.

28. The composition of claim 2, wherein (a) and (b) are contained within the permeabilized cell nucleus.

29. The composition of claim 2, wherein the permeabilized cell nucleus is isolated from a cell.

30. The composition of claim 2, wherein the permeabilized cell nucleus is contained within a cell.

16. A composition comprising:

a permeabilized cell nucleus comprising;

(a) an insertional enzyme complex comprising a transposase enzyme; and

(b) a plurality of tagged nucleic acid fragments, wherein each tagged nucleic acid fragment comprises a first sequencing adapter and a second sequencing adapter;

wherein each tagged nucleic acid fragment is derived from a region of open chromatin.

17. The composition of claim 16, wherein the plurality of tagged nucleic acid fragments correspond to two or more regions of a genome.

18. The composition of claim 16, wherein the plurality of tagged nucleic acid fragments correspond to at least 1,000 regions of a genome.

19. The composition of claim 18, wherein the plurality of tagged nucleic acid fragments correspond to genome-wide regions of open chromatin.

20. The composition of claim 18, wherein the transposase is a Tn transposase.

21. The composition of claim 20, wherein the Tn transposase is a Tn5 transposase.

22. A composition comprising:

a permeabilized cell nucleus comprising;

(a) an insertional enzyme complex comprising a transposase enzyme; and

(b) a plurality of tagged nucleic acid fragments, wherein each tagged nucleic acid fragment comprises a first sequencing adapter and a second sequencing adapter;

wherein the insertional enzyme complex and the tagged nucleic acid fragments are bound to one or more regions of open chromatin within the cell nucleus in a substantially sequence independent manner to form a high affinity macromolecule complex.

23. The composition of claim 22, further comprising a plurality of divalent cations.

24. The composition of claim 23, wherein the divalent cations are not chelated.

25. The composition of claim 23, wherein the divalent cations are selected from the group consisting of:

magnesium, manganese, or any combination thereof.

26. The composition of claim 22, wherein the transposase is a Tn transposase.

27. The composition of claim 26, wherein the Tn transposase is a Tn5 transposase.