Transposition of native chromatin for personal epigenomics

• US 10,059,989 B2
• Filed: 05/20/2014
• Issued: 08/28/2018
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Giresi, Paul, Buenrostro, Jason D., Chang, Howard Y., Greenleaf, William J.
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/784,250
• Publication Number:
  US 20160060691A1
• Time in Patent Office: 1,561 Days
• Field of Search: 435 61, 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 method for analyzing chromatin, comprising:

(a) contacting chromatin from a nucleus of at least one cell with an insertional enzyme complex to produce a plurality of tagged fragments of genomic deoxyribonucleic acid (DNA), wherein said insertional enzyme complex does not comprise an antibody specific to a protein that is part of said chromatin,

(b) performing a nucleic acid assay on a tagged fragment of said plurality of tagged fragments to provide fragment information, and

(c) generating, with the aid of a computer processor, a representation of epigenetic features of a genome region of said at least one cell by mapping said fragment information to said genome region.

2. The method of claim 1, wherein said chromatin is contacted with said insertional enzyme complex within said nucleus of said at least one cell.

3. The method of claim 1, wherein said fragment information comprises one or more members selected from the group consisting of:

a size of said tagged fragment, a position of said tagged fragment in said genome region, a cleavage site of said tagged fragment, a length of said tagged fragment, and a position of said tagged fragment in a defined range of length in said genome region.

4. The method of claim 1, wherein performing said nucleic acid assay on said tagged fragment comprises sequencing said tagged fragment to provide said fragment information, wherein said fragment information comprises a sequence read.

5. The method of claim 1, wherein said epigenetic features include a plurality of members selected from the group consisting of:

chromatin accessibility along said genome region, DNA binding protein occupancy along said genome region, nucleosome-free DNA along said genome region, positioning of nucleosomes along said genome region, and chromatin states along said genome region.

6. The method of claim 4, wherein said fragment information comprises one or more members selected from the group consisting of:

(i) information regarding a cleavage site for said insertional enzyme complex,

(ii) a read length of said sequence read,

(iii) a position of said sequence read in a defined range of length in said genome region, and

(iv) a sequence read abundance.

7. The method of claim 6, wherein said cleavage site is within a nucleosome-free region of said chromatin.

8. The method of claim 1, wherein said at least one cell is a plurality of cells.

9. The method of claim 8, wherein said plurality of cells comprises fewer than 500,000 cells.

10. The method of claim 5, wherein said DNA binding protein is a transcription factor.

11. The method of claim 5, wherein said chromatin states include one or more members selected from the group consisting of:

chromatin that is associated with CCCTC-binding factor, chromatin that has a transcription start site, chromatin that has an enhancer, chromatin that is flanking a promoter, chromatin that is transcribed, and chromatin that is transcriptionally repressed.

12. The method of claim 1, wherein said genome region comprises a regulatory region.

13. The method of claim 1, wherein said insertional enzyme complex comprises a transposase or a human immunodeficiency virus (HIV) integrase.

14. The method of claim 13, wherein said transposase is selected from the group consisting of a prokaryotic transposase, a Tn transposase, a MuA transposase, a Vibhar transposase, HERMES, Ac-Ds, Ascot-1, Bs1, Cin4, Copia, En/Spm, F element, hobo, Hsmar1, Hsmar2, IN (HIV), IS1, IS2, IS3, IS4, IS5, IS6, IS10, IS21, IS30, IS50, IS51, IS150, IS256, IS407, IS427, IS630, IS903, IS911, IS982, IS1031, ISL2, L1, Mariner, P element, Tam3, Tc1, Tc3, Tel, THE-1, Tn/O, TnA, Tn3, Tn5, Tn7, Tn10, Tn552, Tn903, Tol1, Tol2, TnlO, and Tyl.

15. The method of claim 1, wherein said insertional enzyme complex comprises two or more enzymatic moieties.

16. The method of claim 1, wherein said at least one cell is from a blood sample.

17. The method of claim 6, wherein said fragment information comprises said sequence read abundance and said method further comprises normalizing said sequence read abundance based on an insertion sequence preference of said insertional enzyme complex.

18. The method of claim 1, further comprising permeabilizing said at least one cell prior to (a).

19. The method of claim 1, wherein said epigenetic features include chromatin accessibility along said genome region.

20. The method of claim 1, wherein said epigenetic features include DNA binding protein occupancy along said genome region.

21. The method of claim 1, wherein said epigenetic features include nucleosome-free DNA along said genome region.

22. The method of claim 1, wherein said epigenetic features include positioning of nucleosomes along said genome region.

23. The method of claim 1, wherein said epigenetic features include chromatin states along said genome region.

24. The method of claim 14, wherein said Tn5 is a hyperactive Tn5 transposase.