Transposition of native chromatin for personal epigenomics

• US 10,150,995 B1
• Filed: 07/24/2018
• Issued: 12/11/2018
• 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:
  US16/043,874
• Publication Number:
  US 20180355424A1
• Time in Patent Office: 140 Days
• Field of Search: 435 61
• 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 a biological sample, comprising:

(a) contacting chromatin of a genome region of said biological sample with an insertional enzyme complex to produce tagged nucleic acid molecules, wherein said insertional enzyme complex does not comprise an antibody specific to a protein that is part of said chromatin; and

(b) performing a nucleic acid assay on said tagged nucleic acid molecules or derivatives thereof, to provide sequence information of said tagged nucleic acid molecules or derivatives thereof.

2. The method of claim 1, further comprising generating a representation of epigenetic features of said genome region at least in part by mapping said sequence information to said genome region.

3. The method of claim 1, wherein said biological sample is a cell or a nucleus.

4. The method of claim 3, further comprising lysing said cell or said nucleus.

5. The method of claim 3, further comprising permeabilizing said cell or said nucleus to permit said insertional enzyme complex to access said chromatin.

6. The method of claim 5, wherein said permeabilizing is configured to minimally perturb said chromatin of said cell or said nucleus.

7. The method of claim 5, wherein said cell or said nucleus is permeabilized with a reagent configured to permit said insertional enzyme complex to access said chromatin.

8. The method of claim 7, wherein said reagent comprises NP40, digitonin, tween, streptolysin, or a cationic lipid.

9. The method of claim 5, wherein said cell or said nucleus is permeabilized by hypotonic shock or ultrasonication.

10. The method of claim 1, wherein said insertional enzyme comprises a transposase.

11. The method of claim 10, wherein said transposase is a Tn5 transposase or derived from a Tn5 transposase.

12. The method of claim 11, wherein said transposase is a hyperactive Tn5 transposase.

13. The method of claim 1, wherein said insertional enzyme complex comprises a first nucleic acid insert element comprising a first adapter sequence.

14. The method of claim 13, wherein said first adapter sequence comprises a first sequencing adapter sequence.

15. The method of claim 13, wherein said first adapter sequence comprises a barcode sequence.

16. The method of claim 13, wherein said first adapter sequence comprises a first primer sequence.

17. The method of claim 13, wherein said insertional enzyme complex further comprises a second nucleic acid insert element comprising a second adapter sequence.

18. The method of claim 17, wherein said second adapter sequence comprises a second sequencing adapter sequence.

19. The method of claim 17, wherein said second adapter sequence comprises a barcode sequence.

20. The method of claim 17, wherein said second adapter sequence comprises a second primer sequence.

21. The method of claim 1, further comprising subjecting said tagged nucleic acid molecules to one or more reactions.

22. The method of claim 21, wherein said one or more reactions comprise a nucleic acid amplification reaction.

23. The method of claim 22, wherein said nucleic acid amplification reaction is configured to add one or more functional sequences to said tagged nucleic acid molecules or derivatives thereof, wherein said one or more functional sequences are compatible with a selected next generation sequencing platform.