Transposition of native chromatin for personal epigenomics

• US 10,619,207 B2
• Filed: 05/21/2019
• Issued: 04/14/2020
• 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:
  US16/418,889
• Publication Number:
  US 20190309362A1
• Time in Patent Office: 329 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 method for generating a sequencing library from a plurality of cells, comprising:

a) lysing a plurality of cells to provide a plurality of cell nuclei, wherein the plurality of cell nuclei comprises chromatin;

b) contacting a cell nucleus of the plurality of cell nuclei with a transposase complex such that polynucleotides of the cell nucleus are tagmented at regions of open chromatin to produce a plurality of tagged fragments; and

c) performing one or more nucleic acid reactions on the tagged fragment to produce a sequencing library.

2. The method of claim 1, further comprising sequencing the sequencing library to produce a plurality of sequence reads.

3. The method of claim 1, wherein the one or more nucleic acid reactions comprise a nucleic acid amplification reaction.

4. The method of claim 3, 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.

5. The method of claim 1, wherein a tagged fragment of the plurality of tagged fragments comprises a nucleotide sequence corresponding to the tagged fragment.

6. The method of claim 5, wherein the tagged fragment further comprises a primer sequence.

7. The method of claim 1, wherein the transposase complex comprises a Tn5 transposase or a transposase derived from a Tn5 transposase.

8. The method of claim 7, wherein said Tn5 transposase is a hyperactive Tn5 transposase.

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

10. The method of claim 9, wherein said first adapter sequence comprises a first sequencing adapter sequence.

11. The method of claim 9, wherein said first adapter sequence comprises a barcode sequence.

12. The method of claim 9, wherein said first adapter sequence comprises a first primer sequence.

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

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

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

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

17. The method of claim 2, further comprising analyzing the sequence reads to generate a profile of chromatin accessibility along the polynucleotides of the cell nuclei.

18. The method of claim 2, further comprising analyzing the sequence reads to determine a DNA binding protein occupancy for one or more DNA binding protein binding sites in the polynucleotides of the cell nuclei.

19. The method of claim 2, further comprising analyzing the sequence reads to generate an epigenetic map representing one or more epigenetic features of the polynucleotides of the cell nuclei.

20. The method of claim 2, further comprising analyzing the sequence reads to determine a position of one or more transcription start sites (TSSs) in the polynucleotides of the cell nuclei.

21. The method of claim 2, further comprising analyzing the sequence reads to determine a position of one or more nucleosomes in the polynucleotides of the cell nuclei.

22. The method of claim 1, wherein the transposase complex does not comprise an antibody specific to a protein that is part of chromatin.

23. A method for generating a sequencing library from a plurality of cells, comprising:

a) lysing a plurality of cells to isolate a plurality of cell nuclei, wherein the plurality of cell nuclei comprise chromatin;

b) contacting a cell nucleus of the plurality of cell nuclei with a Tn5 transposase complex such that polynucleotides of the cell nucleus are tagmented at regions of open chromatin to produce a plurality of tagged fragments,

wherein the Tn5 transposase complex comprises a first sequencing adapter sequence and a second sequencing adapter sequence,

wherein the Tn5 transposase complex does not comprise an antibody specific to a protein that is part of chromatin,

wherein a tagged fragment of the plurality of tagged fragment comprises

i. a nucleotide sequence corresponding to a region of open chromatin,

ii. the first sequencing adapter sequence, and

iii. the second sequencing adapter sequence; and

c) performing one or more nucleic acid reactions on the tagged fragment to produce a sequencing library.

24. The method of claim 23, further comprising sequencing the sequencing library to produce a plurality of sequence reads.

25. The method of claim 24, further comprising analyzing the sequence reads to generate a profile of chromatin accessibility along the polynucleotides of the cell nuclei.

26. The method of claim 24, further comprising analyzing the sequence reads to determine a DNA binding protein occupancy for one or more DNA binding protein binding sites in the polynucleotides of the cell nuclei.

27. The method of claim 24, further comprising analyzing the sequence reads to generate an epigenetic map representing one or more epigenetic features of the polynucleotides of the cell nuclei.

28. The method of claim 24, further comprising analyzing the sequence reads to determine a position of one or more transcription start sites (TSSs) in the polynucleotides of the cell nuclei.

29. The method of claim 24, further comprising analyzing the sequence reads to determine a position of one or more nucleosomes in the polynucleotides of the cell nuclei.