Methods and systems for analysis of chromatin

• US 10,725,027 B2
• Filed: 04/04/2019
• Issued: 07/28/2020
• Est. Priority Date: 02/12/2018
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Bell, Jason, McDermott, Geoffrey, Meschi, Francesca, Schnall-Levin, Michael, Zheng, Xinying
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US16/375,093
• Publication Number:
  US 20190367969A1
• Time in Patent Office: 481 Days
• Field of Search: None
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C40B 30/04 : by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 50/06 : Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/5032 : on intercellular interactions
• CPC Class Codes: G01N 33/505 : involving T-cells
• CPC Class Codes: G01N 33/5304 : Reaction vessels, e.g. agglutination plates for solid-phase systems G01N33/543
• CPC Class Codes: G01N 33/5306 : Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/5308 : for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/54306 : Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/548 : Carbohydrates, e.g. dextran
• CPC Class Codes: G01N 33/56977 : HLA or MHC typing
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from a single cell. Such polynucleotide processing may be useful for a variety of applications. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins) and chromatin (e.g., accessible chromatin).

Claims

1. A method of analyzing chromatin, comprising:

(a) providing a mixture comprising (i) a cell or nucleus comprising (1) chromatin comprising a template deoxyribonucleic acid (DNA) and (2) a protein, and (ii) a plurality of nucleic acid barcode molecules;

(b) contacting said cell or nucleus with a labelling agent comprising a reporter oligonucleotide such that said labelling agent couples to said protein;

(c) contacting said chromatin with a plurality of transposase complexes, thereby generating a plurality of template DNA fragments;

(d) generating a first barcoded nucleic acid molecule comprising (i) a sequence of a template DNA fragment of said plurality of template DNA fragments and (ii) a sequence of a first nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules; and

(e) generating a second barcoded nucleic acid molecule comprising (i) a sequence of said reporter oligonucleotide and (ii) a sequence of a second nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules.

2. The method of claim 1, wherein a transposase complex of said plurality of transposase complexes comprises (i) a nucleic acid molecule comprising a transposon end sequence, and (ii) a transposase.

3. The method of claim 1, wherein (i) said first nucleic acid barcode molecule comprises a barcode sequence and a first capture sequence configured to couple to said template DNA fragment;

and (ii) said second nucleic acid barcode molecule comprises said barcode sequence and a second capture sequence configured to couple to said reporter oligonucleotide.

4. The method of claim 3, wherein (d) comprises coupling said first capture sequence to said template DNA fragment and synthesizing said first barcoded nucleic acid molecule, wherein said first barcoded nucleic acid molecule comprises said barcode sequence.

5. The method of claim 3, wherein (e) comprises coupling said second capture sequence to said reporter oligonucleotide and synthesizing said second barcoded nucleic acid molecule, wherein said second barcoded nucleic acid molecule comprises said barcode sequence.

6. The method of claim 3, wherein said reporter oligonucleotide comprises a sequence complementary to said second capture sequence.

7. The method of claim 1, further comprising partitioning said mixture into a partition.

8. The method of claim 7, wherein (b) or (c) is performed in said partition.

9. The method of claim 7, wherein (b) is performed prior to said partitioning.

10. The method of claim 7, wherein said partition is an aqueous droplet in an emulsion.

11. The method of claim 7, wherein said partition is a well.

12. The method of claim 1, wherein said cell or nucleus is permeable to said plurality of transposase complexes and wherein said plurality of template DNA fragments is generated in said cell or nucleus.

13. The method of claim 1, wherein said reporter oligonucleotide further comprises an analyte barcode sequence that identifies a presence of said protein and wherein said second barcoded nucleic acid molecule comprises said analyte barcode sequence.

14. The method of claim 1, wherein said reporter oligonucleotide comprises a unique molecule identifier (UMI) sequence.

15. The method of claim 1, wherein said labelling agent is an antibody.

16. The method of claim 1, wherein said protein is a cell surface protein.

17. The method of claim 1, wherein said protein is an intracellular protein.

18. The method of claim 1, wherein said plurality of nucleic acid barcode molecules is attached to a solid support.

19. The method of claim 18, wherein said solid support is a bead.

20. The method of claim 19, wherein said plurality of nucleic acid barcode molecules is releasably attached to said bead.

21. The method of claim 20, further comprising releasing said plurality of nucleic acid barcode molecules from said bead.

22. The method of claim 20, wherein each of said plurality of nucleic acid barcode molecules are releasably attached to said bead through a labile bond.

23. The method of claim 22, wherein said labile bond is selected from the group consisting of a thermally cleavable bond, a chemically labile bond, and a photo-sensitive bond.

24. The method of claim 23, wherein the labile bond comprises a linkage selected from the group consisting of an ester linkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfone linkage, a silyl ester linkage, a glycosidic linkage, a peptide linkage, and a phosphodiester linkage.

25. The method of claim 19, wherein said bead is a gel bead.

26. The method of claim 25, wherein said gel bead is degradable upon application of a stimulus.

27. The method of claim 26, wherein said stimulus is a chemical stimulus.

28. The method of claim 27, wherein said mixture comprises said chemical stimulus.

29. The method of claim 1, further comprising sequencing (i) said first barcoded nucleic acid molecule, a complement thereof, or a derivative thereof or (ii) said second barcoded nucleic acid molecule, a complement thereof, or a derivative thereof.

30. The method of claim 1, wherein said protein is a nuclear membrane protein.