Granted Patent B2

US 12,421,547 · App. 18/324,880 · Granted Sep 23, 2025

Inventors: Christina Fan (Franklin Lakes, NJ); Elisabeth Marie Walczak (Franklin Lakes, NJ)

Assignee: BECTON, DICKINSON AND COMPANY

C12Q1/6876

Abstract

Claims (63)

1. A method, comprising:

providing a plurality of transposed nuclei, wherein the plurality of transposed nuclei is generated by contacting a nuclei suspension derived from a sample comprising a plurality of cells with a transposome,

wherein the transposome comprises a first adaptor, a second adaptor, a double-strand nuclease configured to induce one or more breaks at a structure comprising double-stranded DNA (dsDNA), and a moiety that targets the transposome to the structure comprising dsDNA,

wherein said contacting generates a plurality of dsDNA fragments each comprising the first adaptor and the second adaptor;

wherein the first adaptor comprises a first universal sequence,

wherein the second adaptor comprises a second universal sequence,

wherein the first universal sequence and the second universal sequence are different from each other and comprise at least a portion of a universal primer, or a complement thereof, and

wherein the dsDNA fragments comprise one or more single-stranded regions;

partitioning the plurality of transposed nuclei and a plurality of beads to a plurality of partitions,

wherein at least one partition of said plurality of partitions comprises a single transposed nuclei from said plurality of transposed nuclei and a single bead from said plurality of beads,

wherein a plurality of barcodes are associated with each bead, wherein said barcodes each comprise a cell label and a capture probe, wherein the capture probe is 3′ of the cell label, wherein barcodes associated with the same bead comprise the same cell label sequence, wherein barcodes associated with different beads comprise different cell label sequences, and wherein the capture probe comprises a sequence configured to bind the first universal sequence, or a complement thereof,

conducting a linear amplification reaction in one or more of the plurality of partitions, comprising:

(i) contacting the dsDNA fragments with one or more enzymes configured to fill in the single-stranded regions and generate dsDNA fragments comprising a first DNA strand and a second DNA strand;

(ii) denaturing the dsDNA fragments;

(iii) hybridizing barcodes to each second DNA strand via the capture probe;

(iv) extending said hybridized barcodes to generate barcoded single-stranded deoxyribonucleic acid (ssDNA) fragments;

(v) denaturing to separate said barcoded ssDNA fragments from the second strands; and

(vi) repeating steps (iii)-(v) one or more times to generate a plurality of barcoded ssDNA fragments;

pooling the plurality of barcoded ssDNA fragments of one or more of the plurality of partitions to generate a pool of barcoded ssDNA fragments;

amplifying the pool of barcoded ssDNA fragments using a first primer capable of hybridizing to the barcode and a second primer capable of hybridizing to the second universal sequence generate a plurality of barcoded amplicons.

2. The method of claim 1 , comprising:

obtaining sequencing data of the plurality of barcoded amplicons, or products thereof; and

determining chromatin accessibility of the dsDNA based on the sequences of the plurality of barcoded amplicons, or products thereof, in the sequencing data obtained.

3. The method of claim 1 ,

wherein the first universal sequence comprises the sequence of at least a portion of an Illumina read 1 sequencing primer, or a complement thereof; and/or

wherein the second universal sequence comprises the sequence of at least a portion of an Illumina read 2 sequencing primer, or a complement thereof.

4. The method of claim 1 , wherein each barcode comprises an Illumina P5 sequence, or a complement thereof, located 5′ of the cell label.

5. The method of claim 1 , wherein the cell label is 16 nucleotides in length.

6. The method of claim 1 , wherein the one or more enzymes comprise a polymerase and/or a ligase.

7. The method of claim 1 , wherein none of the plurality of dsDNA fragments comprises an overhang.

8. The method of claim 1 , wherein the first adaptor and/or the second adaptor comprise a 5′ overhang and/or a 3′ overhang, wherein the dsDNA fragments comprise the 5′ overhang and/or the 3′ overhang, and wherein the one or more enzymes fill in the 5′ overhang and/or the 3′ overhang.

9. The method of claim 1 , wherein the second primer comprises a sample index, an Illumina P7 sequence, complements thereof, or any combination thereof.

10. The method of claim 1 , wherein the first adaptor and/or the second adaptor comprises a DNA end sequence of a transposon.

11. The method of claim 1 , wherein the double-strand nuclease comprises a transposase.

12. The method of claim 11 , wherein the transposase is selected from the group comprising Tn5, Tn7, Tn10, Tc3, a mariner transposase, or any combination thereof.

13. The method of claim 1 , wherein the dsDNA is inside the nuclei during the contacting of the dsDNA of the nuclei suspension with the transposome.

14. The method of claim 1 , wherein the plurality of partitions comprise fluid droplets.

15. The method of claim 1 , comprising permeabilizing the nuclei to generate permeabilized nuclei.

16. The method of claim 15 , comprising fixing the plurality of cells prior to permeabilizing the nuclei.

17. The method of claim 1 , wherein the dsDNA is selected from the group consisting of: nuclear DNA, nucleolar DNA, genomic DNA, or any combination thereof.

18. The method of claim 2 , wherein determining the chromatin accessibility of the dsDNA comprises:

aligning the sequences of the plurality of barcoded amplicons to a reference sequence of the dsDNA; and

identifying regions of the dsDNA corresponding to the ends of barcoded amplicons of the plurality of barcoded amplicons to have an accessibility above a threshold.

19. The method of claim 2 , wherein determining the chromatin accessibility of the dsDNA comprises:

aligning the sequences of the plurality of barcoded amplicons to a reference sequence of the dsDNA; and

determining the accessibility of regions of the dsDNA corresponding the ends of barcoded amplicons of the plurality of barcoded amplicons based on the numbers of the barcoded amplicons of the plurality of barcoded amplicons in the sequencing data.

20. The method of claim 2 , comprising, for each unique cell label sequence, which indicates a single cell of the plurality of cells, determining the sequences of the plurality of barcoded amplicons associated with each cell label sequence in the sequencing data, thereby determining the chromatin accessibility of the dsDNA of each cell of the plurality of cells.

21. The method of claim 1 , wherein at least one barcode of the plurality of barcodes is:

immobilized on the bead;

partially immobilized on the bead;

enclosed in the bead; and/or

partially enclosed in the bead.

22. The method of claim 1 , wherein the bead is disruptable upon application of a chemical stimulus, and wherein the method comprises contacting the partitions with the chemical stimulus, thereby releasing the associated barcodes.

23. The method of claim 22 , wherein the chemical stimulus is a reducing agent.

24. The method of claim 1 , wherein the bead comprises a disruptable hydrogel bead.

25. The method of claim 1 ,

wherein the bead comprises a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead, a protein L conjugated bead, an oligo (dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof; and/or

wherein the bead comprises a material selected from the group comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

26. The method of claim 1 , wherein a dsDNA fragment of the plurality of dsDNA fragments is flanked by a first adaptor and a second adaptor.

27. The method of claim 1 , wherein the plurality of cells is a plurality of single cells and/or the sample is a tissue.

28. The method of claim 1 , wherein the moiety comprises an antibody or fragment thereof, an apatmer, or a DNA binding domain.

29. The method of claim 28 , wherein the antibody or fragment thereof specifically binds a histone protein.

30. The method of claim 28 , wherein the DNA binding domain comprises a zinc finger binding domain.

Assignments (2)

Continuity (3)

References Cited (400)