Massively parallel single cell analysis

• US 9,637,799 B2
• Filed: 10/01/2015
• Issued: 05/02/2017
• Est. Priority Date: 08/28/2013
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fan, Christina, Fodor, Stephen P. A., Fu, Glenn, Facer, Geoffrey Richard, Wilhelmy, Julie
• Primary Examiner(s): PRIEST, AARON A
• Application Number:
  US14/872,377
• Publication Number:
  US 20160145683A1
• Time in Patent Office: 579 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• 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 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Claims

1. A droplet comprising:

a. a single bead comprising a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a molecular label sequence, a cellular label sequence, and an oligo dT sequence, and wherein the cellular label sequence of each of the plurality of oligonucleotides is the same and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences; and

b. a single cell.

2. The droplet of claim 1, wherein at least 1,000 of said plurality of oligonucleotides comprise different molecular label sequences.

3. The droplet of claim 1, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

4. The droplet of claim 1, wherein said droplet is encapsulated by an immiscible liquid.

5. The droplet of claim 1, wherein said droplet is in an emulsion.

6. The droplet of claim 5, wherein said emulsion is a water-in-oil emulsion.

7. The droplet of claim 1, wherein the volume of said droplet is between one picoliter and one microliter.

8. The droplet of claim 1, wherein the diameter of said droplet is from 0.001 to 150 microns.

9. The droplet of claim 1, wherein said droplet is an aqueous droplet within a continuous oil phase, or an oil droplet within a continuous aqueous phase, or a droplet within a larger droplet.

10. The droplet of claim 1, wherein said single cell is a cell selected from the group consisting of:

a rare cell, a tumor cell, and an immune cell.

11. The droplet of claim 1, wherein said single cell is a human cell.

12. The droplet of claim 1, wherein said single cell is from a tissue.

13. The droplet of claim 1, wherein said bead is magnetic.

27. The droplet of claim 1, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.

28. The droplet of claim 1, wherein each of the plurality of oligonucleotides further comprises (a) a sample label or (b) a universal label.

29. The droplet of claim 1, wherein the bead is selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, antibody conjugated beads, protein A conjugated beads, protein G conjugated beads, protein NG conjugated beads, protein L conjugated beads, oligodT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, and anti-fluorochrome microbead.

30. The droplet of claim 1, wherein said bead is semi-solid.

31. The droplet of claim 30, wherein said bead comprises a gel.

32. The droplet of claim 31, wherein said bead comprises a hydrogel.

33. The droplet of claim 1, wherein said bead comprises a polymer.

14. A droplet comprising:

a. a single bead comprising a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a molecular label sequence, a cellular label sequence, and an oligo dT sequence, and wherein the cellular label sequence of each of the plurality of oligonucleotides is the same and at least 100 of the plurality of oligonucleotides comprises different molecular label sequences; and

b. a lysate from a single cell.

15. The droplet of claim 14, wherein said lysate comprises nucleic acids targets.

16. The droplet of claim 15, wherein said nucleic acid targets are hybridized to said plurality of oligonucleotides.

34. The droplet of claim 14, wherein said bead is semi-solid.

35. The droplet of claim 34, wherein said bead comprises a gel.

36. The droplet of claim 35, wherein said bead comprises a hydrogel.

37. The droplet of claim 14, wherein said bead comprises a polymer.

41. The method of claim 14, wherein said bead comprises a polymer.

17. A method comprising:

a. introducing a single cell and a single bead into a droplet, wherein said single bead comprises a plurality of oligonucleotides, wherein each of the plurality of oligonucleotide comprises a cellular label sequence, a target binding region, and a molecular label sequence, and wherein the cellular label sequence of each of the plurality of oligonucleotides is the same and at least 100 of the plurality of oligonucleotides comprises different molecular label sequences;

b. lysing said single cell, thereby releasing nucleic acid targets from said cell; and

c. attaching said nucleic acid targets to said plurality of oligonucleotides.

18. The method of claim 17, wherein said droplet spatially isolates said single cell.

19. The method of claim 17, wherein said attaching comprises stochastically attaching said nucleic acid targets to said plurality of oligonucleotides.

20. The method of claim 17, wherein said attaching comprises hybridizing said nucleic acid targets to said plurality of oligonucleotides.

21. The method of claim 17, further comprising reverse transcribing said nucleic acid targets, thereby generating labelled target cDNAs.

22. The method of claim 21, wherein said reverse transcribing occurs in said droplet.

23. The method of claim 17, further comprising amplifying said nucleic acid targets, thereby generating labelled amplicons.

24. The method of claim 23, further comprising determining the number of said labelled amplicons in said cell.

25. The method of claim 24, wherein said determining comprises counting the number of unique molecular label sequences for a given amplicon of said labelled amplicons.

26. The method of claim 17, wherein said nucleic acid targets are RNA.

38. The method of claim 17, wherein said bead is semi-solid.

39. The method of claim 38, wherein said bead comprises a gel.

40. The method of claim 39, wherein said bead comprises a hydrogel.