MASSIVELY PARALLEL SINGLE CELL ANALYSIS

• US 20160145683A1
• Filed: 10/01/2015
• Published: 05/26/2016
• 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
• Granted Patent: US 9,637,799 B2
• Time in Patent Office: Days
• 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 from a population of beads, wherein said bead comprises a plurality of oligonucleotides, each comprising a molecular label, a cellular label, and an oligo dT; and

b. a single cell from a population of cells.

2. The droplet of claim 1, wherein molecular labels of said plurality of oligonucleotides are different.

3. The droplet of claim 1, wherein cellular labels of said plurality of oligonucleotides are the same for said single bead.

4. The droplet of claim 1, wherein beads from said population of beads comprise a different cellular label.

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

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

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

8. The droplet of claim 7, wherein said emulsion is a water-in-oil emulsion.

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

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

11. The droplet of claim 1, wherein said droplet is a compound droplet.

12. 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.

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

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

15. The droplet of claim 1, wherein said population of cells comprises at least two different cell types.

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

17. A droplet comprising:

a. a single bead, wherein said bead comprises a plurality of oligonucleotides, each comprising a molecular label, a cellular label, and an oligo dT; and

b. a lysate from a single cell.

18. The droplet of claim 17, wherein said lysate comprises nucleic acids targets.

19. The droplet of claim 18, wherein said nucleic acid targets are hybridized to said plurality of oligonucleotides.

20. A method comprising:

a. introducing a single cell and a single bead into a droplet, wherein said single bead comprises a plurality of oligonucleotides, each oligonucleotide comprising a cellular label and a molecular label;

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

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

21. The method of claim 20, wherein said droplet is used to spatially isolate said single cell.

22. The method of claim 20, wherein said attaching is stochastic.

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

24. The method of claim 20, further comprising reverse transcribing said nucleic acid targets, thereby generating labelled target cDNAs.

25. The method of claim 24, wherein said reverse transcribing occurs in said droplet.

26. The method of claim 20, further comprising amplifying said nucleic acid targets, thereby generating labelled amplicons.

27. The method of claim 26, further comprising determining the number of said labelled amplicons in said cell.

28. The method of claim 27, wherein said determining comprises counting the number of unique molecular labels for a given amplicon of said labelled amplicons.

29. The method of claim 20, wherein said nucleic acid targets are RNA.