MASSIVELY PARALLEL SINGLE CELL ANALYSIS

• US 20160010151A1
• Filed: 07/15/2015
• Published: 01/14/2016
• 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 9,567,646 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 composition comprising:

a substrate comprising at least 1,000 wells wherein a plurality of wells comprise a single cell from a population of cells and a single bead from a population of beads.

2. The composition of claim 1, wherein said plurality of wells is at least 10% of said at least 1,000 wells.

3. The composition of claim 1, wherein a different plurality of wells contain only a single bead.

4. The composition of claim 1, wherein said population of cells is at least 1,000 cells.

5. The composition of claim 1, wherein said single bead comprises a plurality of oligonucleotides.

6. The composition of claim 4, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

7. The composition of claim 4, wherein said plurality of oligonucleotides each comprises a cellular label, a universal label, a molecular label, and a target-binding region.

8. The composition of claim 4, wherein at least two of said plurality of oligonucleotides have different molecular labels.

9. The composition of claim 4, wherein individual oligonucleotides of said plurality of oligonucleotides have the same cellular label.

10. The composition of claim 1, wherein beads from said population of beads comprise different cellular labels.

11. The composition of claim 1, wherein beads from said plurality of beads in different wells of said plurality of wells comprise different cellular labels.

12. The composition of claim 6, wherein said target-binding region comprises a sequence selected from the group consisting of:

an oligo-dT sequence, a gene-specific sequence, and a random multimer sequence.

13. The composition of claim 1, wherein the diameter of said wells is at least 10 micrometers.

14. The composition of claim 1, wherein said substrate comprises a material selected from the group consisting of:

PDMS, plastic, glass, photoresist, silicon, metal, and optical adhesive, or any combination thereof.

15. The composition of claim 1, wherein the diameter of said at least 1,000 wells is from 1-fold to 2-fold the diameter of said bead.

16. The composition of claim 1, wherein the diameter of said at least 1,000 wells is about equal to the sum of the diameter of said bead and the diameter of said cell.

17. The composition of claim 1, wherein the depth of said at least 1,000 wells is about equal to the sum of the diameter of said bead and the diameter of said cell.

18. The composition of claim 1, wherein the depth of said wells is at least 1-fold the diameter of said bead.

19. The composition 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.

20. The composition of claim 1, wherein said single cell is from a human.

21. The composition of claim 1, wherein said single cell is from a tissue.

22. The composition of claim 1, wherein said single cell is infected with viral polynucleotides.

23. The composition of claim 1, wherein said population of cells comprise different cell types.

24. The composition of claim 1, wherein said substrate comprises about 100,000 wells.

25. The composition of claim 1, wherein the surface between said substrate and said at least 1,000 wells is domed or peaked.

26. The composition of claim 1, wherein said at least 1,000 wells are arranged on said substrate in a hexagonal grid.

27. The composition of claim 1, wherein said single bead is magnetic.

28. A composition comprising:

a substrate comprising at least 1,000 wells wherein a plurality of wells comprise a lysate from a single cell from a population of cells and a single bead from a population of beads.

29. The composition of claim 28, wherein the nucleic acid contents of said lysate from a single cell are associated with a plurality of oligonucleotides attached to said single bead.

30. The composition of claim 29, wherein the said plurality of oligonucleotides comprises a cellular label that is the same for all oligonucleotides attached to said single bead but different from the cellular label for oligonucleotides attached to beads of said population of beads in different wells of said plurality of wells.