MASSIVELY PARALLEL SINGLE CELL ANALYSIS

• US 20180002764A1
• Filed: 03/15/2017
• Published: 01/04/2018
• 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 K., Facer, Geoffrey Richard, Wilhelmy, Julie
• Granted Patent: US 10,954,570 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. (canceled)

2. (canceled)

3. A composition, comprising:

a support attached with a plurality of oligonucleotides and a plurality of peptides, wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, 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.

4. The composition of claim 3, wherein a plurality of peptides comprises a plurality of antibodies.

5. The composition of claim 4, further comprising a single cell, wherein at least one of the plurality of antibodies bind to the surface of the cell.

6. The composition of claim 5, wherein the plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of the single cell.

7. The composition of claim 5, wherein the plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of the single cell via reverse transcription.

8. The composition of claim 3, wherein the 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.

9. The composition of claim 5, wherein the single cell is selected from the group consisting of a rare cell, a tumor cell, a human cell, a cell from a tissue, a tumor cell, an immune cell, a cell infected with viral polynucleotides, and any combination thereof.

10. The composition of claim 3, wherein each of the plurality of oligonucleotides further comprises at least one of:

(a) a sample label sequence; and

(b) a universal label sequence.

11. The composition of claim 3, wherein the support is a particle.

12. The composition of claim 3, wherein the support comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

13. The composition of claim 11, wherein the particle is a bead.

14. The composition of claim 13, 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.

15. The composition of claim 13, wherein the bead comprises hydrogel or a polymer.

16. The composition of claim 13, wherein the bead comprises silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose bead, cellulose bead, polystyrene bead, or any combination thereof.

17. The composition of claim 13, wherein the bead is semi-solid.

18. The composition of claim 3, wherein at least 1,000 of the plurality of oligonucleotides comprise different molecular label sequences.

19. The composition of claim 3, wherein at least 10,000 of the plurality of oligonucleotides comprise different molecular label sequences.

20. The composition of claim 3, wherein the plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

21. The composition of claim 3, wherein the molecular label sequences comprise random sequences.

22. The composition of claim 3, wherein the solid support is coated with the plurality of peptides.