Massively parallel single cell analysis

• US 9,567,646 B2
• Filed: 07/15/2015
• Issued: 02/14/2017
• 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:
  US14/800,526
• Publication Number:
  US 20160010151A1
• Time in Patent Office: 580 Days
• Field of Search: None
• 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 single cell and a single bead,

wherein said single bead comprises a plurality of oligonucleotides,

wherein each of said plurality of oligonucleotides comprises an identical cellular label sequence, a molecular label sequence, and a target-binding region, and

wherein at least 100 of said plurality of oligonucleotides comprise different molecular label sequences.

2. The composition of claim 1, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

3. The composition of claim 1, wherein said plurality of oligonucleotides each comprises a universal label sequence.

4. The composition of claim 1, 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.

5. 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.

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

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

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

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

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

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

16. The composition of claim 1, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of said single cell.

17. The composition of claim 1, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of said single cell via reverse transcription.

18. The composition of claim 1, wherein said bead comprises silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose bead, cellulose bead, polystyrene bead, or any combination thereof.

10. A composition comprising:

a lysate from a single cell and a single bead,

wherein said single bead comprises a plurality of oligonucleotides,

wherein each of said plurality of oligonucleotides comprises an identical cellular label sequence, a molecular label sequence, and a target-binding region, and

wherein at least 100 of said plurality of oligonucleotides comprise different molecular label sequences.

11. The composition of claim 10, wherein the nucleic acid contents of said lysate from a single cell are associated with a plurality of oligonucleotides.

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

15. The composition of claim 10, wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.

19. The composition of claim 10, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of said lysate from a single cell.

20. The composition of claim 10, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of said lysate from a single cell via reverse transcription.

21. The composition of claim 10, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

22. The composition of claim 10, wherein said single cell is a cell selected from the group consisting of a rare cell, a tumor cell, and an immune cell.

23. The composition of claim 10, wherein said single cell is from a human.

24. The composition of claim 10, wherein said single cell is from a tissue.

25. The composition of claim 10, wherein said single cell is infected with viral polynucleotides.

26. The composition of claim 10, wherein said bead is magnetic.

27. The composition of claim 10, wherein said bead comprises silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose bead, cellulose bead, polystyrene bead, or any combination thereof.