Massively parallel single cell analysis

• US 9,567,645 B2
• Filed: 08/28/2014
• 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:
  US14/472,363
• Publication Number:
  US 20150299784A1
• Time in Patent Office: 901 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 method comprising:

associating a single bead with a single cell,

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 method of claim 1, wherein said single cell is selected from the group consisting of a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected with viral polynucleotides, and any combination thereof.

3. The method of claim 1, further comprising lysing said single cell after associating said single bead with said single cell.

4. The method of claim 1, further comprising lysing said single cell before associating said single bead with said single cell.

5. The method of claim 1, wherein said single bead is magnetic.

6. The method of claim 1, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

7. The method of claim 1, wherein said plurality of oligonucleotides each comprise a universal label sequence.

8. The method 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.

9. The method of claim 1, further comprising stochastically labelling target nucleic acid molecules from said single cell.

10. The method of claim 9, wherein said labelling comprises hybridizing said target nucleic acid molecules to the plurality of oligonucleotides on said single bead.

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

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

13. The method of claim 1, wherein said single bead and said single cell are in the same well.

14. The method of claim 1, wherein said single bead and said single cell are in the same droplet.

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

16. The method of claim 10, wherein said target nucleic acid molecules comprise mRNA molecules.

17. The method of claim 16, further comprising reverse transcribing said target nucleic acid molecules, thereby generating labelled target cDNAs.

18. The method of claim 17, wherein said reverse transcribing is done while said plurality of oligonucleotides is on said single bead.

19. The method of claim 17, further comprising performing second strand synthesis on said labelled target cDNAs, thereby generating double-stranded labelled target polynucleotides.

20. The method of claim 19, further comprising amplifying said double-stranded labelled target polynucleotides, thereby generating labelled target amplicons.

21. The method of claim 20, wherein said amplifying is performed with a universal primer and a random multimer primer.

22. The method of claim 20, wherein said amplifying is performed with a universal primer or a gene-specific primer.

23. The method of claim 20, wherein said amplifying comprises conducting a nested PCR reaction.

24. The method of claim 20, wherein said amplifying comprises amplifying the whole transcriptome of said single cell.

25. The method of claim 20, further comprising estimating the number of said target nucleic acid molecules.

26. The method of claim 25, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons.

27. The method of claim 25, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons and at least a portion of said oligonucleotide.

28. The method of claim 25, wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target polynucleotide of said single cell.