Massively parallel single cell analysis

• US 9,598,736 B2
• Filed: 05/27/2016
• Issued: 03/21/2017
• 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:
  US15/167,807
• Publication Number:
  US 20160265069A1
• Time in Patent Office: 298 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 particle,

wherein the particle comprises a plurality of oligonucleotides, 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.

2. The composition of claim 1, wherein the particle is a bead.

3. The composition of claim 2, 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 A/G conjugated beads, protein L conjugated beads, oligodT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.

4. The composition of claim 1, wherein the plurality of oligonucleotides comprises DNA.

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

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

7. The composition of claim 1, wherein the plurality of oligonucleotides comprises at least 10,000 oligonucleotides.

8. The composition of claim 1, wherein each of the plurality of oligonucleotides further comprises at least one of:

(a) a sample label; and

(b) a universal label.

9. The composition of claim 1, wherein the target-binding region comprises a sequence selected from the group consisting of a gene-specific sequence, an oligo-dT sequence, a random multimer, and any combination thereof.

10. The composition of claim 1, wherein the molecular label sequences comprise random sequences.

11. The composition of claim 1, wherein the particle 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.

12. The composition of claim 1, wherein

(a) each of the plurality of oligonucleotides comprises a linker functional group, and

(b) the particle comprises a solid support functional group;

wherein the support functional group and the linker functional group are associated with each other.

13. The composition of claim 12, wherein the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.

14. The composition of claim 1, wherein the cellular label sequences of the plurality of oligonucleotides comprise at least 8 nucleotides.

15. The composition of claim 1, wherein the molecular label sequences of the plurality of oligonucleotides comprise at least 8 nucleotides.

20. The composition of claim 1, wherein the particle is a microparticle or a nanoparticle.

16. A kit comprising a plurality of particles each comprising a plurality of oligonucleotides, 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.

17. The kit of claim 16, wherein the plurality of particles comprises a plurality of beads.

18. The kit of claim 17, wherein the plurality of beads comprises at least 384 beads.

19. The kit of claim 16, further comprising a microwell array, wherein each microwell of the microwell array comprises a particle of the plurality particles.