Massively parallel single cell analysis

• US 10,208,356 B1
• Filed: 10/16/2018
• Issued: 02/19/2019
• Est. Priority Date: 08/28/2013
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: 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:
  US16/161,981
• Publication Number:
  US 20190040474A1
• Time in Patent Office: 126 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. A composition comprising:

a single bead, wherein the bead 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, wherein the cellular label sequence comprises 4-300 nucleotides, wherein the molecular label sequence comprises 4-300 nucleotides, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.

2. The composition of claim 1, further comprising a single cell, or a lysate of a single cell.

3. The composition of claim 2, wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules associated with said single cell.

4. The composition of claim 3, wherein said plurality of oligonucleotides is capable of labeling the individual occurrences of said target molecules associated with said single cell via hybridization of the individual occurrences of said target molecules to the target-binding regions of said plurality of oligonucleotides.

5. The composition of claim 3, wherein said target molecules comprise nucleic acid molecules.

6. The composition of claim 5, wherein said plurality of oligonucleotides is capable of labeling the individual occurrences of said target molecules associated with said single cell via a nucleic acid extension reaction.

7. The composition of claim 6, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.

8. The composition of claim 6, wherein the nucleic acid extension reaction is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.

9. The composition of claim 3, wherein a target molecule of said target molecules comprises a messenger ribonucleic acid (mRNA) molecule.

10. The composition of claim 3, wherein a target molecule of said target molecules comprises a deoxyribonucleic acid (DNA) molecule.

11. The composition of claim 3, wherein a target molecule of said target molecules comprises a sample tag oligonucleotide.

12. The composition of claim 11, wherein the sample tag oligonucleotide is 25-300 nucleotides in length.

13. The composition of claim 1, wherein the molecular label sequence is 4-30 nucleotides in length.

14. The composition of claim 1, wherein the cellular label sequence is 4-30 nucleotides in length.

15. 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 about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.

17. The composition of claim 3, wherein a target molecule of said target molecules is associated with said single cell via a peptide.

18. The composition of claim 17, wherein the peptide comprises an antibody.

19. The composition of claim 18, wherein the peptide is capable of binding to said single cell.

20. The composition of claim 1, comprising a peptide.

21. The composition of claim 20, wherein the peptide comprises an antibody.

22. The composition of claim 20, wherein the peptide is associated with a sample tag oligonucleotide.

23. The composition of claim 22, wherein the plurality of oligonucleotides is capable of labeling the sample tag oligonucleotide.

24. The composition of claim 1, wherein said plurality of oligonucleotides comprises at least 700,000 oligonucleotide.

25. The composition of claim 1, wherein said plurality of oligonucleotides comprises about 1,000,000 oligonucleotide.

26. 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, a target-specific sequence, a multimer sequence, a random multimer sequence, and a complement thereof.

27. The composition of claim 1, wherein said single bead comprises silica gel, Wang resin, Merrifield resin, polydimethylsiloxane (PDMS), polystyrene, glass, controlled pore glass, polypropylene, agarose, gelatin, hydrogel, a paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sephadex, Sepharose, cellulose, nylon, silicone, or a combination thereof.

28. The composition of claim 1, wherein said single bead is a hydrogel bead, a magnetic bead, or a combination thereof.

29. A partition comprising:

a. a composition of claim 1; and

b. a single cell, or a lysate of a single cell.

30. The partition of claim 29, wherein the partition is a well or a droplet.