Massively parallel single cell analysis

• US 10,253,375 B1
• Filed: 10/16/2018
• Issued: 04/09/2019
• 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:
  US16/162,161
• Publication Number:
  US 20190085412A1
• Time in Patent Office: 175 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 method comprising:

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

2. The method of claim 1, wherein the partition is a well or a droplet.

3. The method of claim 1, comprising lysing said single cell after introducing said single bead and said single cell into said partition.

4. The method of claim 1, comprising lysing said single cell before introducing said single bead and said single cell into said partition.

5. The method of claim 1, comprising attaching target molecules associated with said single cell to said plurality of oligonucleotides.

6. The method of claim 5, wherein said attaching comprises hybridizing the target molecules to the target-binding regions of said plurality of oligonucleotides.

7. The method of claim 5, wherein said target molecules comprise nucleic acid molecules.

8. The method of claim 7, wherein said attaching comprises performing a nucleic acid extension reaction on the target molecules attached to said plurality of oligonucleotides.

9. The method of claim 8, wherein the nucleic acid extension reaction comprises a reverse transcription reaction.

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

11. The method of claim 5, wherein said target molecules comprise a messenger ribonucleic acid (mRNA) molecule.

12. The method of claim 5, wherein said target molecules comprise a deoxyribonucleic acid (DNA) molecule.

13. The method of claim 5, wherein said target molecules comprise a sample tag oligonucleotide.

14. The method of claim 13, wherein the sample tag oligonucleotide is 25-300 nucleotides in length.

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

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

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

18. The method of claim 1, wherein about 1,000,000 of said plurality of oligonucleotides comprise different molecular label sequences.

19. The method of claim 13, comprising labeling the sample tag oligonucleotide using an oligonucleotide of said plurality of oligonucleotides.

20. The method of claim 1, comprising labeling target molecules associated with said single cell using said plurality of oligonucleotides.

21. The method of claim 20, wherein labeling target molecules comprises stochastically labeling the target molecules associated with said single cell using said plurality of oligonucleotides.

22. The method of claim 5, wherein a target molecule of said target molecules is associated with said single cell via a peptide.

23. The method of claim 22, wherein the peptide comprises an antibody.

24. The method of claim 22, comprising contacting the peptide with said single cell, whereby the peptide binds to said single cell.

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

26. The method 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.

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

28. The method of claim 23, comprising estimating the number of said target molecules.

29. The method of claim 28, wherein said estimating comprises counting the number of unique molecular label sequences associated with a distinct target molecule of said target molecules associated with said single cell.

30. The method of claim 13, comprising determining an origin of said single cell based on the sequence of the sample tag oligonucleotide, or a portion thereof.