MASSIVELY PARALLEL SINGLE CELL ANALYSIS

• US 20180291470A1
• Filed: 06/19/2018
• Published: 10/11/2018
• 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
• Granted Patent: US 11,618,929 B2
• Time in Patent Office: 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 bead and a plurality of barcode oligonucleotides immobilized to the bead, wherein each of the plurality of barcode oligonucleotides comprises a first barcode sequence and a first random sequence, wherein the first barcode sequences in the plurality of nucleic acid barcode molecules are identical.

2. The composition of claim 1, wherein the bead is solid or semi-solid.

3. The composition of claim 1, wherein the bead is a hydrogel bead.

4. The composition of claim 1, wherein the plurality of barcode oligonucleotides is non-covalently coupled to the bead.

5. The composition of claim 1, wherein the plurality of barcode oligonucleotides is attached to the bead via a linker.

6. The composition of claim 1, wherein the plurality of barcode oligonucleotides is covalently linked to the bead.

7. The composition of claim 1, wherein the plurality of barcode oligonucleotides is releasably associated with the bead.

8. The composition of claim 6, wherein the plurality of barcode oligonucleotides is covalently linked to the bead through a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof.

9. The composition of claim 1, wherein the first random sequence is configured to hybridize to a target molecule.

10. The composition of claim 1, wherein each of the plurality of barcode oligonucleotide comprises a molecular label, and wherein the molecule label comprises the first random sequence.

11. The composition of claim 10, wherein at least 30% of the plurality of barcode oligonucleotide comprise molecular labels with different sequences.

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

13. The composition of claim 10, wherein each of the plurality of barcode oligonucleotides comprising a second random sequence configured to hybridize to a target molecule.

14. The composition of claim 1, wherein each of the plurality of barcode oligonucleotides comprises an oligodT sequence.

15. The composition of claim 1, wherein each of the plurality of barcode oligonucleotides comprises one or more of a primer binding site, a universal primer, a sample label sequence, a universal label sequence.

21. A partition, comprising a composition of claim 1 and a single cell.

22. The partition of claim 21, wherein the partition is a microwell.

23. The partition of claim 21, wherein the partition is a droplet.

24. The partition of claim 21, wherein the partition has a volume of between about 1 microliter and about 1 nanoliter.

25. The partition of claim 21, wherein the single cell is selected from the group consisting of a rare cell, a tumor cell, a human cell, a cell from a tissue, a tumor cell, an immune cell, a cell infected with viral polynucleotides, and a combination thereof.

26. An emulsion, comprising a composition of claim 1.

16. A barcoding library comprising a plurality of compositions each comprising a bead and a plurality of barcode oligonucleotides immobilized to the bead, wherein each of the plurality of barcode oligonucleotides comprises a first barcode sequence and a first random sequence, wherein the first barcode sequences in the plurality of nucleic acid barcode molecules immobilized to the same bead are identical, and wherein the first barcode sequences are different between two or more beads in the plurality of compositions.

17. The library of claim 16, wherein the beads of the plurality of compositions comprise at least 10,000 different first barcode sequences coupled to different beads in the beads.

18. The library of claim 16, wherein the bead is a hydrogel bead.

19. The library of claim 16, wherein the plurality of barcode oligonucleotides is releasably coupled to the hydrogel bead.

20. The library of claim 16, wherein each of the plurality of barcode oligonucleotide comprises a molecular label, and wherein the molecule label comprises the first random sequence.

27. A kit, comprising

a barcoding library of claim 16;

an agent for extension; and

a plurality of primers.

28. The kit of claim 27, further comprising a microwell array or a microfluidic chip.

29. The kit of claim 27, wherein the agent for extension comprises a reverse transcriptase with terminal transferase activity.

30. The kit of claim 27, further comprising an agent for amplification.