Massively parallel single cell analysis

Patent metadata

Field	Value
Publication number	US-9567646-B2
Application number	US-201514800526-A
Country	US
Kind code	B2
Filing date	Jul 15, 2015
Priority date	Aug 28, 2013
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

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).

First claim

What is claimed is:

1. A composition comprising:
a single cell and a single bead,
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 composition of claim 1 , wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

3. The composition of claim 1 , wherein said plurality of oligonucleotides each comprises a universal label sequence.

4. 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, and a random multimer sequence.

5. The composition of claim 1 , wherein said single cell is a cell selected from the group consisting of a rare cell, a tumor cell, and an immune cell.

6. The composition of claim 1 , wherein said single cell is from a human.

7. The composition of claim 1 , wherein said single cell is from a tissue.

8. The composition of claim 1 , wherein said single cell is infected with viral polynucleotides.

9. The composition of claim 1 , wherein said single bead is magnetic.

10. A composition comprising:
a lysate from a single cell and a single bead,
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.

11. The composition of claim 10 , wherein the nucleic acid contents of said lysate from a single cell are associated with a plurality of oligonucleotides.

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

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

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

15. The composition of claim 10 , wherein at least 10,000 of said plurality of oligonucleotides comprise different molecular label sequences.

16. The composition of claim 1 , wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of said single cell.

17. The composition of claim 1 , wherein said plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of said single cell via reverse transcription.

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

19. The composition of claim 10 , wherein said plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of said lysate from a single cell.

20. The composition of claim 10 , wherein said plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of said lysate from a single cell via reverse transcription.

21. The composition of claim 10 , wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

22. The composition of claim 10 , wherein said single cell is a cell selected from the group consisting of a rare cell, a tumor cell, and an immune cell.

23. The composition of claim 10 , wherein said single cell is from a human.

24. The composition of claim 10 , wherein said single cell is from a tissue.

25. The composition of claim 10 , wherein said single cell is infected with viral polynucleotides.

26. The composition of claim 10 , wherein said bead is magnetic.

27. The composition of claim 10 , wherein said bead comprises silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose bead, cellulose bead, polystyrene bead, or any combination thereof.

Assignees

• Cellular Res Inc

Inventors

• Fan Christina
• Fodor Stephen P A
• Fu Glenn
• Facer Geoffrey Richard
• Wilhelmy Julie

Classifications

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• B01L3/50857

  using arrays or bundles of open capillaries for holding samples · CPC title

• C12Q2535/101

  Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators · CPC title