Massively parallel single cell analysis

Patent metadata

Field	Value
Publication number	US-9567645-B2
Application number	US-201414472363-A
Country	US
Kind code	B2
Filing date	Aug 28, 2014
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 method comprising:
associating a single bead with a single cell,
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 method of claim 1 , wherein said single cell is selected from the group consisting of a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected with viral polynucleotides, and any combination thereof.

3. The method of claim 1 , further comprising lysing said single cell after associating said single bead with said single cell.

4. The method of claim 1 , further comprising lysing said single cell before associating said single bead with said single cell.

5. The method of claim 1 , wherein said single bead is magnetic.

6. The method of claim 1 , wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

7. The method of claim 1 , wherein said plurality of oligonucleotides each comprise a universal label sequence.

8. 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, and a random multimer sequence.

9. The method of claim 1 , further comprising stochastically labelling target nucleic acid molecules from said single cell.

10. The method of claim 9 , wherein said labelling comprises hybridizing said target nucleic acid molecules to the plurality of oligonucleotides on said single bead.

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

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

13. The method of claim 1 , wherein said single bead and said single cell are in the same well.

14. The method of claim 1 , wherein said single bead and said single cell are in the same droplet.

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

16. The method of claim 10 , wherein said target nucleic acid molecules comprise mRNA molecules.

17. The method of claim 16 , further comprising reverse transcribing said target nucleic acid molecules, thereby generating labelled target cDNAs.

18. The method of claim 17 , wherein said reverse transcribing is done while said plurality of oligonucleotides is on said single bead.

19. The method of claim 17 , further comprising performing second strand synthesis on said labelled target cDNAs, thereby generating double-stranded labelled target polynucleotides.

20. The method of claim 19 , further comprising amplifying said double-stranded labelled target polynucleotides, thereby generating labelled target amplicons.

21. The method of claim 20 , wherein said amplifying is performed with a universal primer and a random multimer primer.

22. The method of claim 20 , wherein said amplifying is performed with a universal primer or a gene-specific primer.

23. The method of claim 20 , wherein said amplifying comprises conducting a nested PCR reaction.

24. The method of claim 20 , wherein said amplifying comprises amplifying the whole transcriptome of said single cell.

25. The method of claim 20 , further comprising estimating the number of said target nucleic acid molecules.

26. The method of claim 25 , wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons.

27. The method of claim 25 , wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons and at least a portion of said oligonucleotide.

28. The method of claim 25 , wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target polynucleotide of said single cell.

Assignees

• Cellular Res Inc

Inventors

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

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title