Massively parallel single cell analysis

Patent metadata

Field	Value
Publication number	US-11618929-B2
Application number	US-201816012584-A
Country	US
Kind code	B2
Filing date	Jun 19, 2018
Priority date	Aug 28, 2013
Publication date	Apr 4, 2023
Grant date	Apr 4, 2023

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 reverse transcriptase and a plurality of beads each coupled thereto a plurality of barcode oligonucleotides,
wherein each of the plurality of barcode oligonucleotides comprises a first partition-specific barcode sequence, a molecular barcode sequence, and a target-binding region that is bound with a target nucleic acid molecule; and
wherein the partition-specific barcode sequences in the plurality of barcode oligonucleotides coupled to the same bead are identical and the partition-specific barcode sequences are different in the plurality of barcode oligonucleotides coupled to different beads in the plurality of beads, and at least 1000 molecular barcode sequences in the plurality of barcode oligonucleotides coupled to the same bead are different.

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.

9. The composition of claim 1 , wherein at least 30% of the plurality of barcode oligonucleotides comprise different molecular barcode sequences.

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

11. 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, and a universal label sequence.

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

13. The partition of claim 12 , wherein the partition is a microwell or a droplet.

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

15. A kit, comprising
a barcoding library of claim 1 ;
an agent for extension; and
a plurality of primers.

16. The kit of claim 15 , further comprising a microwell array or a microfluidic chip.

17. The kit of claim 15 , wherein the agent for extension comprises a reverse transcriptase with terminal transferase activity.

18. The kit of claim 15 , further comprising an agent for amplification.

19. The composition of claim 8 , wherein the cleavable linkage is selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linkage, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof.

20. The composition of claim 1 , wherein at least 100,000 molecular barcode sequences in the plurality of barcode oligonucleotides coupled to the same bead are different.

21. The composition of claim 1 , wherein the target-binding region comprises an oligodT sequence.

22. The composition of claim 1 , wherein the target-binding region is configured to act a primer for DNA and/or RNA synthesis.

23. The composition of claim 1 , wherein the target nucleic acid molecule is cellular nucleic acid.

24. The composition of claim 1 , wherein the target nucleic acid molecule is mRNA.

25. The composition of claim 1 , wherein the target nucleic acid molecule is genomic DNA or viral DNA.

26. The composition of claim 1 , wherein the target nucleic acid molecule is rRNA, tRNA, ncRNA, lncRNA, siRNA, microRNA, or miRNA.

27. The composition of claim 1 , wherein the target nucleic acid molecules that are bound to the target binding regions of the plurality of barcode oligonucleotides coupled to the same bead are from the same cell.

28. The composition of claim 1 , wherein the target nucleic acid molecules that are bound to the target binding regions of the plurality of barcode oligonucleotides coupled to different beads are from different cells.

29. The composition of claim 1 , wherein the plurality of beads comprises at least 18 beads.

30. The composition of claim 1 , wherein the plurality of beads comprises at least 175 beads.

Assignees

• Cellular Res Inc
• Becton Dickinson Co

Inventors

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

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• B01L2300/0829

  Multi-well plates; Microtitration plates · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• B01L7/52

  with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples · CPC title

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title