Massively parallel single cell analysis

Patent metadata

Field	Value
Publication number	US-10927419-B2
Application number	US-201815987851-A
Country	US
Kind code	B2
Filing date	May 23, 2018
Priority date	Aug 28, 2013
Publication date	Feb 23, 2021
Grant date	Feb 23, 2021

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 for processing messenger ribonucleic acid (mRNA) molecules from a single cell, comprising:
(a) partitioning a plurality of cells and a plurality of beads in a plurality of partitions, wherein a partition of said plurality of partitions comprises a single cell from said plurality of cells and a single bead from said plurality of beads,
wherein said single bead comprises a plurality of nucleic acid barcode molecules each comprising a first barcode sequence a second barcode sequence, a RNA priming sequence,
wherein the first barcode sequence of each of the plurality of nucleic acid barcode molecules is the same; and wherein at least 1000 nucleic acid barcode molecules of the plurality of said nucleic acid barcode molecules comprise different second barcode sequences, wherein at least 1000 of the plurality of beads comprises a plurality of nucleic acid barcode molecules comprising first barcode sequences that are different across the at least 1000 beads;

(b) in said partition comprising said single cell and said single bead, releasing messenger ribonucleic acid (mRNA) molecules from said single cell, thereby said mRNA molecules attach to the nucleic acid barcode molecules via the RNA priming sequence;
(c) subjecting said mRNA molecules to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules each comprising said first barcode sequence and a second barcode sequence; and
(d) subjecting said cDNA molecules to one or more reactions to generate a set of nucleic acid molecules for nucleic acid sequencing.

2. The method of claim 1 , wherein (c) is performed in said partition comprising said single cell and said single bead, and wherein subsequent to (c), said cDNA molecules, or derivatives thereof, are removed from said given partition.

3. The method of claim 1 , wherein, prior to (c), said mRNA molecules are removed from said partition comprising said single cell and said single bead.

4. The method of claim 1 , wherein each of said plurality of nucleic acid barcode molecules comprises a universal primer sequence.

5. The method of claim 1 , wherein said single bead is a single magnetic bead, and wherein said plurality of nucleic acid barcode molecules are attached to said single magnetic bead.

6. The method of claim 1 , wherein said single bead comprises hydrogel.

7. The method of claim 6 , wherein said plurality of nucleic acid barcode molecules are covalently or non-covalently attached to the single bead.

8. The method of claim 1 , wherein said RNA priming sequence is an oligo(dT) sequence.

9. The method of claim 8 , wherein in step (b) said mRNA molecules attach to the oligo(dT) sequence of said nucleic acid barcode molecules by hybridization.

10. The method of claim 1 , wherein the first barcode sequences of at least 10,000 of said plurality of beads are different across said at least 10,000 beads.

11. The method of claim 1 , wherein said different first barcode sequences are capable of distinguishing nucleic acid molecules in different partitions comprising said 1,000 beads.

12. The method of claim 9 , further comprising, prior to (c), (i) pooling said mRNA molecules attached to said nucleic acid barcode molecules and (ii) performing said one or more reactions in bulk.

13. The method of claim 1 , wherein said one or more reactions comprise nucleic acid amplification that generates amplified products from said plurality of cDNA molecules.

14. The method of claim 1 , comprising performing said nucleic acid sequencing on said set of nucleic acid molecules, or derivatives thereof, to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said first barcode sequence.

15. The method of claim 1 , wherein said partition comprises a droplet.

16. The method of claim 1 , wherein in step (b) said mRNA molecules attach to the RNA priming sequence of said nucleic acid barcode molecules by hybridization.

17. The method of claim 1 , wherein said partition comprises a well.

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

• C12N15/1075

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

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title