Methods for analyzing nucleic acids from single cells

Patent metadata

Field	Value
Publication number	US-10240197-B1
Application number	US-201816194047-A
Country	US
Kind code	B1
Filing date	Nov 16, 2018
Priority date	Aug 20, 2009
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

Abstract

Aspects of the present invention include analyzing nucleic acids from single cells using methods that include using tagged polynucleotides containing multiplex identifier sequences.

First claim

What is claimed:

1. A method of counting nucleic acids in a sample, the method comprising:
(a) providing a sample comprising a plurality of cells, wherein a cell of the plurality of cells comprises a plurality of sample polynucleotides;
(b) generating a plurality of tagged polynucleotides from the plurality of sample polynucleotides of said cell and a plurality of oligonucleotide tags, wherein a tagged polynucleotide of the plurality of tagged polynucleotides comprises:
(i) a sample sequence from a sample polynucleotide of the plurality of sample polynucleotides;
(ii) a first tag sequence distinguishing said sample polynucleotide from sample polynucleotides from other cells; and
(iii) a second tag sequence distinguishing said sample polynucleotide from other sample polynucleotides from said cell;

(c) sequencing the tagged polynucleotide to determine the sample sequence, the first tag sequence, and the second tag sequence; and
(d) using the first tag sequence and the second tag sequence to count a number of sample polynucleotides in said plurality of sample polynucleotides of said cell.

2. The method of claim 1 , wherein the method further comprises amplifying the plurality of tagged polynucleotides prior to the sequencing step (c).

3. The method of claim 1 , wherein the plurality of sample polynucleotides is selected from DNA and RNA.

4. The method of claim 1 , wherein the plurality of sample polynucleotides comprises mRNA.

5. The method of claim 1 , wherein the plurality of tagged polynucleotides is generated through at least one ligation reaction.

6. The method of claim 1 , wherein an oligonucleotide tag of said plurality of oligonucleotide tags comprises said first tag sequence and said second tag sequence.

7. The method of claim 1 , wherein an oligonucleotide tag of said plurality of oligonucleotide tags comprises a sequence that is configured to hybridize to said sample polynucleotide.

8. The method of claim 1 , wherein the plurality of tagged polynucleotides is generated by (i) hybridizing an oligonucleotide tag of said plurality of oligonucleotide tags to said sample polynucleotide and (ii) extending said oligonucleotide tag or said sample polynucleotide or both.

9. The method of claim 1 , wherein the plurality of tagged polynucleotides is generated through at least one linear amplification reaction.

10. The method of claim 1 , wherein the plurality of tagged polynucleotides is generated through at least one reverse transcription reaction.

11. The method of claim 1 , wherein the plurality of tagged polynucleotides is generated through at least one polymerase chain reaction (PCR).

12. The method of claim 1 , wherein substantially every sample polynucleotide of said plurality of sample polynucleotides is associated with the same first tag sequence.

13. The method of claim 1 , wherein at least 90 percent of said plurality of sample polynucleotides is associated with a unique second tag sequence.

14. The method of claim 1 , wherein at least 95 percent of said plurality of sample polynucleotides is associated with a unique second tag sequence.

15. The method of claim 1 , wherein at least 99 percent of said plurality of sample polynucleotides is associated with a unique second tag sequence.

16. The method of claim 1 , wherein substantially every sample polynucleotide of said plurality of sample polynucleotides is associated with a unique second tag sequence.

17. The method of claim 1 , wherein the number of different second tag sequences is larger than the number of sample polynucleotides.

18. The method of claim 17 , wherein the number of different second tag sequences is at least ten times the number of sample polynucleotides.

19. The method of claim 18 , wherein the number of different second tag sequences is at least one hundred times the number of sample polynucleotides.

20. The method of claim 1 , wherein said plurality of sample polynucleotides are randomly associated with said plurality of oligonucleotide tags to generate said plurality of tagged polynucleotides.

21. The method of claim 1 , wherein step (c) comprises hybridizing said tagged polynucleotide to a solid support.

22. The method of claim 20 , wherein said solid support is a bead.

23. The method of claim 1 , wherein step (d) comprises using said first tag sequence to distinguish (i) the number of sample polynucleotides having said sample sequence from said cell from (ii) the number of sample polynucleotides having said sample sequence from other cells.

24. The method of claim 1 , wherein step (d) comprises determining the number of different second tag sequences associated with said sample sequence, thereby estimating the number of sample polynucleotides having said sample sequence from said cell.

25. The method of claim 1 , wherein said plurality of sample polynucleotides comprises substantially all mRNA molecules of said cell.

26. The method of claim 1 , wherein said plurality of sample polynucleotides comprises a subset of polynucleotides of said cell having the same sequence.

Assignees

• 10X Genomics Inc

Inventors

• Brenner Sydney
• Mikawa Gi
• Osborne Robert
• Slatter Andrew

Classifications

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title