Methods for analyzing nucleic acids from single cells

• US 10,280,459 B1
• Filed: 01/29/2019
• Issued: 05/07/2019
• Est. Priority Date: 08/20/2009
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Brenner, Sydney, Mikawa, Gi, Osborne, Robert, Slatter, Andrew
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US16/261,268
• Publication Number:
  US 20190153533A1
• Time in Patent Office: 98 Days
• Field of Search: 506 16
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

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

Claims

1. A method for multiplexed analysis of nucleic acids from single cells, the method comprising:

(a) providing a sample comprising a plurality of cells, wherein a single cell of the plurality of cells comprises a plurality of sample polynucleotides;

(b) performing primer extension to generate a plurality of tagged polynucleotides from said plurality of sample polynucleotides 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 originating from other single cells; and

(iii) a second tag sequence distinguishing said sample polynucleotide from other sample polynucleotides originating from said same single cell;

(c) amplifying said tagged polynucleotide, thereby generating a plurality of amplified polynucleotides; and

(d) sequencing said plurality of amplified polynucleotides to determine sequences of the amplified polynucleotides corresponding to the sample sequence, the first tag sequence, and the second tag sequence of the tagged polynucleotide; and

(e) using the sequences determined in step (d) to count sample polynucleotides for multiple different sample polynucleotides of multiple different single cells of said plurality of cells.

2. The method of claim 1, wherein the plurality of sample polynucleotides comprise messenger RNA (mRNA).

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

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

5. The method of claim 1, wherein step (b) comprises (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.

6. The method of claim 2, wherein step (b) comprises (i) hybridizing an oligonucleotide tag of said plurality of oligonucleotide tags to said mRNA and (ii) extending said oligonucleotide tag using said mRNA as a template to generate complementary DNA (cDNA).

7. The method of claim 6, wherein said hybridizing comprises hybridizing a poly-dT sequence of said oligonucleotide tag to a poly-dA sequence of said mRNA.

8. The method of claim 1, wherein step (b) comprises randomly associating said plurality of sample polynucleotides with said plurality of oligonucleotide tags.

9. The method of claim 1, wherein said plurality of oligonucleotide tags comprise second tag sequences that are random sequences.

10. The method of claim 1, wherein step (c) comprises generating said plurality of amplified polynucleotides using polymerase chain reaction (PCR).

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

12. The method of claim 1, wherein at least 90 percent of said plurality of tagged polynucleotides have a second tag sequence that is different from second tag sequences of the other tagged polynucleotides.

13. The method of claim 1, wherein substantially every one of said plurality of tagged polynucleotides has a second tag sequence that is different from second tag sequences of the other tagged polynucleotides.

14. The method of claim 1, wherein said plurality of oligonucleotide tags comprises a number of different second tag sequences that is larger than the number of sample polynucleotides.

15. The method of claim 14, wherein said plurality of oligonucleotide tags comprises a number of different second tag sequences that is at least ten times the number of sample polynucleotides.

16. The method of claim 15, wherein said plurality of oligonucleotide tags comprises a number of different second tag sequences that is at least one hundred times the number of sample polynucleotides.

17. The method of claim 1, wherein said plurality of oligonucleotide tags comprises at least 200,000 different second tag sequences.

18. The method of claim 1, wherein an oligonucleotides tag of said plurality of oligonucleotide tags comprises a sequencing adaptor.

19. The method of claim 18, wherein step (d) comprises hybridizing said amplified polynucleotides or derivatives thereof to a solid support via said sequencing adaptor or derivative thereof.

20. The method of claim 19, wherein said solid support is a bead.

21. The method of claim 1, wherein step (e) comprises using second tag sequences of said plurality of amplified polynucleotides to determine that said plurality of amplified polynucleotides are amplified from said tagged polynucleotide.

22. The method of claim 1, wherein step (e) comprises using sample sequences, first tag sequences, and second tag sequences of said plurality of amplified polynucleotides to determine that said plurality of amplified polynucleotides are amplified from said tagged polynucleotide.

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

24. The method of claim 1, wherein step (e) comprises using second tag sequences of said plurality of amplified polynucleotides to provide a digital count of said sample polynucleotides.

25. The method of claim 1, wherein said plurality of oligonucleotide tags are generated by combinatorial synthesis from a defined set of subunits.

26. The method of claim 1, wherein said plurality of sample polynucleotides are pooled with sample polynucleotides from other single cells of said plurality of cells prior to the generating of step (b).

27. The method of claim 1, wherein said plurality of tagged polynucleotides are pooled with tagged polynucleotides from other single cells of said plurality of cells prior to the amplifying of step (c).

28. The method of claim 1, wherein said plurality of amplified polynucleotides are pooled with amplified polynucleotides from other single cells of said plurality of cells prior to the sequencing of step (d).

29. The method of claim 1, wherein step (e) comprises using first tag sequences of said plurality of amplified polynucleotides to correlate the sample sequences of said plurality of amplified polynucleotides with the single cell from which the sample sequences are derived based on amplified polynucleotides from the same cell having the same first tag sequence.

30. The method of claim 1, wherein step (e) comprises using second tag sequences of said plurality of amplified polynucleotides to correlate the sample sequences of said plurality of amplified polynucleotides with the sample polynucleotide from which the sample sequences are derived based on amplified polynucleotides from the same sample polynucleotide having the same second tag sequence.