Methods for analyzing nucleic acids from single cells

• US 10,155,981 B2
• Filed: 08/15/2017
• Issued: 12/18/2018
• 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:
  US15/677,957
• Publication Number:
  US 20180080075A1
• Time in Patent Office: 490 Days
• Field of Search: 506 4
• 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 of analyzing nucleic acids from a plurality of single cells, the method comprising:

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

(b) generating a plurality of tagged polynucleotides from the plurality of sample polynucleotides, wherein each tagged polynucleotide comprises;

(i) a sequence from a sample polynucleotide of the plurality of sample polynucleotides; and

(ii) a multiplex identifier (MID) sequence comprising;

I. a first tag sequence associated with the single cell from which the sample polynucleotide is derived, wherein the first tag sequence is a different sequence for different single cells in the plurality of single cells; and

II. a second tag sequence distinguishing the sample polynucleotide from other sample polynucleotides derived from the same single cell;

(c) sequencing the plurality of tagged polynucleotides to obtain a plurality of identified polynucleotide sequences;

(d) using the first tag sequence to correlate the identified polynucleotide sequence with the single cell from which the identified polynucleotide sequence is derived; and

(e) using the second tag sequence to correlate the identified polynucleotide sequence with the sample polynucleotide from which the identified polynucleotide sequence is derived.

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

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

4. The method of claim 3, wherein the sample polynucleotides comprise mRNA.

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

6. The method of claim 1, wherein the tagged polynucleotides are generated through PCR or linear amplification of the plurality of sample polynucleotides using an adapter sequence comprising the MID sequence and an amplification primer.